ADRENERGIC AND CHOLINERGIC REGULATION OF PHOSPHOLIPID RELEASE IN SUBLINGUAL SALIVARY
GLAND IN VITRO
BRONISLAW L. SLOMIANY, SHIPRA SENGUPTA, ZOLTAN FEKETE, VARAHABHOTLA L. N. MURTY and AMALIA SLOMIANY
Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. [TeL (201) 456-7052]
(Received 20 November 1990)
Abstract--l. The role of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion was investigated using rat sublingual acinar cells maintained in the presence of [3H]choline.
2. The release of [3H]choline containing phospholipids was enhanced by fl-adrenerglc agonist, isoproterenol to a greater extent than the cholinerglc agonist, pilocarpine.
3. The effect of isoproterenol on phospholipid release was observed even after 5 min and by 30 min a 1.7-fold increase in secretion occurred, whereas pilocarpine evoked 1.2-fold increase by 30 min with no discernible effect in 5 min. The isoproterenol effect was blocked by alprenolol, and an inhibition of pilocarpine effect was observed with atropine.
4. In the absence of mediators, 82% of secreted labeled phospholipids were represented by phosphatidyl- choline (PC), 4.3% by lysophosphatidylcholine (LPC) and 12.1% by sphingomyeline (Sph), those secreted in the presence of isoproterenol showed significantly lower (37%) content of LPC, while those secreted in response to pilocarpine were substantially richer in LPC (2.5 times) and Sph (27%), and contained less (17%) PC.
5. The results provide first evidence for the involvement of adrenergic and cholinergic mediators in the regulation of salivary phospholipid secretion.
INTRODUCTION
Among the components of silivary gland secretions which play an important role in functional properties of saliva are lipids (Dirksen, 1979; Slomiany et al., 1985, 1987, 1989). The elevated levels of lipids in saliva are associated with the high incidence of caries, and the development of plaque, calculus and peri- odontal disease (Murty et al., 1984; Slomiany et al., 1989). A high level of salivary lipids is also a charac- teristic feature of periodontal disease-prone animal (Murty et al., 1984). The lipids are known to facilitate the penetration of oral mucosa by lipophilic sub- stances and are capable of altering the interaction of salivary proteins and glycoproteins with calcium (Johnson, 1976; Vogel and Belcourt, 1980; Slomiany et al., 1989). Some of the salivary lipids, i.e. lysophosphatidylcholine (LPC) also affect the glyco- syltransferase activity associated with the cariogenic potential of bacteria (Schachtele et al., 1978). Fur- thermore, since the initial interaction of bacteria with oral tissue involves the formation of hydrophobic bonds which are stabilized by lipid-rich environment, the elevated level of lipids in saliva may be conducive to the facile adherence of bacteria in the oral environ- ment of caries-susceptible individuals. Hence, the factors controlling the lipid levels in saliva may be directly relevant to the maintenance of the health of oral mucosa and teeth.
Delineation of the nature of changes in properties of saliva influenced by lipids requires thorough knowledge of the processes leading to salivary lipids elaboration. While studies show that salivary glands
have a considerable capacity for the rapid bio- synthesis of phospholipids and glycolipids (Pritchard, 1970; Slomiany et al., 1983, Zdebska et al., 1983), little is known about effectors mediating their se- cretion. In this study, the sublingual acinar cells in culture were used to determine the effects of cholin- ergic and adrenergic mediators on the secretion of choline-containing phospholipids.
MATERIALS AND METHODS
Materials
Male Sprague-Dawley rats (150-200 g) were purchased from Charles River Lab., Wilmington, MA. Tissue culture medium components were supplied by Grand Island Bio- logical Co., Grand Island, NY, and [methyl-3H]choline chloride (80 Ci/mmol) by New England Nuclear, Boston, MA. Pilocarpine chloride, atropine sulfate, l-isoproterenol bitartrate, alprenolol, phosphatidylcholine (PC), LPC and sphingomyelin (Sph) were from Sigma Chemical Co., St Louis, MO. Silica Gel 60 thin-layer plates were obtained from Merck. All other chemicals were of reagent grade.
Cell preparation The animals were fasted overnight, killed by cervical
dislocation, and the sublingual glands dissected. The glands trimmed of fat and connective tissue were cut into small pieces and minced by passage through a 50 mesh metal grid. The minced tissue was suspended in 5 vol. of ice-cold Dulbecco's modified Eagles' minimal essential medium (DMEM), containing gentamyein (50/~g/ml) and fun#zone (0.I mg/ml). The acinar cells were gently dispersed with a glass homogenizer and were settled by centrifugation at 300 g for 5 min. The cells were washed twice with DMEM and resuspended in the medium to a concentration of
969
970 BRONISLAW L, SI,OML~hq¢ et aL
Table 1. Effect of incubation time on the incorporation of [3H]choline into the cellular phospholipids in sublingual salivary
gland cells in culture
[3H]Choline incorporation (% total) Incubation period (hr) PC LPC Sph
Values represent means+SD of four separate experiments performed for each time interval in duplicate, and expressed as percent of total cell lipid radiolabel.
5 x l0 s cell/ml (Wu-Wang et al., 1991). The viability of such prepared cells was > 98% as determined by the Trypan Blue exclusion method.
Incubation o f cells
One milliliter aliquots of the cell suspensions were trans- ferred to DMEM in culture dishes containing [3H]choline (10 ttCi/ml), and incubated under 95% 02-5% CO2 atmos- phere in a tissue culture incubator. Following 3 hr incu- bation, the cells were centrifuged at 300 g for 5 min, washed twice with fresh medium and resuspended in DMEM. After 5rain equilibration period, 15/zl of the appropriate ago- nists, antagonists or, as control, their diluents were added and incubated at 37°C for up to 1 hr. Antagonists were added immediately prior to the addition of agonists and in the experiments with isoproterenol, sodium ascorbate ( lmM) was included as an antioxidant (Brown and Longmore, 1981). At the end of incubation period, the cells were centrifuged at 300 g for 5 rain and washed three times with fresh medium. The medium and washes were com- bined, dialyzed against distilled water and used for the isolation of [3H]choline labeled released phospholipids, and for the determination of lactate dehydrogenase (Fanestil and Barrows, 1965). The pelleted cells, following washing, were subjected to cell-associated [3H]choline-containing phospholipid analysis.
Phospholipid analysis
Extraction of lipids from the prepared medium cells samples was performed with chloroform-methanol [2: 1, v/v; Slomiany et al. (1986)]. The extracts were filtered through a grade F sintered glass funnel, and the lipids contained in the filtrates were separated on silicic acid columns into neutral lipid, glycolipid, and phospholipid fractions (Murty et al., 1985), Aliquots of phospholipid fraction, eluted from the columns with methanol, were counted to determine the total [3H]choline containing phos- pholipid content, while the remainder was analyzed by
thin-layer chromatography using consecutive developments in chloroform-methanol-water (65:35:8, by vol.) followed by chloroform-methanol-acetic acid water (80:18:3:3, by vol.). Bands co-migrating with PC, LPC and Sph standards were visualized by iodine vapors, scraped and recovered from the gel by elution with chloroform-methanol (1:4, v/v) and methanol Bilski et al., 1987). Following removal of the solvents, the individual phospholipids were analyzed for radioactivity by scintillation spectrometry.
Statistics
All experiments were carried out in duplicate, and the results are expressed as means _+ SD. Student's t-test was used to determine significance, and P values of 0.05 or less were considered significant.
RESULTS
Incubation of rat sublingual salivary gland cells in the presence of [3H]choline led to the incorporation of the label into phosphatidylcholine, lysophosphatidyl- choline and sphingomyelin. The incorporat ion of the marker into three phospholipids increased steadily with incubation time with over 90% of the lipid label at any time being incorporated into PC (Table 1). During the course of incubation the incorporation values for LPC did not exceed 3% of the [3H]choline labeled lipids, while that for Sph reached the value of 3.8%. Since the viability of sublingual acini in culture as measured by the Trypan Blue uptake remained over 98% up to 4 hr, and the lactate dehydrogenase assays indicated little if any (1%) cellular damage within this period, the labeling time of 3 hr was used and the release of radiolabeled phospholipids into the medium in response to various mediators was assayed after 30 rain o f incubation.
Under normal conditions, in the absence of any mediators, the release of choline-containing phospho- lipids during the incubation period of 30rain amounted to 1.97% of the total cellular [3H]choline containing phospholipid (Table 2). Addit ion of the /~-adrenergic agonist, isoproterenol, caused a dose dependent stimulation in phospholipid secretion, which reached maximum value of 1.7-fold increase over the control at 1 0 # M isoproterenol concen- tration. This stimulatory effect was inhibited by /~-adrenergic antagonist, alprenolol, but not by cholinergic antagonist, atropine (Table 2). In contrast
Table 2. Effect of/~-adrenergic and cholinergic mediators on the [3H]choline-containing phospholipid secretion by sublingual salivary gland cells in culture
Cells were labeled for 3 hr with [3H]choline, washed and exposed for 30 rain to various mediators. At the end of incubation, the medium was collected and analyzed for radiolabel phospholopids. The incorporation of [3H]choline into the cellular phospholipids at the end of 3 hr incubation was 43,450 + 4120/assay. Values represent means =l= $D of four separate experiments performed in duplicate. * P < 0.05.
Phospholipid secretion in salivary gland
Table 3. Effect of p-adrenargic mediator on the [~H]cholin¢-containing phospholipid distribution in the secretion of sublingual salivary gland cells in culture
[3H]Choline incorporation (% total) Effector PC LPC Sph None (control) 82.1 ± 6.6 4.3 ± 0.6 12.1 ± 1.5 Isoproterenol (10/~M) 86.2_+7.3 2.7±0.4* 10.4± 1,9 Alprenolol (50/~ M) 80.7 ± 8.1 4.8 ± 0.7 11.3 ± 1,4 Isoproterenol (10 ItM) + alprenolol (50/zM) 84.5 ± 7.6 3,7 ± 0.5 10.8 ± 1.2 Isoproterenol (10/~M) + atropine (40/JM) 85.3±7.7 3,1 ±0.4* 10.2± 1.3 Cells were labeled for 3 hr with 13H]choline, washed and exposed for 30 min to various mediators.
At the end of incubation, the medium was removed and analyzed for radiolabeled phospholipid distribution. Values represent means ± SD of four separate experiments performed in duplicate.
971
to isoproterenol, the effect of which on the secretion was already observed (l.3-fold increase) after 5 min, pilocarpine a cholinergic agonist appeared to cause much slower response. At the optimal concentration of 0.2/~M, pilocarpine evoked only 1.2-fold increase in phospholipid secretion during the 30 rain incu- bation period with no discernible effect in 5 min. Partial inhibition of pilocarpine effect was observed with atropine, while fl-adrenergic antagonist, al- prenolol, had no effect.
The effect of fl-adrenergic mediator on the choline- containing phospholipid distribution in the secretion of sublingual salivary gland cells in culture is pre- sented in Table 3, and the data on the effect of cholinergic mediator are summarized in Table 4. In the absence of any mediators, 82.1% of the released [3H]choline labeled lipids were represented by PC, 4.3% by LPC, and 12.1% by Sph. Isoproterenol caused a marked (37%) decrease in the release of LPC and evoked consistently higher, although not statistically significant responses in the secretion of PC and lower in Sph. This effect of isoproterenol was countered by its antagonist alprenolol, but not by atropine (Table 3). In the case of pilocarpine, a cholinergic agonist, the secreted [3H]choline contain- ing phospholipids showed a significant (2.5-fold) increase in LPC and Sph (27%) and a decrease (17%) in PC. The pilocarpine effect on the secretory phos- pholipid composition was not altered by//-adrenergic antagonist, alprenolol, while inhibition of pilocarpine effect was obtained with atropine (Table 4).
DISCUSSION
Although the presence of lipids in saliva has been recognized of importance to a variety of processes occurring in the oral cavity that determine the ability to resist caries and periodontal disease, the factors controlling their levels in secretions of salivary glands are not well explored (Slomiany et al., 1989). Gener-
ally, the control and salivary secretion is mainly derived through sympathetic and parasympathetic innervation (Garrett, 1982; Baum, 1987), and the acinar cells of salivary glands possess receptors capable of responding to signals from either system (Martinez, 1987). The parasympathetic nerve stimu- lation leads to increase in fluid secretion, while sym- pathetic nerve stimulation appears to have a greater effect on the macromolecular consituents of saliva (Baum, 1987). Indeed, studies show that acinar cells of salivary glands in vitro respond to stimulation of fl-adrenergic agonists with increase in mucin and amylase secretion (Quissell and Barzen, 1980; McPberson et al., 1986; Bradbury and McPberson, 1987). An increase in PC secretion by lung epithelial cells in culture in response to adrenergic and cholin- ergic mediators has also been reported (Brown and Longmore, 1981; Sano et al., 1985).
In the present study, using acinar cells of sub- lingual salivary gland, we have examined the release of choline-containing phospholipids in the presence of fl-adrenergic and cholinergic effectors. The data revealed that the cells maintained in the presence of [3H]choline incorporate over 90% of the phospho- lipid label into PC with the rest being distributed among Sph and LPC. In the absence of mediators, the release of [3H]choline-containing phospholipids into the incubation medium over 30 min period aver- aged about 2% of the total cellular [3H]choline- labeled phospholipids.
Both cholinergic and fl-adrenergic mediators stimulated independently an increase in the release of [3H]choline-labeled phospholipids. This increase in the labeled phospholipids release apparently was the effect of acinar cells responding to the mediators employed rather than the result of cellular damage, as demonstrated by lactate dehydrogenase assay. The maximum stimulation of phospholipid secretion with fl-adrenergic agonist, occurred at isoproterenol con- centration of 10 #m at which a 1.7-fold increase in
Table 4. Effect of cholinergic mediator on the [3H]choline-containing phospholipid distribution in the secretion of sublingual salivary gland cells in culture
Cells were labeled for 3 hr with [3H]choline, washed and exposed for 30 rain to various mediators. At the end of incubation, the medium was removed and analyzed for radiolabed phospholipid distribution. Values represent means + SD of four separate experiments performed in duplicate.
972 BRONISLAW L. SLOML~ et al.
[3H]choline-containing phospholipids over the con- trol was observed. Pilocarpine, a cholinergic agonist, at its optimal dose of 0.2/~M evoked only 1.2-fold increase in the secretion and in contrast to iso- proterenol its effect was not discernible as early. A similar situation was observed with lung epithelial cells were phospholipid secretion in response to fl-adrenergic stimulation was already demonstrable 2.5 min following stimulation (Brown and Longmore, 1981).
The action of each agonist, furthermore, was sensi- tive to inhibition by its appropriate antagonist but not by the opposing antagonist. Neither pilocarpine nor isoproterenol caused cross-stimulation. The ob- served variation in response to the two types of agonists could be related to the differences in the mechanism of their action. The fl-adrenergic ago- nists, such as isoproterenol, act through the acti- vation of cAMP-dependent protein kinase signalling pathway, while the action of cholinergic agonists, such as pilocarpine, involves phosphoinositide- mediated protein kinase C signal transduction system (Sano et al., 1985; Baum, 1987).
Our data as to the distribution of the [3H]choline containing phospholipids, secreted in response to cholinergic and/~-adrenergic effectors, revealed that in addition to the differences in secretory responses, each mediator evoked distinctly different response with respect to the composition of the released [3H]choline-containing phospholipids. Those released in response to isoproterenol stimulation exhibited consistently higher content of PC and showed a significant decrease in LPC as compared to the controls. On the other hand, the phospholipids re- leased in response to pilocarpine were markedly enriched in LPC, showed lower content of PC and a moderate increase in Sph. The changes in the composition of the secreted phospholipids were, however, reversed with the use of the respective antagonist.
The enrichment of the secreted phospholipids in LPC in response to piiocarpine suggests that the cholinergic stimulation of the acinar cells leads to protein kinase C dependent activation of phospho- lipase A2, and hence the increase in LPC generation. That protein kinase C is involved in the regulation of the expression of phospholipase A2 for the break- down of PC has been demonstrated (Exton, 1990). In contrast, the observed decrease in LPC content in the secretion evoked by isoproterenol may be due to the /~-adrenergic mediated inhibitory effect of cAMP- dependent protein kinase on phospholipase A2, as this kinase and protein kinase C are known to phosphorylate different sites on the same protein with opposing effects (Cohen, 1988).
Acknowledgements--Supported by USPHS Grant No. DE05666-12 from the National Institute of Dental Research and Grant No. AA05858-09 from the National Institute of Alcoholism and Alcohol Abuse, NIH.
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