1
Department of Molecular Medicine and Surgery
Karolinska Institutet, Stockholm, Sweden
New insights in parathyroid
hormone secretion
- with focus on exocytosis and calcium signaling
Ming Lu
Stockholm 2010
2
Supervisors
Robert Bränström, Associate Professor
Department of Molecular Medicine and Surgery
Karolinska Institutet, Stockholm, Sweden
Lars-Ove Farnebo, Professor
Department of Molecular Medicine and Surgery
Karolinska Institutet, Stockholm, Sweden
Lars Forsberg, Ph.D
Department of Molecular Medicine and Surgery
Karolinska Institutet, Stockholm, Sweden
Faculty opponent
Peter Stålberg, Associate Professor
Department of Surgical Sciences
Uppsala University, Uppsala, Sweden
Examination board
Bo Rydqvist, Professor
Department of Physiology and Pharmacology
Karolinska Institutet, Stockholm, Sweden
Olle Kämpe, Professor
Department of Medical Sciences
Uppsala University, Uppsala, Sweden
Per Uhlén, Associate Professor
Department of Medical Biochemistry and Biophysics
Karolinska Institutet, Stockholm, Sweden
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet. Printed by Repro Print AB 2010.
Copyright © Ming Lu, 2010
ISBN 978-91-7409-881-5
3
世上唯一不能倒退的是时间，别在意过去，抓住现在，规划未来 No one can turn back the clock, so don’t regret the past, hold the presence and look forward To My Family
- 送给爱我的家人
4
Thesis defense: Wednesday May 19, 2010 at 9 AM Location: L8:00, CMM, Karolinska University Hospital, Stockholm, Sweden
5
Abstract
The parathyroid is distinguished from other endocrine systems since its primary stimuli
inhibit hormone secretion. It is generally considered that extracellular Ca2+
binds to a
membrane receptor (calcium sensing receptor, or CaSR), and via second messengers
modulate parathyroid hormone (PTH) secretion. However, the intracellular signaling
pathway is not completely characterized, but intracellular Ca2+
concentration (Ca2+
i) is
set aside as a centre mediator for this regulation.
In regulated hormone secretion, or exocytosis, synaptosomal-associated proteins
(i.e. SNAPs and others) are one of the key-players in hormone vesicle and membrane
fusion. These proteins also refered to as SNARE-proteins are widly expressed in
neurons and neuroendocrine cells. In paper I, immunohistochemistry and Western blot
were used to investigate expression of SNARE-proteins, i.e. SNAP-25, SNAP-23,
Syntaxin1 and VAMP in normal and pathological human parathyroid tissues. SNAP-25
and Syntaxin1 were absent in normal parathyroid, but expressed in 20% of chief cell
adenoma and 45% of parathyroid carcinomas. SNAP-23 and VAMP were expressed in
all parathyroid samples, indicating that SNAP-23 and VAMP, rather than SNAP-25
and Syntaxin1, play a central role in exocytosis in human parathyroid cell.
Ca2+
i has a pivotal role in the stimulation-secretion coupling in parathyroid cell,
and study II and III focuse on this process. Increased levels of extracellular Ca2+
triggers Ca2+
influx in parathyroid cells, but the mechanism for this influx is not
completely understood. Using patch-clamp technique and Ca2+
i measurements (Fura-2),
we show the presence of store operated calcium current in human parathyroid and that
this is constituted by TRPC1, STIM1 and Orai1 ternary complex (Paper II).
In addition, calmodulin and calmodulin dependent protein kinase II (CaMKII),
two important proteins in Ca2+
i handling, are demonstrated in human parathyroid cells.
Blocking of calmodulin and CaMKII regulated activity results in an increase of PTH
secretion that was disassociated from Ca2+
i. Our findings suggest that calmodulin and
CaMKII are involved in PTH secretion (Paper III). Interesting from a clinical
perspective is that a negative correlation was seen between serum calcium and
phosphorylated CaMKII protein level.
Finaly, in paper IV, we show that at least two types of Ca2+
-activated K+
channels, i.e. KSK and KBK channel, are expressed in the parathyroid cell. These
channels are sensitive to specific peptide toxin blockers (apamin, charybdotoxin) and
activator (NS-11021). Modulation of these channels affects the membrane potential,
and hence the PTH secretion.
In summary, the stimulation-secretion coupling in the parathyroid cell is complex and
in some aspects unique. This thesis characterizes different parts of this process, from
Ca2+
i handling and influx, ion channel events to exocytosis. In vivo, all parts may
influcence each other and regulate PTH secretion.
6
List of Publications
This thesis is base on the following papers, which are referred to by their Roman
numbers in the text:
I. Lu M, Forsberg L, Höög A, Juhlin CC, Vukojević V, Larsson C,
Conigrave AD, Delbridge LW, Gill A, Bark C, Farnebo LO, Bränström R.
Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25,
Syntaxin1 and VAMP in human parathyroid tissue.
Molecular and Cellular Endocrinology 2008:287, 72-80.
II. Lu M, Bränström R, Berglund E, Höög A, Björklund P, Westin G, Larsson
C, Farnebo LO, Forsberg L.
Expression and association of TRPC subtypes with Orai1 and STIM1 in
human parathyroid.
Journal of Molecular Endocrinology 2010: 44, 285-294
III. Lu M, Berglund E, Larsson C, Höög A, Farnebo LO, Bränström R.
Involvement of Calmodulin and Calmodulin dependent protein kinase II
(CaMKII) in human parathyroid hormone secretion.
Submitted.
IV. Lu M, Forsberg L, Mun HC, Vukojevic V, Berggren PO, Conigrave AD,
Larsson C, Farnebo LO, Bränström R.
Expression of Ca2+
-activated K+ channels in human parathyroid cell:
potential role in hormone secretion.
Manuscript.
7
Related Publication
Zhang F, Dey D, Bränström R, Forsberg L, Lu M, Zhang QM, Sjöholm A.
BLX-1002, a novel thiazolidinedione with no PPAR affinity, stimulates AMP-activated
protein kinase activity, raises cytosolic Ca2+
, and enhances glucose-stimulated insulin
secretion in a PI3K-dependent manner.
American Journal of Physiology: Cell Physiology 2009:296, C346-354.
8
Table of content
Introduction ............................................................................................................... 11
Anatomy and histology of parathyroid gland .................................................... 12
Biosynthesis and metabolism of parathyroid hormone (PTH) ........................... 13
Biological effects of PTH ................................................................................. 14
Diseases in parathyroid glands ......................................................................... 16
Primary hyperparathyroidism
Secondary hyperparathyroidism
Hypoparathyroidism
Calcium signaling ....................................................................................................... 17
Ca2+
mobilization and Ca2+
influx .................................................................... 17
Ca2+
binding proteins ....................................................................................... 19
Hormone secretion – exocytosis ................................................................................. 20
Regulation of PTH secretion ..................................................................................... 21
Calcium sensing receptor (CaSR)..................................................................... 21
Vitamin D ........................................................................................................ 23
Phosphate ........................................................................................................ 24
Fibroblast growth factor 23 and α-klotho ......................................................... 24
Other regulators, like divalent cations, Lithium, ATP, estrogen, and prolactin .. 26
Aim of study ............................................................................................................... 28
Materials and methods............................................................................................... 29
Human parathyroid specimens ......................................................................... 29
Immunohistochemistry .................................................................................... 29
Western blot .................................................................................................... 30
Protein complex immunoprecipitation (Co-IP) ................................................ 31
Reverse transcription (RT) polymerase chain reaction (PCR) and
quantitative real-time PCR (qRT-PCR) ............................................................ 31
Human parathyroid cell preparation ................................................................. 33
Hormone secretion ........................................................................................... 34
Intracellular Ca2+
measurements with Fura-2 ................................................... 34
Electrophysiology technique ............................................................................ 36
9
Results and discussion ............................................................................................... 38
Paper I - Heterogeneous expression of SNARE proteins SNAP-23,
SNAP-25, Syntaxin1 and VAMP in human parathyroid tissue ......................... 38
Paper II - Expression and association of TRPC subtypes with Orai1 and
STIM1 in human parathyroid ........................................................................... 40
Paper III - Involvement of calmodulin and calmodulin dependent kinase II
(CaMKII) in parathyroid hormone secretion .................................................... 41
Paper IV - Expression of Ca2+
-activated K+ channels in human parathyroid cell:
potential role in hormone secretion ................................................................... 42
Conclusion ................................................................................................................. 44
摘要............................................................................................................................ 46
Acknowledgements .................................................................................................... 47
References .................................................................................................................. 51
10
Abbreviations
ATP Adenosine-5'-triphosphate
Ca2+
e Extracellular Ca2+
concentration
Ca2+
i Intracellular Ca2+
concentration
CaMKII Calmodulin-dependent protein kinases II
cAMP Cyclic adenosine monophosphate
CaSR Calcium sensing receptor
Co-IP Protein complex immunoprecipitation
ER Endoplasmic reticulum
FGF23 Fibroblast growth factor 23
IP3 Inositol-1,4,5- trisphosphate
KBK channel Big conductance Ca2+
activated K+ channels
KSK channel Small conductance Ca2+
activated K+ channels
Orai1 Calcium release-activated calcium channel protein 1
pHPT Primary hyperparathyroidism
PKA, PKC Protein kinase A, and C
PLA2, PLC, PLD Phospholipase A2, C and D
PTH Parathyroid hormone
qRT-PCR Quantitative real-time polymerase chain reaction
RT-PCR Reverse transcription polymerase chain reaction
RYRs Ryanodine receptors
SERCA Sarco (endo) plasmic reticulum Ca2+
-ATPase
sHPT Secondary hyperparathyroidism
SNAP-23/25 Synaptosomal-associated protein 23/25
SNARE Soluble NSF Attachment Protein receptors
SOCS Store operated calcium channels
STIM1 Stromal interaction molecule 1
TRP channel Transient receptor potential channel
VAMP2 Vesicle-associated membrane protein 2 (Synaptobrevin 2)
VOCCs Voltage operated calcium channels
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Introduction
There is a tiny endocrine organ in the neck called parathyroid gland. It produces
parathyroid hormone (PTH), an important hormone for calcium regulation in our body.
Loss or gain of function in this organ leads to multiple clinical features. However, the
parathyroid gland has been unobserved until middle of 18th century. Richard Owen, a
conservator of Hunterian Museum at the Royal College of Surgeons of England, gave
the first description of parathyroid gland in 1852. By discovery in Indian rhinoceros, he
reported ‘a small compact yellow glandular body was attached to the thyroid at the
point where the veins emerge’ (Rhys Evans et al. 2004). That small gland was named
as parathyroid by Ivar V. Sandström, a postgraduate student in Uppsala, Sweden in
1877 (Carney 1996; Eknoyan 1995). It was observed that removal of parathyroid
glands led to ‘tetany’ in both animals and human, such as symptoms of involuntary
systemic muscles contraction known as Chvostek’s sign, Trousseau’s sign and
carpopedal spasm (Pool 1907; Vincent 1904), but the reason was not clear at that
moment. One claim was that parathyroid glands might be responsible for cleaning of
toxic substances, e.g. dimethylguanidine. The idea was that after removal of
parathyroid glands, accumulation of toxicants in the body caused tetany. However, the
scientific proof for this idea was poor. Simultaneously, more and more evidences
showed that tetany was associated with low serum calcium, and that the parathyroid
glands were involved in regulation of serum calcium since both calcium supplement
and injection of parathyroid extracts prevented tetany (Branham 1908; Collip 1925;
Shepardson 1927; Stewart & Percival 1927; Willard 1935).
Besides calcium, phosphate was also observed to be regulated by PTH. It was figured
out lately that not only parathyroid but also kidney and bone were involved the
maintenance of serum calcium and phosphate (Ellsworth 1932). In the late decades of
1900’s, many studies were done to understand the secretion of PTH. It was found that
calcium was the main regulator of PTH secretion. Within a narrow range, PTH is
negatively regulated by calcium. A break trough in the understanding of parathyroid
physiology was the identification and cloning of calcium sensing receptor (CaSR)
(Brown et al. 1993). CaSR are located in cell membrane, facing the calcium sensing
epitope towards the extracellular space whereby sensing small changes in circulating
calcium concentration. CaSR is a G-protein linked receptor, and acts via a
phospholipase C (PLC) pathway including production of inositol-1,4.5-trisphosphate
(IP3) and release of Ca2+
from intracellular stores (Brown 1991). Intracellular Ca2+
12
(Ca2+
i) has been shown to have a pivotal role in regulation of PTH secretion (Shoback
et al. 1984). The central role of CaSR in the stimulus-secretion coupling is well
understood since mutations in CaSR cause changes in PTH secretion. Mutations in
CaSR in the population are rare and can only explain a minority of parathyroid
diseases. However, the finding of CaSR has provided a new target for treatment of
parathyroid diseases (Brown 2007). Another breakthrough in parathyroid gland
physiology was the cloning of PTH/PTHrP (parathyroid hormone related peptide)
receptor by Juppner in 1991 and Schipani in 1993 (Juppner et al. 1991; Schipani et al.
1993). The discovery of PTH receptor on bone and kidney enables us to know more
about the interaction of PTH and its target organs (Potts 2005).
Anatomy and histology of parathyroid gland
Anatomy
Parathyroid glands are usually composed of four glands, two superior glands and two
inferior glands; the location is on the backside of the thyroid gland in the neck (Figure
1). The superior glands arise from the fourth endodermal pharyngeal pouches, and the
inferior glands originate from the third endodermal pharyngeal pouches. The weight of
each gland is on average 40-50 mg. The location and the number of the glands may
vary in normal population. For example, they can be located down in the thymus or be
five to eight glands in a normal person. Their color is yellow-brown. In theory, they are
easily distinguished from the reddish structure of thyroid; nevertheless, they are
sometimes hard to recognize from thyroid and fat in reality. It is high demand for the
experience of the surgeon for identification (Gardner & Shoback 2007).
Figure 1. Location of parathyroid glands.
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Histology
Histologically, normal parathyroid glands are composed of epithelial cells and
stromal fat. Stromal fat is around 30% at age of 25 and decreases with aging. The
epithelial cells include chief cells and oxyphilic cells, and majority is chief cells. The
chief cells are small (8 µm), light, centrally round nucleus, and contain intracellular
fat. Some of them have extremely clear cytoplasm and well-defined cell membrane
due to abundant cytosolic glycogen, namely water clear cells. Chief cells are PTH
producing cells in parathyroid glands. Oxyphilic cells are usually slightly larger (12
µm) with acidophilic cytoplasm due to large amounts of mitochondria, which is
distinguished from smaller and weakly acidophilic cytoplasm of chief cells. Their
numbers increase with age. The number of oxyphilic cells is much less than chief
cells in normal parathyroid and their function are still not very clear. However,
oxyphilic cells was found to release PTH in sHPT patients (Rudberg et al. 1986;
Tanaka et al. 1996) and PTH producing oxyphilic cell adenomas are reported from
time to time (Allen & Thorburn 1981; Bedetti et al. 1984; Zhou et al. 2003), therefore,
it is also considered as functional cells.
Figure 2. Transmission Electron Microscopy image of normal parathyroid cell
(1: oxyphilic cell, 2: chief cell)
.
Biosynthesis and metabolism of PTH
Mature PTH is an 84-amino-acid long peptide. Its gene is located on chromosome 11.
The same as other hormone, PTH is initially synthesized as prepro-PTH with 115
14
amino acids in the rough endoplasm reticulum. After the first cleavage removing 25-
amino-acid on the N-terminal, it becomes pro-PTH. The leader sequence of pro-PTH
serves as a signal transporting pro-PTH to Golgi apparatus. Then a second cleavage
with six amino acids on the N-terminal results in the formation of mature PTH. After
mature PTH leaves the Golgi apparatus, it is stored in secretory-vesicles and waiting
for secretion signal (Habener 1981).
Full length PTH (1-84) has a half-life of 2-4 minutes in blood. After released from
parathyroid glands into blood circulation, it is further cleaved in liver and kidney at
the 33–34 and 36–37 positions to produce an amino terminal fragment and a carboxyl
terminal fragment. The amino terminal fragment (1-34) is the biologically active part
of PTH. It binds to PTH receptor and activates cell signaling in target tissues. The
active fragments only constitute small part of circulating PTH and soon are degraded,
whereas the carboxyl terminal fragments consist of majority of circulating PTH with
longer life. They are mainly cleared by kidney, so they accumulate in renal failure.
Instead of acting with PTH receptor, they have their own receptors but the
physiological importance is not completely understood.
The first immunoassay of PTH was invented by Berson and Yalow in 1963 (Berson
et al. 1963). After decade’s development, the measurement of intact PTH has become
precise and fast. Current assays compose of two-site immunoradiometric assay or
immunochemiluminescent assay techniques, by which the normal range for PTH is
approximately 10–60 ng/L (1–6 pmol/L).
Biological effects of PTH
Calcium is a key intracellular messenger and co-factor of various enzymes. It plays
diverse roles in multiple organs. It modulates neuromuscular excitability,
proliferation, apoptosis, exocytosis and so on. Calcium levels vary greatly between
extracellular and different cellular compartments in the human body; ranging from
1.15-1.35 mmol/L in blood (reference value at Karolinska University Hospital).
Maintenance of serum calcium level and calcium gradient in cells are very important
for the physiological and pathophysiological functions of our body. PTH and Vitamin
D are the main regulators of blood calcium level.
15
When serum calcium is low, it gives a positive signal to parathyroid gland resulting in
an increase of PTH secretion. The release of PTH causes mobilization of calcium
from bone and calcium absorption from kidney and intestine. The increased blood
calcium in turn gives a negative feedback to parathyroid gland and inhibits PTH
secretion. This response feedback system controls serum calcium within a narrow
range (Figure 3).
An increase of PTH affects renal tubular cells within minutes. It facilitates calcium
influx into distal renal tubules by transporting and activating dihydropyrine-sensitive
Ca2+
channel in apical plasma membrane (Bacskai & Friedman 1990; Lau &
Bourdeau 1995). It promotes phosphate excretion by decreasing type II Na+/Pi co-
transporter activity and protein content at the apical brush-border membrane of
proximal tubules (Kempson et al. 1995; Murer et al. 1996).
It was originally believed that the effect of PTH on intestinal calcium absorption was
only indirect. PTH stimulates the biosynthesis of 1α-hydroxylase in kidney resulting
in increased production of active Vitamin D metabolite 1,25(OH)2D3 (1,25-
dihydroxycholecalciferol) (Henry et al. 1974), which directly enhances both calcium
and phosphate reabsorption through the small intestine (Norman 1979). However,
recent studies found the expression of functional PTH receptor in intestine; and more
evidence indicates that PTH may have a direct effect on calcium transport and uptake
in intestine (Nemere & Larsson 2002).
Bone is also a direct target of PTH. The effect of PTH on bone is dualistic, including
both anabolic and catabolic effect. High level of PTH causes bone loss and
intermittent administration induces bone remodeling. PTH enhances proliferation and
differentiation of osteoblast cells responsible for bone formation. But it can also
stimulate ostoclast cells through activation of Receptor Activator for Nuclear Factor
B Ligand/Receptor Activator for Nuclear Factor B (RANKL/RANK) signaling which
is required for osteoclastogenesis, resulting in increase of calcium mobilization (Qin
et al. 2004). It is the reason why hyperparathyroidism patients develop osteoporosis
and osteitis fibrosa cystica. However, intermittent administration of PTH induces
bone reconstruction and rarely causes bone resorption, therefore it has been approved
for treatment of osteoporosis.
16
The action of PTH on target organ is mediated by type 1 PTH receptor, a G protein
receptor that is highly expressed in kidney and bone. Interaction of PTH and PTH
receptor activates adenylate cyclase/cAMP/protein kinas A (PKA) pathway or
phospholipase C/protein kinase C (PKC) pathway, which in turn regulate down-
stream biological actions (Schluter 1999).
Figure 3. Simplified model of human calcium homeostasis.
Diseases in parathyroid glands
Primary Hyperparathyroidism
Hyperparathyroidism refers to excess PTH secretion. The cause of primary
hyperparathyroidism (pHPT) is parathyroid adenoma (80-90%), hyperplasia (10-20%)
and carcinoma (<1%). The prevalence is about 1 to 4 cases per 1000 of the general
population, but can be much higher in the elderly population above the age of 50
(Nilsson et al. 2002). The sex ratio between women and men is about 3:1 (Heath
1989). The peak is among menopause women. It has been reported the prevalence in
menopause women reaches 3.4% in Sweden (Lundgren et al. 2002; Lundgren et al.
1997; Palmer et al. 1988). It is the most common cause of hypercalcemia in
unselected patients.
17
Overproduction of PTH increases mobilization and absorption of calcium from bone
and increases renal excretion of phosphate, resulting in hypercalcemia and
hypophosphatemia. Clinic features include osteoporosis, peptic ulcer, pancreatitis,
hypertension, anxiety, kidney stone, and so on. However, many patients lack
symptoms and some of them have normal level of calcium.
Secondary hyperparathyroidism
One important cause for secondary hyperparathyroidism (sHPT) is vitamin D
deficiency; it is a common complication of chronic renal failure. Initially, reduction
of Vitamin D and low ionized Ca2+
stimulate PTH synthesis and release. As disease
progresses, Vitamin D receptor and CaSR decrease in parathyroid gland (Kifor et al.
1996), promoting proliferation of parathyroid gland (Tokumoto et al. 2005; Yano et
al. 2000) and resulting in insensitivity of parathyroid to calcium, which further
enhances secretion of PTH. Moreover, high phosphate and uremia can induce
hyperplasia of parathyroid gland independently.
Hypoparathyroidism
Hypoparathyroidism refers to impairment of PTH secretion. It is mostly a
complication to thyroidectomy. Primary hypoparathyroidism is rare and due to
autoimmune destruction of parathyroid gland. It may also be due to activating
mutations in CaSR. Hypocalcemia and hyperphosphatemia are major laboratory
findings, and clinical magnificence is hypocalcemia tetany. Supplements of calcium
and vitamin D are fundamental treatment.
Calcium signaling
Calcium mobilization and calcium influx
Calcium is a critical divalent ion involved in all cellular processes. It regulates
contraction, secretion, apoptosis, proliferation, differentiation, fertilization, and so on.
Normally, extracellular free calcium ion concentration around 1.2 mM, whereas
calcium concentration within the cell (intracellular Ca2+
concentration, or Ca2+
i) can
be as low as 100 nM in resting stage and rise to 1-10 M when cell is excited.
Increased level of Ca2+
i is the result of calcium mobilization from ER and calcium
influx from extracellular spaces. This action involves efforts of several ion channels,
18
exchangers and second messengers and consumes lots of energy (Clapham 1995;
Clapham 2007).
Within calcium signaling, SERCA pump on ER helps to take in Ca2+
and channels
with IP3 receptor or Ryanodine receptors are transporting Ca2+
out to cytosol. IP3
receptors usually are expressed in non-excitable cells and are activated by IP3,
whereas calcium or Cyclic ADP Ribose activates Ryanodine receptors in excitable
cells. Calcium permeable channels in plasma membrane mediate calcium influx,
including voltage-operated calcium channels (VOCCs) and voltage-independent
calcium channel. VOCCs are found in excitable cell and are activated by membrane
depolarization. Voltage-independent calcium channels comprise three channels. 1)
Ligand-gated ionotropic channels are channels operated by ligands (chemical
messenger), e.g. ATP, serotonin, Gamma-aminobutyric acid (GABA). They are
usually non-selective channels for Ca2+
permeation. 2) Transient receptor potential
channel (TRP) is a big non-selective cation channels family composed of three
homologous: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin) and three
distantly related subfamilies (TRPA (ankyrin), namely, TRPP (polycystin) and
TRPML (mucolipin)) in mammals. Most of TRP channels are Ca2+
permeable
channels (Inoue 2005; Inoue et al. 2003; Pedersen et al. 2005). They are widely
expressed in body and are activated by a variety of stimulus. For example, TRPV1
channel is activated by heat and capsaicin, TRPV4 is stimulated by osmolarity and
TRPC channels are regulated by PLC signaling cascades activated by G-protein
coupled receptor or tyrosine-kinase receptor. 3) Store operated calcium channels
(SOCs) are activated by the depletion of intracellular calcium store. This is the
dominant calcium entry pathway in non-excitable cells, such as epithelial cells,
hepatic cells and blood cells. Two well established components of SOCs are STIM1
(Stromal interaction molecule 1) located on ER membrane and Orai1 (Calcium
release-activated calcium channel protein 1) on plasma membrane (Frischauf et al.
2008; Hewavitharana et al. 2007). STIM1 senses decrease of Ca2+
in ER and drives
ER membrane to plasma membrane (Zhang et al. 2005). The binding of STIM and
Orai1 facilitates the opening of Orai1 channels and calcium influx (Soboloff et al.
2006). Recently, TRP channels, especially TRPC1 channel, are also considered as
components of SOC channels. The interaction of TRPC channel, STIM1 and Orai1
has been reported in many cells types (Cheng et al. 2008; Ong et al. 2007).
Thapsigargin, a specific blocker of SERCA, is the most common tool for
19
investigation of SOCs. Moreover, Na+/Ca
2+ exchanger and plasma membrane calcium
ATP pump (PMCA) are pumps for moving out Ca2+
.
Figure 4. Schematic illustration of calcium signaling.
Calcium binding proteins
There are many Ca2+
binding proteins involved in calcium signaling. They may act as
Ca2+
sensors or receptors for signal transduction or may exist as Ca2+
buffers.
Depending of the type of Ca2+
binding domain, they are classified into EF-hand,
annexin and C2 region. EF hand proteins, for example Troponin C modulates muscle
contraction, and calmodulin regulates several enzymes (e.g. CaMKI, II and IV,
phosphodiesterase) involved in most cellular processes, such as exocytosis, muscle
contraction, ion channel activation, and cell proliferation. Calbindin-D9K and D28K
regulate calcium transport in intestine and kidney, and S100 is involved in cell
growth. Annexin proteins modulate phospholipase-A2 (PLA2) inhibition and ion
channel activity. C2 domain proteins, for example, Rab3A regulates secretory
vesicles trafficking, and synaptotagmin regulates exocytosis. Some proteins, such as
calsaquestrin, calreticulin, bind to calcium as storage (Clapham 1995; Niki et al.
1996).
20
Hormone secretion-exocytosis
The process of packing proteins into vesicles and releasing it out to the extracellular
space is called exocytosis. Generally, exocytosis can be categorized into regulated
exocytosis and constitutive exocytosis. Constitutive exocytosis occurs in all cells. It
spontaneously and continuously transports proteins from the Golgi apparatus to the
plasma membrane. However, in neurons, as well as in endocrine cells, proteins are
stored in secretory vesicles and released only in response to stimulation of certain
external signal, namely regulated exocytosis (Alberts et al. 2007). The same as in
other endocrine cells, parathyroid hormone was observed to be stored in large
secretory vesicles waiting for release using electron microscopy by Setoguti and
colleges. The same authors also showed that PTH is rapidly released by exocytosis
when serum calcium was decreased (Setoguti et al. 1981; Setoguti et al. 1988).
Exocytotic process includes vesicle trafficking, tethering, docking, priming and
fusion, which involve several proteins and protein-protein interactions. Among them,
SNARE complex, munc-18 proteins and small GTP binding protein Rab3A are
essential for Ca2+
-regulated exocytosis. SNARE refers to Soluble N-ethylmaleimide-
sensitive factor Attachment protein REceptors. It is a big membrane-bound proteins
family categorized into vesicle-SNARE (v-SNARE) on vesicle membrane and target-
SNARE (t-SNARE) on plasma membrane. The formation of a stable 4-α-helix
SNAREs complex is the core-step driving vesicle fusion. The most well established
SNARE complex consists of t-SNARE proteins SNAP-25 and Syntaxin1A and v-
SNARE protein Synaptobrevin (VAMP). Protein Rab3A transports vesicles to
docking site, and then the disassociation of munc-18 proteins from Syntaxin1A
facilitates the reversible contact of SNARE complex to plasma membrane. Following
by Ca2+
influx, Ca2+
binding to synaptotagmin, a calcium sensor promotes assembly
of SNAREs into 4-α-helix bundles and accelerates SNARE-catalyzed fusion (Chicka
et al. 2008; Gerst 1999; Tang et al. 2006). The components of SNAREs proteins vary
in different cell types.
21
Figure 5. Model of Ca2+
regulated exocytosis.
Regulation of parathyroid hormone secretion
Calcium sensing receptor
Serum calcium is the major physiological regulator of PTH secretion. In contrast to
other endocrine cells, low calcium stimulates PTH secretion, whereas high calcium
inhibits. The calcium-PTH curve is very steep between 1.1 mM to 1.3 mM, indicating
that parathyroid cells are extremely sensitive to the change in Ca2+
e level (Brown
1991). The high Ca2+
sensitivity enables us to control serum calcium within a tight
range. Practically, a calcium-PTH set point (calcium required for half-maximal
inhibition of PTH release) has been used to evaluate the individual Ca2+
sensitivity. It
has been shown cultured normal human parathyroid cells have a set-point close to 1.0
mM, and around 1.1-1.3 mM in vivo (Brown 1991). The set-point is shifted to the
right in hyperparathyroidism which means decreased Ca2+
sensitivity and shifted to
left in hypoparathyroidism indicating increased Ca2+
sensitivity. The Ca2+
sensitivity
varies between individuals. CaSR was discovered as an important factor for the
regulation of Ca2+
sensitivity in parathyroid cells (Brown et al. 1993). CaSR senses
small increase of Ca2+
e resulting in hundreds times rise of Ca2+
i which downstream
leads to inhibition of PTH secretion.
The CaSR is a member of subfamily C of G-protein coupled receptors. The gene is
located on chromosome 3q13.3-2.1. The molecular weight of the protein is 120 kDa,
and is coupled to Gαq-PLCβ complex (Brown et al. 1993). Activation of CaSR
enhances hydrolysis of phosphoinositides resulting in increased generation of IP3 and
diacylglycerol (DAG). IP3 binds to IP3 receptor in endoplasmatic reticulum inducing
22
transient calcium release from intracellular stores, followed by sustained influx of
extracellular calcium. The pathway of calcium influx is still unclear in parathyroid
cells. It has been widely debated if there are voltage gated calcium channels or not.
Although two studies have shown the expression of dihydropyridine-sensitive L-type
calcium channels in parathyroid cells (Chang et al. 2001; Yokoyama et al. 2009), K+-
induced depolarization reduces Ca2+
i (Shoback & Brown 1984b) which do not
support the present of VOCCs. On the contrary, thapsigargin, an inhibitor of SERCA,
induces a rapid increase of Ca2+
i and lowered PTH secretion, which indicate the
existence of store operated calcium entry (Ferzandi & MacGregor 1997; Shoback et
al. 1995).
Calcium is not the only ligand for CaSR. CaSR can also be activated by a number of
divalent or trivalent cations and various amino acids. Increased Ca2+
i due to the
activation of CaSR is strongly correlated to decreased PTH secretion (Shoback et al.
1983). Notable, in endocrine cells as well as in neurons, an increased Ca2+
i generally
promotes hormone secretion. It is unknown why increased Ca2+
i causes an inhibition
of PTH secretion in parathyroid cells. By using the model of permeabilizing cells,
Oetting and his colleagues found that increased Ca2+
i induced PTH secretion after
abolishment the effect of membrane potential, which implied that membrane potential
is a critical regulator for PTH secretion (Oetting et al. 1987). Our group has
previously showed that high Ca2+
e results in hyperpolarization, whereas low Ca2+
e
leads to depolarization in human parathyroid cells, most likely via Ca2+
-activated K+
channels (Valimaki et al. 2003).
Besides PLC, PLA2 and PLD signaling cascades are also activated by CaSR in
parathyroid cells (Kifor et al. 1997). The phosphorylation and activation of PLA2
generates arachidonic acid that inhibits PTH secretion via the 12- and 15-
lipoxygenase pathway (Bourdeau et al. 1992). Activation of CaSR also couples to Giα
inactivating adenylyl cyclase and reducing the production cAMP, thereby decreasing
PTH secretion (Brown et al. 1990; Brown et al. 1978; Shoback & Brown 1984a).
CaSR is known as the most important receptor for calcium regulated PTH secretion.
This statement rests on several findings; 1) Familial Hypocalciuric Hypercalcemia
(FHH), which is characterized by hypercalcemia, lower urinary calcium excretion
(hypocalciuria) and normal or increased PTH levels, is associated with inactivated
mutations of CaSR gene. 2) expression of CaSR was found to be reduced in pHPT
23
and sHPT at both mRNA and protein level (Farnebo et al. 1997; Farnebo et al. 1998).
3) reduction of CaSR is also associated with right-shift of calcium-PTH set point in
hyperparathyroidism (Corbetta et al. 2000; Kaneko et al. 1999). 4) activating
antibody binds to CaSR results in hypoparathyroidism (Goswami et al. 2004). 5)
pharmacological activation of CaSR using calcimimetic agonists (Cinacalcet or R-
568) have dose-dependent effect in reducing PTH secretion and serum calcium, and
have been used for treatment of both primary and secondary hyperparathyroidism
(Lindberg et al. 2005; Peacock et al. 2005; Shoback et al. 2003; Torres 2006). All
this point to the central role of CaSR in the parathyroid physiology. However, it has
been shown that changes of CaSR directly contributes to enhanced or impaired PTH
secretion, but it has not been possible to correlate the level of CaSR expression with
individual Ca2+
sensitivity (Cetani et al. 2000; Corbetta et al. 2000).
Vitamin D
Another important regulator of PTH secretion is the active form of Vitamin D. Vitamin
D in our body comes from skin and food. When skin is exposed to sunlight, 7-
dehydrocholesterol, a derivative of cholesterol, is converted by UVB light to vitamin-
D3. However, vitamin-D3 has low biological activity. In order to be functional, it needs
to be hydroxylated to 25(OH)D3 (25-hydroxycholecalciferol, calcidiol) by the enzyme
25-hydroxylase in the liver, and then further converted into 1,25(OH)2D3 (calcitriol) by
the enzyme 1α-hydroxylase in the kidney. Calcitriol, the most active from of vitamin D,
is critical for maintenance of blood calcium and phosphate via interaction with
intestine, kidney and bone. In case of kidney failure, inefficient 1α-hydroxylase reduces
the production of calcitriol resulting in low blood calcium. The following compensative
increase of PTH secretion is named secondary hyperparathyroidism (sHPT). Besides
regulating blood calcium, calcitriol directly suppresses PTH synthesis and secretion by
binding Vitamin D receptor on the 5’-untranslated region of PTH gene and down-
regulating PTH gene transcription (Brown et al. 1995; Demay et al. 1992). It has been
shown that calcitriol inhibits PTH secretion in cultured parathyroid cells from sHPT
patients in a dose-dependent manner and independent of vitamin D receptor
polymorphisms (Alvarez-Hernandez et al. 2003). Calcitriol also has a similar action on
PTH secretion, but is a hundred times less active than calcitriol (Ritter et al. 2006).
Clinically, analogs are an essential treatment for sHPT, especially in the early stage.
24
Phosphate
Phosphate is a positive regulator of PTH secretion. It has been found that there are three
phosphate binding proteins on 3’-UTR of PTH mRNA: AU-rich binding factor
(AUF1), N-ras(Unr) and KH-splicing regulatory protein (KSRP). High phosphate
enhanced the binding of AUF1 and Unr to PTH mRNA, which increases mRNA
stability. In low serum phosphate, KSRP recruited exosome to PTH mRMA facilitating
PTH mRNA degradation (Levi et al. 2006; Nechama et al. 2008; Sela-Brown et al.
2000). But it is still unclear that how parathyroid cells recognize phosphate. A
complementary DNA encoding Na+-Pi co-transporter, namely PiT-1, was cloned in rat
parathyroid tissue in 1998 (Tatsumi et al. 1998). Low phosphate diet increased
expression of PiT1, whereas high phosphate diet inhibited, and calcitriol also raised the
expression of PiT1, indicating that PiT1 was contributing to both phosphate and
calcitriol regulation in parathyroid. But there is no evidence that phosphate needs to be
transported into cells to exert its function. So PiT1 can be a sensor of phosphate
(Miyamoto et al. 2000; Miyamoto et al. 1999). Phosphate can also indirectly regulates
PTH secretion via production of fibroblast growth factor 23 (FGF23) from bone cells
(see discussed as below).
Fibroblast growth factor 23 and α-Klotho
Fibroblast growth factor 23 (FGF23) is a hormone released from osteblast and
osteocytes cells in bone. It is very critical for phosphate and vitamin D metabolism. It
stimulates urinary phosphate excretion and reduces production of calcitriol by
suppression of 1α-hydroxylase activity in the kidney. Ablation of FGF23 gene causes
hyperphosphatemia, hypercalcemia, high serum calcitriol, and suppressed PTH
(Shimada et al. 2004). Active mutation of FGF23 in human causes autosomal dominant
hypophosphatemic rickets (Shimada et al. 2002). Over-production of FGF23 by tumor
is the main reason for tumor-induced osteomalacia (Shimada et al. 2001). Recently, it
was found that parathyroid gland is also a target of FGF23. FGF23 receptor type 1 and
3 are found in parathyroid gland (Ben-Dov et al. 2007). Serum FGF23 was increased in
pHPT mouse model and the level was significantly correlated with serum PTH and
calcium. The level of FGF23 decreased after parathyroidectomy (Kawata et al. 2007).
But the level of serum FGF23 in HPT patients is still controversial (Kobayashi et al.
2006; Tebben et al. 2004). In vitro study on bovine parathyroid cells has shown that
25
FGF23 reduced PTH mRNA level and inhibited PTH secretion in a dose-dependent
manner (Krajisnik et al. 2007).
Klotho gene was first described in 1997 by Kuro-o and his colleagues. Mutant mice
showed multiple aging-related disorders, including short lifespan, infertility,
arteriosclerosis, osteoporosis, age-related skin changes etc. The name was given
according to Geek mythology in which klotho is a goddess who spins the thread of life
(Kuro-o et al. 1997). Interestingly, hyperphosphatemia, high serum calcitriol and
hypercalcemia were also found in klotho mutant mice, which indicate that klotho is
involved in calcium and phosphate homeostasis. Later on, α-Klotho, an isoform of
Klotho, was found to be important for PTH secretion and calcium reabsorption in
kidney. In response to low Ca2+
e, it recruits Na+/K
+-ATPase from ER to cell surface and
induces PTH secretion in parathyroid glands (Imura et al. 2007). In kidney, recruitment
of α-Klotho activates TRPV5 channels that increases Ca2+
intake from lumen into distal
convoluted tubule cell, then Ca2+
is further absorbed into blood by Na+-Ca
2+ exchanger
(Chang et al. 2005). It has reported that the expression of α-Klotho was reduced in
pHPT patients, and the level of α-Klotho was inversely correlated to serum calcium, i.e.
higher level of serum calcium, lower expression of α-Klotho. But there is no correlation
between PTH level and α-Klotho expression (Bjorklund et al. 2008). Distinguished
from CaSR, α-Klotho/Na+/K
+ATPase pathway may be more important for low Ca
2+
stimulated PTH secretion.
The fact that the α-Klotho deficient mice showed all phenotypes of FGF23-null mice
indicated an association between FGF23 and α-Klotho (Kuro-o et al. 1997; Shimada et
al. 2004). Using renal homogenate, Urakawa et al. was for the first time to show that α-
Klotho binds to FGF23. They also found that the binding of α-Klotho was essential for
the activation of FGF23 signaling since FGF receptor 1 converts to a receptor specific
for FGF23only in the presence of α-Klotho (Urakawa et al. 2006). Supplement of
FGF23 did not reduce serum phosphate in FGF23(-/-)/klotho(-/-) and klotho(-/-) mice,
nevertheless, it improved serum phosphate in wild-type and FGF23(-/-) mice, which
further proved that FGF23 regulated phosphate homeostasis dependent on α-Klotho
(Nakatani et al. 2009).
26
Other regulators
Divalent cations (Mg2+
, Mn2+
, Ba2+
, and Sr2+
)
Besides Ca2+
, many other divalent cations also influenced PTH secretion. Mg2+
was
found to have duplicate effect on Ca2+
regulated PTH secretion. When Mg2+
was less
than 0.8mM, it impaired low Ca2+
stimulated PTH release. 0.8-1 mM Mg2+
gave the
maximal stimulation of PTH secretion, and when it was higher than 1 mM, it
progressively inhibits PTH release (Takatsuki et al. 1980). When compared the
efficiency on PTH secretion, Mn2+
>Ca2+
=Sr2+
>Mg2+
. Ba2+
has not obvious effect at
concentration of 0.5-3.25 mM (Wallace & Scarpa 1982). The inhibition of PTH
secretion by other cations was not due to Ca2+
influx because Sr2+
, Mg2+
and Ba2+
were
only evoked rapid transient but no sustained increased in Ca2+
i (Nemeth & Scarpa
1987). Mn2+
also induced transient increased in Ca2+
i, but followed by a decrease of
Ca2+
uptake (Johansson et al. 1988). Similarly, those ions were found to increase IP3,
IP2, and IP1 in parathyroid cells (Shoback et al. 1988). Therefore, there it is likely that
membrane receptors are activated by divalent cations and induced IP3 related Ca2+
mobilization. The mechanism is still not clear.
Lithium
After the first case of Lithium associated hyperparathyroidism was reported in 1973
(Garfinkel et al. 1973), several studies have further conformed the stimulation effect of
Lithium on PTH secretion (Birnbaum et al. 1988; Graze 1981; Saxe et al. 1995). The
stimulation of PTH is likely due to decreased Ca2+
e sensitivity on receptor level (Brown
1981; McHenry et al. 1991), since Lithium do not change the level of Ca2+
i and cAMP
(Wallace & Scarpa 1983). Preincubation with LiCl caused a dose-dependent right-shift
of ‘calcium set point’ in bovine parathyroid cells (Brown 1981). Interestingly, CaSR
agonist, normalizes the hypercalcemia and hyperparathyroidism induced by lithium
(Sloand & Shelly 2006).
Adenosine-5'-triphosphate
Adenosine-5'-triphosphate (ATP) and its non-metabolizable analog Adenosine 5′-O-(3-
thio) triphosphate (ATPγS) were found to inhibit PTH secretion (Nemeth & Kosz
27
1989). When applied to parathyroid cell, they caused a transient increase of Ca2+
i due
to mobilization of intracellular calcium store, which may suggest the presence of
purinoceptor P2 receptor (Nemeth & Kosz 1989). But more studies are needed to
further elucidate this pathway.
Estrogen
The prevalence of hyperparathyroidism is increased in postmenopausal women, which
indicates a possible association between estrogen and parathyroid disease. Estrogen
receptor α and β has been demonstrated in human parathyroid cells, and it was
predicted an increased expression in pathological parathyroid tissue compared with
normal tissue (Wong et al. 2002). In vitro studies show that physiologic concentration
of estrogen or estrogen receptor modulators increased PTH secretion (Duarte et al.
1988; Greenberg et al. 1987). In vivo, estrogen replacement improved calcium
sensitivity of parathyroid glands (Boucher et al. 1989; Zofkova et al. 1993).
Prolactin
Many studies have shown that hyperprolactinemia is associated with increased bone
loss (Palmer et al. 1988). This correlation is not fully elucidated. Some studies have
shown it may be due to hypogonadism, others found that prolactin has direct effects on
vitamin D and calcium metabolism, whereas, others suggests that prolactin has its
mode of action on bone directly (Seriwatanachai et al. 2008). From our perspective, it
is interesting that one study showed that prolactin directly stimulates PTH secretion in
bovine parathyroid cells (Magliola & Forte 1984).
28
Aim of the study
The overall aim of the thesis was to enhance the understanding of stimulus-secretion
coupling in human parathyroid.
More specific, the aims for individual papers were:
1. to characterize the exocytotic proteins in human parathyroid, with special
focus on SNAP-23, SNAP-25, Syntaxin1 and VAMP (paper I).
2. to identify the mechanism of Ca2+
entry and Ca2+
handling proteins in human
parathyroid cells (paper II and III).
3. to investigate the role of Ca2+
-activated K+ channel and membrane potential in
relation to PTH secretion (paper IV).
29
Materials and Methods
Human parathyroid specimens
All molecular analysis in this study was done on human parathyroid samples. All
parathyroid tissues were classified according to the World Health Organization (WHO)
guideline (DeLellis RA et al. 2004). In paper I, besides seven parathyroid adenomas
and fifteen normal parathyroids that were collected from the Mater Private Hospital,
North Sydney, NSW, Australia or the Royal North Shore Hospital, St. Leonards, NSW,
Australia and seven parathyroid carcinomas that were collected world widely, all other
parathyroid tissues were obtained from ‘Endocrine BioBank’ in the Department of
Oncology and Pathology at Karolinska University Hospital. The same twenty
parathyroid adenomas from Karolinska University Hospital were used in Paper II, III
and IV for protein and gene expression analysis. All parathyroid adenomas used for
PTH secretion study, electrophysiological study and Ca2+
i measurement in paper II, III
and IV were gotten freshly from surgery at Karolinska University Hospital between
2006 and 2009. All samples collection was under patient’s consent and approved by
local ethic committees.
Immunohistochemistry (Paper I and IV)
Immunohistochemistry (IHC) is a method to study the spatial expression pattern of a
certain protein within a tissue or other cellular structures. The process includes the use
of an antibody specifically binding to one antigen and then visualization of the antigen-
antibody complex by fluorescent dye, enzyme or radioactive element.
Generally, IHC is performed in formalin fixed paraffin-embedded tissue sections.
Paraffin-embedded sections provide a clear tissue structure and cell morphology, but
some antigens are easily degraded by formalin fixation and paraffin embedding.
Therefore, frozen tissue section is required in certain analysis. The tissues are freshly
frozen, then cut and fixed with cold acetone or formalin. Frozen secretions conserve
more antigens, but poor morphology reduces the specificity and quality. Antigen
retrieval is a process specifically used for Paraffin-embedded sections, in which
sections are heated with Citrate or EDTA or digested with enzyme to break the protein
30
cross-linking formed by formalin fixation and unmask antigenic binding sites. Before
adding the antibody, fat-free milk or normal serum is usually applied to reduce the
background staining. The detecting method can be either direct or indirect. When using
the direct method, the indicator (fluorescent dye, biotin or Peroxidase) is conjugated to
an antibody, including only one step staining. The indirect method includes an
unlabeled primary antibody that binds to antigens, a conjugated secondary antibody that
reacts to primary antibody, and then a third layer with a complex recognizing the
secondary antibody. The indirect method is thereby more specific and provide higher
signal compared with direct method.
The method used in Paper I is a
classical indirect method called ABC.
The technique involves three layers:
unlabeled primary antibody,
biotinylated secondary antibody and a
complex of avidin-biotin peroxidase.
The signal is visualized by DAB
because DAB is converted to insoluble
brown substance by peroxidase in the
presence of H2O2 (hydrogen peroxide).
Figure 6. Illustration of ABC method.
Western blot (paper I-IV)
Western blot is also a method to expression by using an antigen-antibody reaction.
Instead of tissue sections, cell extracts were used for Western blot. SDS is a detergent
that can denature multiple proteins into linear structure and coated with negative
charges. Polyacrylamide gel contains tunnels with different diameters, which allow
proteins going through by their sizes under the same electric current. Therefore, after
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), proteins are
separated on gel according to their molecular weight. Separated proteins were then
blotted on nitrocellulose or Polyvinylidene fluoride (PVDF) membranes for antigen-
antibody reaction. Generally, nitrocellulose membrane is easier to handle since it wets
naturally and gets high signal and low background. PVDF membrane, on the other
hand, needs to be prewetted in methanol and is sensitive to SDS level on the gel.
31
Detection of the protein includes a primary antibody specific to the protein of interest, a
secondary antibody conjugated with an indicator or substrate interacting with indicator.
The commonly used indicator is horseradish peroxidase (HRP). An Enhanced luminol-
based chemiluminescent (ECL) converts the HRP signal into light and detects by film.
The representativity of Western blot is very much depending on the specificity and
sensitivity of the primary antibody. In general, monoclonal antibody is more specific
than polyclonal antibody.
Protein complex immunoprecipitation (Co-IP) (Paper II)
Co-IP is a powerful method used to study of intact protein protein interaction. In order
to evaluate suspected protein interaction, an antibody that targets on a known protein of
the study protein complex is mixed with cell or tissue lysis buffer. After incubation, a
complex of antibody against proteins of interest is formed. Protein A or G agarose is
then added to capture the complex. Proteins that interact with the antigen-antibody
complex are also pulled down after centrifugation. Free proteins are washed way.
Precipitated products are finally analyzed by immunoblotting to verify the interacted
proteins. To further confirm the results, experiments are repeated by using antibodies
identified other proteins in the complex. The proteins interaction will be questioned if
antibody against any membrane of the complex cannot precipitate other proteins in the
complex.
Reverse transcription (RT) polymerase chain reaction (PCR) and quantitative real-
time (qRT) PCR (Paper II and III)
Polymerase chain reaction (PCR) is a technique to amplify a certain region of DNA
strand, which can provide quantitative or qualititative information about a specific
gene. The reaction includes denaturation, annealing and extension. Double strand DNA
is denatured into single strand at 94-98C. When temperature goes down to 50-65
C,
primers will bind to DNA template and the DNA polymerase can subsequently
facilitate the duplication of the sequence of interest. After that, when temperature is
raised to 68-75C, the optimal working temperature for polymerase, primers are
continuously extended by DNA polymerase. The same cycles are usually repeated for
30-45 times resulting in millions of copies of DNA fragment of interest. If real time
monitoring the process, the PCR reaction progress can artificially be divided into three
32
phases: exponential phase in which reaction goes quickly because of sufficient working
materials, linear phase in which amplification starts to slow down, finally plateau phase
in which reaction has stopped and products start to degrade.
RT-PCR and qRT-PCR were
the two most widely used PCR
for gene analysis. In RT-PCR,
single strand RNA is firstly
reverse transcribed into its
complementary DNA (cDNA)
using reverse transcriptase and
primers, then cDNA is used as
template to amplify specific
DNA region by traditional
PCR. The PCR products were
detected using agarose gel
electrophoresis and ethidium
bromide or Gelred. By this
method, we verify the
expression of target gene. The
product size or sequence
analysis of the PCR products
helps to verify the correct
products. Because the agarose
gel only detects the endpoint
(plateau phrase) products, it
only provides quality of the
gene and is imprecise to predict
the quantity of templates.
qRT-PCR, on other hand, provides information of the whole PCR proceeding, which is
more accurate for DNA or RNA quantification. The data collection of qRT-PCR is
fluorescence based. Two commonly used chemistries for detection are Taqman probe
and SYBR green. SYBR green directly binds to double-stranded DNA and emits light.
TaqMan probes are oligonucleotides with a high energy reporter dye on the 5' end and a
33
low energy quencher dye at the 3' end. Before reaction, two dyes sit together by which
the emission of reporter dye is suppressed by quencher dye. During PCR, when DNA
replication reaches to the TaqMan probes bonded on template, the 5'-nuclease of
polymerase cleaves the probe. The release of reporter from quencher increases
fluorescent emission of reporter. The signal increases in direct proportion to the amount
of PCR product in the reaction. SYBR green dye, on the other hand, binds to any
double-stranded DNA molecular including primer-dimers and other non-specific
reaction products, which may lead to an overestimate of the PCR product. Therefore,
Taqman method is a more precise and advance method compared to SYBR green.
Figure 8. Illustration of TaqMan qRT-PCR.
Human Parathyroid cells preparation
In this thesis, primary cultured human parathyroid cells were used for hormone
secretion, intracellular calcium measurement and electrophysiological studies. Fresh
human parathyroid tissue was put into MEM medium (Hank’s) right after dissection
and transported to the lab for isolation within one hour. Tissue was cut into small pieces
and digested in 1.5 mg/ml Type II collagenase for 30 min, then triturated with syringe.
After washed twice with MEM medium, cells were placed and cultured within
DMEM/F-12 medium with supplement of 10% fetal calf serum and 1% antibiotic.
Cultivation of parathyroid cells has been a challenge because cells quickly lose their
secretion property during culture. In my hands, cells completely lose their ability to
release PTH on the third week of normal tissue culture. Type I collagenase and other
cultured mediums, e.g. DMEM, RPMI-1640, and SFM (serum-free keratinocyte)
34
supplied with growth factor, have been used for isolation and culture by other groups.
SFM is a serum free medium containing only 0.09 mM Ca2+
. It has been shown to
provide functional attached culture for up to 2 months, but we did not use it for our
culture because of the poor attachment of the cells in SFM. The DMEM/F-12 medium
used in our study has a more physiological level of calcium (1.05 mM Ca2+
) and was
shown to give functional suspension culture for three months (Kanai et al. 2009).
Hormone secretion (Paper II, III and IV)
There are two ways to assess hormone secretion. One is batch incubation of cells on
tissue culture dishes. Intervenors are directly applied to cells after cell attached and
supernatant is collected for hormone measurement at certain time points. The weakness
of this method is the inconstant amount of cells which makes it imprecise to compare
samples. Another method that is used in this thesis is cell perfusion. Cells are put in a
column between gel filters. Chemicals are added to cells by a perfusion system and
samples are collected at certain times. In this method, results are more reliable and
comparable because of the constant number of cells.
Figure 9. Illustration of parathyroid cell preparation and hormone secretion.
Ca2+
i measurement with Fura-2 (Paper II, III and IV)
In this study, intracellular calcium was detected by fluorescence measurement using
Fura-2. Fura-2 is a commonly used fluorescent indicator of Ca2+
. With the binding of
acetoxymethyl (AM) esters, Fura-2 is transported into cells. After hydrolysis by
35
intracellular esterases, Fura-2 is free
in the intracellular space. Fura-2 has
excitation wavelength at 340 nm and
380 nm. As shown in figure, when it
is free from Ca2+
, excitation is equal
at both wavelengths. When it binds
to Ca2+
, the excitation signal at 340
nm increases in a Ca2+
dependent
manner, whereas signals at 380 nm
are reduced. Therefore, measurement
of fluorescence at two wavelengths gives information of Ca2+
i independent of cytosolic
dye concentration, cell thickness and excitation light intensity (Hayashi & Miyata 1994;
Takahashi et al. 1999; Tsien et al. 1985).
The equipments required for this method include an inverted microscope with a 40×
objective; a perfusion system with heating system which keeps cells at 37C; xenon arc
lamp for light source; monochromaters for selecting wavelength; a shutter controlling
the duration of excitation pulses; a charge-coupled device (CCD) camera detecting
fluorescence signal and
image processor to
convert fluorescent
image to digital number
displayed by computer.
The working flowchart
is shown as Figure 11
(Hayashi & Miyata
1994).
Figure 11. Working flowchart of Ca2+
i measurement.
However, we have to consider some limitations when using a fluorescent dye. The
common problem is Ca2+
insensitivity. The main reason is Fura-2/AM doesn’t
completely convert to free dye in the cells which results in a high signal of fluorescence
but insensitive to the change of calcium concentration. Furthermore, longer loading
36
time can cause compartmentation which means that Fura-2 is accumulating in
intracellular organelles. High concentration of dye can buffer Ca2+
i lowering Ca2+
i or
delaying Ca2+
transit. Bleaching and autofluorescence can also reduce the reliability of
Ca2+
i measurement.
Electrophysiological technique (Paper II and IV)
Patch clamp technique is one of the electrophysiological methods used for the study of
ion channels. It was developed by Erwin Neher and Bert Sakmann in 1970’s (Neher &
Sakmann 1976; Neher et al. 1978).
In this method, a glass micropipette with a flat small tip (around 1 µm) is used as an
electrode. By direct contact and additional suction, a high resistance seal (> 1 GΩ), is
formed between micropipette and cell plasma membrane. The high resistance seal
provides reduced noise and stable recording. Proper buffer that depends on the purpose
of measurement and recording configuration is filled in the pipette. A chloride silver
wire contacts with buffer and conducts electrical current to the amplifier. The way for
recording is either voltage-clamp or current-clamp. In voltage-clamp, the voltage is
fixed and the changes on current are observed, for example recording of ion channel
current. The other way around, current-clamp monitors the changes in voltage while
current is constant, such as the measurement of membrane potential.
Five patch techniques can be used for different purposes. As shown in Figure 12, on-
cell patch is the fundamental configuration from which the other four configurations are
derived. In on-cell patch, the pipette is tightly sealed to the cell membrane without
damage. This method allows us to measure the current from one or few ion channels
within pipette. If deeper suction was given after gigaseal to break the membrane
between pipette and cells, we get whole-cell configuration. Instead of single channel,
whole-cell patch record multiple ion channels on the entire cell. Since there is an open
connection between cell and pipette, the intracellular content is dialyzed by pipette
solution. Therefore, it will not be a good choice for the study of channel activated
through second messengers. To avoid this problem, other type of whole-cell patch
called perforated-patch can be used. Instead of breaking the membrane violently, small
amounts of antibiotic, such as amphothericin-B or gramicidin, are used to create small
pores for monovalent ions going through and generating current. This method reduces
37
the dialysis of cell contents, but also creates some problems. The antibiotic in pipette
makes it difficult to get gigaseal to cell membrane, it takes a long time to get
perforation, contact may be lost or rupture of the membrane may occur. After on-cell
patch is formed, if the pipette is quickly pulled out from the cell, cell membrane on the
tip of pipette is parted from the cell and sticks onto pipette, the inner surface of cell
membrane will be exposed to the bath solution. This configuration is called inside-out
patch. It is useful to study channels activated by inner ligand, such as ATP-dependent
K+ channel. If the pipette is slowly pulled out from the cell after whole-cell patch, the
cell membrane around pipette can be ripped out and then form a bulb on the top of
pipette. That is called outside-out patch. This method reduces the effect of second
messengers and provides better way to study ion channels activated on outer face.
In this thesis, whole-cell patch was used for measurement of voltage operated calcium
channel, store operated calcium channel (Paper II) and Ca2+
-activated K+ channel and
perforated-patch was performed to measure membrane potential (Paper IV).
Figure 12. Illustration of patch-clamp technique and configurations.
38
Results and discussion
Paper I - Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25,
Syntaxin1 and VAMP in human parathyroid tissue
Like in other endocrine cells, parathyroid hormone is stored in vesicles and released via
exocytosis by stimulation of external signal. Exocytosis is a complicated process
requiring participation of several proteins. SNAREs are the central players in
membrane fusion. According to the location, SNAREs are classified into two groups:
v-SNAREs and t-SNAREs. v-SNAREs include VAMP family and are located on
vesicle membrane. t-SNAREs are composed of SNAP and syntaxin families located on
plasma membrane. It is well established that the formation of SNAREs complex by
SNAP-25, Syntaxin1 and VAMP2 is essential for neurotransmitter and insulin secretion
in synapse and pancreatic β-cells (Chen & Scheller 2001). The exocytotic machinery
has so far not been investigated in parathyroid cells. Instead of triggering by increased
Ca2+
i, parathyroid hormone is released when serum calcium is decreased. Therefore, it
is possible that parathyroid cells have a different setup of exocytotic proteins.
To answer this question, we first analyzed the expression of SNAP-25, Syntaxin1 and
VAMP proteins in 20 human parathyroid chief cell adenomas and a pool of three
normal tissues using Western blot analysis. Surprisingly, we did not detect any signal
of SNAP-25 and Syntaxin1 in normal parathyroid tissues. And only 5 out of 20
adenomas did show the expression of SNAP-25 and Syntaxin1 proteins. The absence of
SNAP-25 protein in parathyroid tissues indicated that SNAP-25 is not responsible for
hormone secretion machinery in general. We therefore extended our search and turned
to SNAP-23, a homologue that shares 59% identity of SNAP-25. In contrast to SNAP-
25, which requires relatively high Ca2+
(1 M) to trigging the formation of SNARE
complex, SNAP-23 is able to mediate vesicle docking and fusion at resting condition
(100 nM Ca2+
) (Chieregatti et al. 2004).
Using a specific anti-SNAP-23 antibody, we detected the expression of SNAP-23 in all
parathyroid adenoma and a pool of normal tissues (Figure 1, paper I). An antibody
recognized VAMP1-3 proteins also detected clear signals in all adenoma and normal
parathyroid tissues. These data implied that SNAP-23 and VAMP are more likely to be
39
involved in parathyroid hormone secretion.
To further evaluate the possible role of SNAP-25 and SNAP-23 in human parathyroid
cells, a total of 51 parathyroid specimens, which included 27 chief cell adenomas, 15
normal parathyroid and 9 carcinomas, were used for immunostaining of SNAP-25,
SNAP-23 and PTH. Similar to Western blot analysis, immunohistochemical
investigation showed even expression of SNAP-23 in all examined samples. SNAP-23
was expressed both in the plasma membrane and in the cytoplasm, whereas SNAP-25
is mainly accumulated in plasma membrane (Figure 4, paper I). Parallel to Western
blot, immunoreactivity of SNAP-25 was also absent in all normal parathyroids.
Interestingly, the expression pattern of SNAP-25 in parathyroid adenoma varied.
Thirteen out of them were totally blank, five out of them were widely positive, three
out of them showed nodular expression, while six of them only has staining in single
cells. No correlation was found between SNAP-25 expression and PTH
immunoreactivity. Neither was there a correlation between serum calcium, tumor
weight and plasma PTH level and SNAP-25 level, implying lack of function of SNAP-
25 in parathyroid physiology and pathophysiology. Notably, SNAP-25 has higher
expressions in carcinomas (45%) compared with adenomas (20%). The specific
expression of SNAP-25 in tumor may indicate low differentiation of tumor since
increased SNAP-25 has been shown in human undifferentiated colon and rectum
carcinomas and small cell lung carcinomas with poor prognosis (Grabowski et al. 2004;
Graff et al. 2001). Another hypothesis of increased SNAP-25 is the amplification of
SNAP-25 gene (located in chromosome 20p11). However, published genetic analysis
papers did not show a chromosomal unbalance in chromosome 20 in parathyroid
adenomas and carcinomas (Kytola et al. 2000; Palanisamy et al. 1998). The nodular
expression of SNAP-25 in a few cases suggested the occurrence of an additional
genetic hit during tumorigenesis.
In summary, in contrast to other endocrine cells, SNAP-23 and VAMP play a role in
calcium regulated parathyroid hormone secretion. SNAP-25 and Syntaxin1, on the
other hand, may serve an undefined tumor related function in parathyroid cells.
40
Paper II - Expression and association of TRPC subtypes with Orai1 and STIM1 in
human parathyroid
It has been well established that increased Ca2+
e induces increase of Ca2+
i characterized
by a transient spark followed by a steady-state elevation in parathyroid cells (LeBoff et
al. 1985). Using radioactive labeled Ca2+
, it has been shown that the first transient spark
is due to calcium release from intracellular calcium stores activated by increased IP3
following CaSR activation, while the second phase is dependent on calcium influx
(Wallfelt et al. 1985). However, it is still unclear what type of calcium channel that
mediates calcium influx in parathyroid cells.
The first question was if there is voltage operated and or voltage insensitive calcium
channels in parathyroid cells. Therefore, patch-clamp studies were performed to
investigate possible presence of voltage-gated calcium channels. However, step
depolarization from -70 mV to 70 mV on whole cell configuration did not trigger any
detectable current neither in normal cells nor in cells from parathyroid adenoma. As a
control, on the other hand, the same protocol evoked a clear inward current in mouse
pancreatic β-cells. Besides voltage-gated calcium channel, store operated calcium entry
is another way for calcium influx, which occurs after emptying of intracellular calcium
stores. Thapsigargin, an inhibitor of SERCA, is a reliable tool to deplete calcium store
and trigger calcium entry. To begin with, 2 M thapsigargin was applied to single
parathyroid cells when performing voltage-ramps. The use of ramps demonstrates that
no voltage-gated channels are activated. Our results show a marked increase of current
after exposure to thapasigargin. In parallel, Fura-2 measurements showed a larger rise
of Ca2+
i at 1.5 mM Ca2+
e after addition of thapsigargin. 2-APB, a store operated
calcium channel blocker, totally blocked thapsigargin induced Ca2+
i increase. This
finding implies the presence of store operated calcium influx in human parathyroid
cells.
Recent findings in other cell types show that TRPC channels, Orai1 and STIM1 are key
players in store operated calcium entry (Ambudkar et al. 2007; Potier & Trebak 2008).
In TRPC family, besides of TRPC2 which is known as pseudogene, all other members
TRPC1, 3-7 have been shown to be activated by stored depletion (Salido et al. 2009).
Screening of normal and adenoma parathyroid tissue using RT-PCR showed expression
of TRPC1, 4 and 6, but not TRPC3, 5 and 7. The level of TRPC1 expression was
41
significantly higher than that of TRPC4 and 6, detected by qRT-PCR. However, no
statistical comparisons of these genes could be made between normal and adenomas.
qRT-PCR and Western-blot showed the general expression of STIM1 and Orai1.
Finally, co-IP gave strong evidence of the interaction between TRPC1, STIM1 and
Orai1.
In conclusion, we show that store operated calcium channels composed by TRPC1-
STIM1-Orai1 complex mostly likely are involved in mediating calcium influx in
human parathyroid.
Paper III - Involvement of calmodulin and calmodulin dependent protein kinase II
(CaMKII) in human parathyroid hormone secretion
CaSR senses small elevation in Ca2+
e and converts to several folds increase of Ca2+
i
inhibiting PTH secretion via Ca2+
binding proteins pathway. Among the numerous of
Ca2+
binding proteins described in the literature, calmodulin is the most ubiquitous
protein mediating many Ca2+
regulated cellular functions by binding to different protein
targets. One of the best-described targets and also shown to be involved in exocytosis is
calmodulin dependent protein kinase II (CaMKII)(Wang 2008). Both calmodulin and
CaMKII have been found in parathyroid cells in the early 1980’s and 1990’s (Brown et
al. 1981; Kato et al. 1991; Kinder et al. 1987; Oldham et al. 1982). However, the
correlation between function of calmodulin and CaMKII is still unclear.
Since the technical limitation in the earlier years, calmodulin and CaMKII were
identified indirectly by detection of calmodulin dependent phosphodiesterase activity.
Therefore, to further demonstrate their expression, we performed RT-PCR with primers
specific recognized calmodulin isoforms 1-3 and four CaMKII subunits mRNA and
Western-blot with specific antibodies against those proteins. Our results clearly show
the expression of calmodulin and CaMKII in human normal and adenomatous
parathyroid cells at both mRNA and protein levels (Figure 1, paper III).
To investigate potential association between calmodulin/CaMKII and PTH secretion,
calmodulin and CaMKII antagonists, Calmidazolium and KN-62, were used to block
Calmodulin and CaMKII activity, respectively. Exposing parathyroid cell suspension to
1 µM and 10 µM Calmidazolium caused 309.4% and 260130% increase of PTH
42
secretion, respectively. KN-62 resulted in a 344% increase of PTH release (Figure 2,
paper III). This finding suggests that calmodulin and CaMKII are involved in PTH
secretion.
As stated above, Ca2+
i is negatively related to PTH secretion. Therefore, Ca2+
i
measurement was done to investigate the mechanism of calmodulin and CaMKII
regulated PTH secretion. Our studies shows that calmodulin and CaMKII regulates
PTH secretion regardless of Ca2+
i. since Calmidazolium induced an increase of Ca2+
i,
and 10 µM KN-62 has no effect on Ca2+
i (Figure 3, paper III). It also indicates that
Ca2+
i may not be pivotal regulator in stimulus-secretion coupling in parathyroid.
Calmodulin and/or CaMKII may directly or indirectly interact with exocytotic proteins
and influences exocytosis.
CaMKII needs to be phosphorylated in order to be physiological active (pCaMKII).
Reviewing a cohort of 20 chief cell parathyroid adenomas for calmodulin and
pCaMKII, we found that calmodulin was equally distributed in all adenomas. There is
no correlation between S-Ca2+
, S-PTH, tumor weight and calmodulin protein level.
However, a negative correlation was shown between the level of pCaMKII and S-Ca2+
.
In vitro studies also found the reduction of pCaMKII after treated with 2.5 mM Ca2+
. It
is concluded that high Ca2+
e suppress the phosphorylation of CaMKII.
In conclusion, this study demonstrates the expression of calmodulin and CaMKII in
human parathyroid, and that these proteins are involved in PTH secretion. Reduction of
phosphorylation of CaMKII at high Ca2+
e may have pathophysiological implication that
needs to be further addressed.
Paper IV - Expression of Ca2+
-activated K+ channels in human parathyroid cell:
potential role in hormone secretion
Membrane potential is a key player of hormone secretion in neurons and
neuroendocrine cells. In pancreatic -cells, for example, it has been well known that
membrane depolarization induced by the closure of ATP-dependent K+ channels give
rise to insulin secretion (Ashcroft 2000). Similarly, membrane depolarization increases
of PTH release in parathyroid cells (Dempster et al. 1982). Wallfelt and co-workers
found that elevated cytoplasmatic Ca2+
triggered K+ efflux when monitoring by K
+
43
tractor 86R (Wallfelt et al. 1985). Taken together, it suggests the existence of Ca2+
activated K+ permeability. This hypothesis was latterly confirmed by our group by
detection of Ca2+
-activated K+ current using patch-clamp (Valimaki et al. 2003).
Distinguishably, high Ca2+
o, which inhibits PTH secretion also hyperpolarize
parathyroid cell membrane (Valimaki et al. 2003). In paper IV, we show that
parathyroid express at least two types of Ca2+
-activated K+ channels in human
parathyroid cell membrane, namely the big conductance Ca2+
-activated K+ channels
(KBK channel) and small conductance Ca2+
-activated K+ channels (KSK channel) by
applying specific channel blockers (Figure 1, paper IV). The presence of these channels
was further verified by Western blot and immunocytochemistry.
When we investigated the relationship of Ca2+
i and Ca2+
-activated K+ current, we found
that adenomas cells had significantly lager K+ current at 0.3 µM than normal
parathyroid cells. However, Western blot analysis could not detect any difference in
KBK channel protein level.
Using perforated-patch technique, we show that both high extracellular K+
concentration and KBK channel blocker toxins lead to depolarization of parathyroid cell,
while KBK channel activator (NS-11021) caused hyperpolarization. Using a perfusion
chamber, exposing parathyroid cells to KBK channel blockers (TEA and charybdotoxin)
induce PTH secretion at 1.0 and 1.2 mM Ca2+
e, but no KSK channel blocker (apamin).
The KBK channel activator NS-11021 showed an inhibition of PTH secretion at 0.5-1.5
mM Ca2+
e without alterations in Ca2+
i.
In summary; we conclude that parathyroid cells, normal and adenoma, express Ca2+
-
activated K+ channels. The majority of Ca
2+-activated K
+ channel current can be
assigned to KSK and KBK channel. Moreover, these channels contribute in maintaining
the membrane potential in the parathyroid cell, where opening of these channels
hyperpolarize the membrane and vice verse. Membrane potential seems to be, at least
partly, a Ca2+
i independent modulator of PTH secretion. Our finding that parathyroid
adenoma cells have higher K+ current compared to normal cells is potentially very
interesting, even though we are not able to verify this finding on protein level.
However, there are several explanations for our incongruent finding. Even so, one may
speculate that an increased K+ channel activity could result in a more hyperpolarized
cell. Down-stream in the signaling pathway this could decrease PTH secretion. Since
44
KBK channel gene is located in chromosome 10q22, a genomic area which has not been
shown to harbor chromosomal unbalance (Kytola et al. 2000; Palanisamy et al. 1998),
it is plausible that an increased activity represents a compensatory mechanism for a
decrease PTH secretion in parathyroid adenoma.
Conclusions
The aim of this thesis was to characterize the stimulus-secretion coupling in human
parathyroid. The major findings in summary:
1. In contrast to other endocrine cells, SNAP-23 and VAMP are widely
expressed in normal and adenoma parathyroid tissue. It is likely that these
proteins play a role in calcium regulated hormone secretion. SNAP-25 and
Syntaxin1, on the other hand, may serve an undefined tumor related function
in parathyroid cells.
2. Store operated calcium channels composed by TRPC1-STIM1-Orai1 complex
mostly likely are involved in mediating calcium influx in human parathyroid.
Calmodulin and CaMKII are involved in Ca2+
regulated PTH secretion. High
calcium suppresses the phosphorylation of CaMKII, which may have a
pathophysiological implication.
3. At least two types of Ca2+
-activated K+ channels, namely KBK and KSK channels
are present in the human parathyroid cell. These channels contribute in
maintaining the membrane potential in the parathyroid cell, and membrane
potential seems to be, at least partly, a Ca2+
i independent modulator of PTH
secretion.
45
Figure 13. Summary of main findings in the thesis.
46
摘要 甲状旁腺是一个独特的内分泌腺体，因为离子钙抑制激素分泌，而不象在别的
内分泌腺体内刺激激素分泌。它的机制一般认为是，当离子钙结合到细胞膜上的钙
敏感受体，激活了一系列的第二信史从而影响甲状旁腺激素分泌。其中细胞内钙离
子被认为在这激素分泌过程中起着中心的作用，然而许多细胞内的信号机制仍未完
全清楚。
胞吐是激素分泌的主要方式，在调节性胞吐中，突触小体相关蛋白,比如 SNAP
蛋白在激素囊泡和细胞膜融合过程中着关键作用。突触小体相关蛋白广泛存在于神
经和神经内分泌细胞。在第一篇文章中，我们通过免疫组化和蛋白印迹探索突触小
体相关蛋白 SNAP-23,SNAP-25, Syntaxin1 和 VAMP 在人甲状旁腺正常和病理组织的
表达。结果发现 SNAP25 和 Syntaxin1 不存在于正常甲状旁腺内，但表达在 20%的主
细胞腺瘤和 45%的甲状旁腺癌里。反之 SNAP-23和 VAMP 则存在于所有的甲状旁腺标
本里。这些发现暗示蛋白 SNAP-23 和 VAMP，而不是 SNAP-25 和 Syntaxin1，在人甲
状旁腺细胞胞吐中起主要作用。
细胞内钙离子在甲状旁腺激素分泌中起着重要作用，课题 II 和 III 侧重于研
究这个过程。在甲状旁腺中，当细胞外钙离子升高时，钙离子的内流就增加，但机
制并不清楚。使用膜片钳和细胞内钙离子测定技术，我们发现在甲状旁腺细胞中存
在钙池操纵性钙通道，蛋白 TRPC1,STIM1 和 Orai1 组成的复合体可能是这个通道的
主要元素。
另外，两个在钙信号通道中很重要的钙结合蛋白 calmodulin 和 calmodulin 依
赖性激酶也被发现存在于人甲状旁腺中。当他们失活时，甲状旁腺激素分泌增加，
而这个作用与细胞内钙离子的高低没有直接联系。我们的发现首次表明了钙结合蛋
白 calmodulin 和 calmodulin 依赖性激酶参与了甲状旁腺激素分泌的调解。有趣的
是我们还发现了在腺瘤病人中血钙水平和腺瘤组织中磷酸化的 calmodulin 依赖性
激酶水平成反比关系，这也许暗示着一定的临床意义。
最后在文章四，我们发现了甲状旁腺细胞至少存在两种钙激活性钾离子通道，
分别是大电导型和小电导型钙激活性钾离子通道。这些通道对相应的钙激活性钾离
子通道的激活剂和拮抗剂都很敏感。这些通道的开放和关闭直接影响到膜电位的改
变，并因而影响甲状旁腺激素分泌。
概括而言，甲状旁腺细胞的刺激-激素分泌复杂而特别。这本论文从钙内流，
信号传递，离子通道和胞吐不同的方面分别阐述了这个过程。但在体内，这些不同
的部分也许互相影响，共同调节甲状旁腺激素分泌。
47
Acknowledgment
I would like to express my sincere gratitude to all the people who have been involved in
my postgraduate study. Without you, my life in the past four years cannot be so
wonderful. In particular, I would like to thank:
Robert Bränström, my main supervisor, for accepting me as a PhD student and
guiding me into scientific field, for enthusiasm and intelligence in parathyroid research.
Thank you for always encouraging me and giving me time and space to develop
myself, for all the time spent on my projects and thesis.
Lars-Ove Farnebo, my co-supervisor, for financial support for my PhD study, for
broad knowledge in research, for patiently correction and discussion for my
manuscripts and thesis.
Lars-Forsberg, my co-supervisor, for helping me adapting to Swedish life and
research work, for helping me to improve my oral presentation skills. You are like an
elder brother giving me lots of encouragements and advice. My earlier life in Sweden
became much easier with your help.
Zhao Bian, who introduce me to Karolinska Institute, for providing me the opportunity
to study abroad, for trust and encouragements, for personal concern and supports for
my family.
Xia Ning, supervisor of my Master degree in China, for warm greeting cards in every
New Year, for always generously expressing your supports, for help whenever I need.
Other parathyroid coworkers
Catharina Larsson, for organizing wonderful parathyroid meeting, for kindness and
deep knowledge in parathyroid research, for all brilliant comments to my manuscripts.
Anders Höög, the super expert in endocrine pathology and master of Endocrine
Biobank, for arrangement of study samples, for help in reading immunohistochemistry
slides. Lisa Ånfalk, for taking care of Endocrine BioBank.
48
Inga-Lena Nilsson, for sharing your epidemiological study related to parathyroid
tumor. Jörgen Nordenström, for brilliant idea and interesting discussion in
parathyroid meeting, for introduce me to Robert.
Christofer C Juhlin, an outstanding Ph.D, I am very impressed in your thesis. Thank
you for establishing the immunohistochemistry lab and technique helps. Felix
Haglund, for interesting presentation and collaboration. Jamileh Hashemi and
Luqman Sulaiman, big parathyroid adenoma workers, for common interest and
interesting discussion.
Erik Berglund, an enthusiasm medical student and next PhD in our lab, for help and
discussion in patch-clamp and intracellular Ca2+
measurement, for all nice chats.
Collaborators in other lab, Vladana Vukojević, for help in Fluorescent
immunocytochemistry, for kindness and nice collaboration. Christina Bark, for
knowledge in exocytosis, for advice in primer design.
Collaborators from other Universities: Arthur D. Conigrave, Leigh W. Delbridge,
Anthony Gill, Hee-Chang Mun from University of Sydney, Australia and Peyman
Björklund, Gunnar Westin from Uppsala University, for common interest and fruitful
collaborations.
Thank you for all surgeons who have helped me for samples collection in Section of
Endocrine Surgery, Karolinska University Hospital. Thank you for all the patients who
have been involved in my studies.
Other members in surgical research: Bertil Hamberger, Martin Bäckdahl, Marja
Thorén for kindness and interest, Ulla Enberg, a nice lady with strong will, for your
optimism attitude, for organizing nice group parties and delicious dinners in your small
cottage, for tasty cakes for Fika, for nice companion and tips for baking, wish you
endless happiness in another world. Cristina Volpe, a nice roommate and friend during
last period of my PhD study, for so many fun chats and nice companion for thesis
writing. Jan Zedenius, for organizing the wine seminar, for your vast knowledge in
tumor genetic and wine. All the former and present members of wine seminar between
2006 to present, for interesting presentation, discussion and wine tasting.
49
Other former members of our research group: Jussi Sane and Viljam Lindqvist, for
kindness and companion, for technique help for Patch clamp. Cecilia Laurell for
kindly letting me your apartment when I was homeless.
Former and present, Corridor mates in L5:02, L8:01 and L1, thank you for kindness,
help and friendly working environment, Members in Professor Catharina
Larsson’s group: Stefano, Andrii, Anastasios, Lui, Nimrod， David ;
Professor Gunnar Norstedt and members in his group, especially, Yin-Choy (for
humors and always help for my methodological problems), Fahad, Mattias, Anenisia,
Roxana, Cecilia, Louisa, Diego, Irina, Hong-yan, ZiCai; Professor Lars Tenenius
and his group members, especially Yu Ming, Agneta, for nice chats and
encouragements; Professor Tomas Ekström and his group members, Monira,
Zahidul, Mohsen and Anna-Maria; Professor Kerstin Brismar and her group
members: Jacob, Vivek, Stina, Katrin, Michael, Jing; Professor Per-Olof Berggren
(for inviting our group moving to such nice work place) and all his members: Lisa,
Martin and Tilo (all for help in Ca2+
i measurements), Ingo, Barbara, Pilar,
Sergei, Irina, Gian-Carlo, Luo-Sheng, Andrea, Erwin, Jantje, SengEun, Essam,
Shao-Nian, Jia, Yue, Xiao-Lei, Lina, Slavena, Rebecka, Lola, Karin, Ulrika,
Elisabetta (for helpful advice in primary cell culture and hormone secretion and
nice lunch chats), Chris, Christopher, Dominic, Gustavo, Yusuf, Lars, Alan,
Patrick, Aarti, Craig (for help on many scientific questions, for nice collaboration),
Annika and Yvonne for help in the lab, Hannelore for showing perfusion experiment
and issues in laboratory safety.
People in Administration board, Katarina Breitholtz, Christina Bremer, Kerstin
Florell, Anne-Marie Richardsson, Helena Nässén, especially, Ann-Britt Wikström,
for kindness and administrative help for my PhD study. Lennart Helleday and
Thomas Westerberg, for IT maintenance.
My friends: Lin Rui, my dear friend, for accompany in the special time of my life,
watching the birth of my son. Hu Jin and his family, thank for all time spending
together. Zhao Yun-Gang (for always patient ly listening and answering
my idiot scientific questions and helps in molecular biological methods), Xie Hong
and their son, for nice trip in Germany. My neighbors, Sun Ying, Qiu Ping and their
50
families, for good company and charing happiness and secretes. Li Xue-Lian, latifa
bikndarne and Linh Dang for all nice chats and dinners. Xu Hong and Ma Zhu-
Heng family, for invited dinners in your house. Other friends, Liu Tong, Zheng Hui-
Yan, Wei Tian-Ling, Jiao Wei, Meng Chun-Xia, Lin Hui-Qiong, Zhou Ying, Wu
Liang, Tu Dan, Zhang Yun, Zhang Xiao, Li Zheng-Hong, Zhang Dong-Ying,
Jiang Li-Ying. My friends in China, Huang Bo, for teaching me English and
encourage me to come to Sweden, you will be in my memory forever.
My colleagues in my home country, Zhao Jin-Min, President of Hospital, Liu Hong，
chief of department, Huang Hong, Yang Xi, Sun Wen, Qin Bao-Yu, Yao Yan-Bing,
thank all for supports.
我的家人,在此要感谢父母培养了我的个性和引导我走上医学的道路，特别
感谢母亲给我生活上的关心和帮助，感谢弟弟陆泉和弟媳宪春对我的理解，支
持和这几年中对母亲的照顾。感谢一直关心和帮助我们家人的亲戚朋友。感谢
婆婆在我需要的时候，来到瑞典帮我带儿子，支持我的学业。
感谢我的丈夫Robin为了我和我们的儿子来到瑞典， 感谢你多年来对我的督
促和给我们无私的爱。感谢上天给了我一个聪明可爱的儿子典典，你的来到带
给我无限的希望和快乐。
最后，我还要感谢基督教会的沈牧师以及所有给过我丈夫和家庭帮助的人。
Thank you for the First Affiliated Hospital of Guangxi Medical University and
Department of education of GuangXi province, NanNing, Peoples Republic of China
for funding my PhD study within 2006-2007.
The study was financially supported by grants from Swedish Research Council, the
Novo Nordisk Foundation, Funds of Karolinska Institutet, the Tore Nilsson Foundation,
the Thuring Foundation, the Jeansson Foundations, the Åke Wiberg Foundation, the
Göran Gustavsson Foundation for Research in Natural Sciences and Medicine, Magn.
Bergvall Foundations, the Stockholm County Council, the Swedish Cancer Foundation,
and the Knut and Alice Wallenberg Foundation.
51
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