Pflfigers Arch (1992) 420:127-135 Journal

of Physiology �9 Springer-Verlag 1992

Angiotensin II and acetylcholine differentially activate mobilization of inositol phosphates in Xenopus laevis ovarian follicles Paige Lacy, Rosalind P. Murray-Mclntosh, and James E. A. McIntosh

Department of Obstetrics and Gynaecology, University of Otago, Wellington School of Medicine, P.O. Box 7343, Wellington, New Zealand

Received April 8, 1991/Received after revision August 9, 1991/Accepted September 23, 1991

Abstract. Angiotensin II (AII) evokes a Ca 2 § CI- current in Xenopus laevis ovarian follicles that ap- pears to involve a pertussis-toxin-sensitive G protein me- diating phosphoinositide hydrolysis and Ca 2 + mobiliza- tion from intracellular stores. Follicle responses to AII closely resemble the two-component response stimulated by acetylcholine (ACh) in this tissue. Intraoocyte in- jections of phytic acid, heparin, and inositol 1,4,5- trisphosphate [Ins(1,4,5)P3], acting as inhibitors of Ins(l,4,5)Pa-induced Ca/ +-release, resulted in loss of responsiveness to AII and ACh. As previously reported for ACh [Moriarty et al. (1988) Proc Natl Acad Sci USA 85:8865-8869], pertussis toxin and microinjected GTP[TS ] were found to inhibit follicle responses to AII, implying the involvement of a G protein. However, ACh and AII responses differ strikingly in the way they mobilize inositol phosphates and in densitization charac- teristics. We have previously been unable to find signifi- cant increases in inositol phosphates after 60 min stimu- lation (with Li +) by AII, although ACh potently activated increases in these [Mclntosh and McIntosh (1990) Arch Biochem Biophys 283: 135-140]. In the present paper, AII was found to activate rapid increases in inositol bis- and trisphosphates after 1 min stimulation without Li § ACh and AII also exerted different actions on follicle adenylate-cyclase-dependent responses. We conclude that at least two separate inositol-phosphate-linked receptor mechanisms may exist in ovarian follicles, resulting from involvement of one or more pertussis-toxin-sensitive G protein(s).

Key words: Angiotensin II - Oocytes - Low-molecular- mass heparin - Inositol phosphates - Acetylcholine

Introduction

Angiotensin II (AII) is a peptide hormone that regulates many physiological functions, including those related to

Offprint requests to: R. P. Murray-McIntosh

the control of blood pressure and maintenance of water and salt balance (for reviews, see [33, 34]). In target tissues such as smooth muscle, adrenal and pituitary glands, and liver, AII receptors are coupled to phosphoinositide hydrolysis and Ca 2+ mobilization from intracellular stores. Recently, AII binding sites have been charac- terized in mammalian granulosa cells, suggesting the presence of a local ovarian renin/angiotensin system [17, 42].

We have established that Xenopus laevis ovarian fol- licles express an endogenous response to AII, which can be detected using conventional electrophysiological tech- niques [20]. These cells are useful for the study of AII actions in ovarian tissue, because their large size (1 mm diameter) readily permits microinjection and electro- physiological measurements. Ovarian follicles from X. laevis consist of a large yolk-filled oocyte surrounded by a layer of follicle cells, with gap junctions mediating intercellular communication [6, 47]. This tissue therefore provides the opportunity for study of receptor activation in one cell type influencing responses in adjoining cells.

The underlying mechanism and function of the re- sponses of toad follicles to AII are largely unkown, although the depolarizing response to AII has a marked similarity to the muscarinic cholinergic response in this tissue [19, 23]. Follicles respond to acetylcholine (ACh) stimulation by hydrolysing phosphatidylinositol 4,5-bis- phosphate in the membrane to generate the second mes- senger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [22, 23, 30], which activates Ca 2 § release from intracellular stores [23]. Mobilization of Ca 2 § from intracellular stores in the oocyte by Ins(1,4,5)P3 causes membrane depolariza- tion by opening Ca 2 +-dependent C1- channels [15]. In- sights into the mechanism and function of the AII re- sponse can be generated by comparing AII stimulation with the well-known ACh response and messenger-RNA- induced receptor responses in injected follicles (e.g. [22, 23, 29]). In this way, we have expanded our investigations on the receptor signalling pathway activated by AII and we discuss its implications for the functional role of this hormone in ovarian tissue.

128

Materials and methods

Materials. Synthetic human angiotensin II, epinephrine, low-molec- ular-mass heparin ( 4 - 6 kDa), neomycin sulphate, and phytic acid were purchased from Sigma. Adenosine, atropine, and 4,4'- diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) were ob- tained from Serva. Guanosine 5'-([7-thio]triphosphate) (GTP[yS]) was from Boehringer Mannheim. Pertussis toxin (Sigma) was made up fresh as a stock in sterile 0.1 M sodium phosphate (pH 7.0) containing 0.05% bovine serum albumin, before addition to the incubation medium. Samples of HPLC-purified inositol 1,4,5- trisphosphate [Ins(l,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)Pj were generously supplied by Dr. Robin Irvine, AFRC Institute of Animal Physiology and Genetics Research, Cambridge, England.

Xenopus laevis toads were kindly donated by the Universities of Canterbury and Auckland, New Zealand, and also purchased from NASCO, Ft Atkinson, WN, USA. Toads were maintained at 1 8 - 20~ on a 14 h/10 h light/dark cycle.

Preparation offollicles. Stage 5 and 6 follicles (with investing follicu- lar layers) were collected and measured from toads anaesthetized with 0.2% 3-aminobenzoic acid (Sigma) or ice. Following removal from the toad, follicles were placed in sterile modified Barths' solu- tion containing 88 mM NaC1, 1 mM KC1, 2.4mM NaHCOa, 0.33 mM Ca(NO3)2, 0.41 mM CaCI/, 0.82 mM MgSO4, ]0 mM HEPES, 20 ~tg/ml each of penicillin and streptomycin, 50 U/ml nystatin, buffered to pH 7.5, and supplemented with 2 mM sodium pyruvate as an energy source. Responses could usually be obtained from follicles for 4 - 5 days after removal with storage at room temperature (17-25 ~ C). Eleven toads were used during the course of these experiments.

Electrophysiological measurements. In membrane potential re- cordings, individual follicles were placed in a 50-gl or 125-p.1 per- fusion bath for measurement by a single electrode (1 - 10 Mg2 resis- tance) against a Ag/AgC1 bath electrode as previously described [20]. The bath was continuously perfused with frog Ringer's solution (115 mM NaC1, 2 mM KC1, 1.8 mM CaCI2, and 5 mM HEPES, buffered to pH 7.4) at a flow rate of approximately 1 ml/min. In some experiments, perfusion was halted 4 - 5 min before application of drugs (5-10 p.1) by a micropipette. Otherwise, drugs were dis- solved in frog Ringer's solution and perfused across the follicle during measurement. Perfused drugs reached the bath within 5 s, while washout from the bath was complete within 3 min as judged by visual inspection of dye application. Only those follicles maintaining resting membrane potentials of at least - 35 mV were used in single- electrode recordings. Electrode measurements were recorded with a W-P Instruments Ltd. model 750 amplifier, the output of which went to a MacLab data acquisition system (ADI Ltd., Dunedin, New Zealand) and an Apple Macintosh computer.

In voltage-clamp experiments follicles were clamped at - 60 mV using a conventional two-electrode assembly (Dagan 8500 voltage- clamp apparatus, Dagan Corp., Minneapolis, Minn., USA) con- nected to the same MacLab system described above. The resistance of electrodes used in voltage clamping were 1 - 5 M~2. For the determination of voltage/current relationships in follicles before and after application of drugs, the MacLab signal generator from the Scope application was used to generate a "ramp" in the clamp voltage applied to the follicle from - / 0 0 mV to +20 mV over 5 s [7]. The amplitudes of follicle responses, recorded in both membrane voltage and voltage clamping traces, were measured at the peak of depolarization unless otherwise mentioned.

approximately 500 nl [6], within 15 rain of injection. Substances with molecular masses below i kDa injected into the oocyte cytoplasm were assumed to diffuse into the surrounding follicular cells via gap junctions that prevent the movement of molecules larger than approximately 1 kDa [6].

Determination of inositol phosphate accumulation. The method used for extraction of inositol phosphates from Xenopus follicles was essentially as previously described [24], with some modifications. Briefly, groups of about 20 follicles were microinjected with 50 nl [3H]inositol (57.9 Ci/mmol, NEN Research Products, Du Pont) and incubated for 18 h before stimulation by 1 - 2 BM angiotensin II or I mM acetylcholine for 1 -20ra in in modified Barths' solution without LiC1 added. Stimulations were terminated by addition of ice-cold perchloric acid to 5% (w/v) to the follicles and freezing the cells in a mixture of solid CO2 and methanol. After thawing, cells were disrupted by sonication and centrifuged. The supernatants were extracted and neutralized with 1:1 (v/v) trichlorotri- fluoroethane/tri-n-octylamine mixture [12]. Radiolabelled inositol phosphates in the extracts were then separated by ion-exchange chromatography on Bio-Rad AG l-X8 formate-form resin [3]. Eluted peaks were identified by their coincidental elution times relative to tritiated inositol phosphate standards (NEN Research Products, Du Pont).

Statistical analysis. All uncertainties are quoted as means _+ SEM in these experiments. Data were analysed using the Mann-Whitney U-test and Student's t-test unless otherwise indicated.

Results

A I I s t imula tes a depo la r i z ing cu r ren t in Xenopus follicles, which closely resembles the we l l -known ace ty lchol ine (ACh) response in these cells [7, 8, 23]. Some differences exist in the a p p e a r a n c e s o f these two responses when they are app l i ed at m a x i m a l doses, tha t is, a t 1 IaM A I I and 5 0 - 1 0 0 g M ACh, as de t e rmined f rom dose / r e sponse curves [20]. In vo l t age -c l amp recordings , the d u r a t i o n o f the follicle response was 45% shor te r for A I I (243 _+ 11 s, n = 36) c o m p a r e d wi th A C h (443 + 26 s, n = 35). But the amp l i t ude o f the A I I response ( - 6 3 4 _+ 37 nA, n = 57; 8 toads ) was f o u n d to be 30% larger t han tha t o f the A C h response ( - 4 3 8 ___ 30 nA, n = 46; 6 toads) .

I n c u b a t i o n o f follicles for 15 min wi th the musca r in ic recep tor inh ib i to r a t rop ine (6 g M ) d id no t b lock follicle responses to A I I (1 laM), as cu r ren t amp l i t udes in un t r ea t ed cells were no t s ignif icant ly d i f f e ren t ( - 4 4 5 _ 27 nA, n = 5) f rom those o f a t rop ine - t r ea t ed follicles ( - 5 1 4 _ 73 nA, P = 0.27, n = 5, results no t shown). As expected [19], this dose o f a t rop ine comple t e ly b locked A C h - e v o k e d (50 g M ) currents , wi th con t ro l amp l i t udes measu r ing - 5 8 3 _+ 93 n A (n = 6) c o m p a r e d to - 2 9 + 9 n A in a t r o p i n e - t r e a t e d cells (P < 0.02, n = 3, results no t shown). These results rule ou t the poss ib i l i ty tha t A I I m a y be b ind ing to or o therwise in te rac t ing wi th chol inerg ic receptors on the follicle.

Microinjection of substances. Glass capillaries (1.5 mm outer diame- ter) were pulled and their tips broken back to a diameter of 1 0 - 25 gm. These were back-filled with paraffin oil and connected to a 1 to t0-gl Drummond micropipette mounted on a W-P Instruments Ltd. micromanipulator. Follicles were routinely injected with 50 nl under a Nikon dissecting microscope. Injected material was assumed to be evenly distributed throughout the available follicle volume,

A H stimulates the follicle Ca 2 +-dependent CI- current

Fol l ic les tha t were p r e i n c u b a t e d for 10 min wi th Ca 2 +- free f rog Ringer con ta in ing 0.1 m M E G T A a n d 18 m M M g 2 +, a d d e d to p reven t cell de t e r i o r a t i on resul t ing f rom dep le t ion o f Ca 2 + f rom m e d i a [8], d id no t lose respons ive-

1 gM All

400 nA 1

C o n t r o ~ - -

1 min

4oo

=- 0 - t o

- 400 ve

Control

i

-80 i i

- 6 0 - 4 0

Clamping voltage (mV)

i i

- 2 0 0

Fig. 1. The current/voltage (I/V) characteristics of the response to angiotensin II (AII, 1 gM) as assessed using the voltage-ramp method. The trace (inset) indicates the times (arrows) before and during the AII-evoked depolarization at which the voltage ramp was applied. The bar above the trace shows the duration of hormone perfusion. The intersection of the control and test ramps indicates the reversal potential of the response, approximately -20 mV in this case. A representative ramp experiment is shown here (n = 6, traced from the original plot for clarity)

129

c 0

8 "6

v

Q.

E

B.

- 1200 - (8)

(4)

- 800 ~

- 400

0 Uninjected H20- injected

m m

500 n A l i

3 min

(4)

(3)

50 pmol 100 pmol GTP~S GTP~3 Fig. 2. Effect of intraoocyte injections of GTP[TS] on the follicle response to AII. Amplitudes of AII (1 gM) responses from uninjected and water-injected (50 nl) follicles did not differ in their mean maximal responses. The bar graph shows the mean maximal response to agonist 4- SEM. The inset shows representative traces from control and GTP[TS]-injected follicles. The small bar above each trace indicates the addition of 1 ttM AII. The mean maximal response in uninjected follicles was - 823 • 145 nA, while for water- injected follicles it was - 932 4- 100 nA. The mean maximal response for follicles injected with 50 pmol GTP[yS] was - 347 _+ 72 nA, while for follicles injected with 100 pmol GTP[TS] it was -164 4- 66 nA. Significance has been marked as *** P < 0.01, ** P < 0.02, and * P < 0.05 for all figures

hess to 1 gM AII in either membrane potential (n = 6, 2 toads) or voltage-clamp recordings (n = 14, 3 toads, data not shown). Preincubation of follicles for 10 rain with 18 m M Mn 2+, a Ca 2+ channel blocker, added to normal frog Ringer also failed to block the response to 1 gM AII (n = 4, results not shown). As found in the follicle cholinergic response [8, 28], these results indicate that AI I responses involve the release of Ca 2 + f rom intra- cellular stores.

Activation of Ca 2 + release in the follicle during stimu- lation by AI I would be expected to evoke the oocyte Ca 2 +-dependent C1- effiux [1, 26]. In order to determine the ion likely to carry the AII-evoked current in the follicles, the reversal potential (Vr) of the response was analysed by voltage ramp. The intersection of the control and test voltage/current curves, shown in Fig. 1, rep- resents the Vr of the response. The average Vr of the AII response 1 rain after the start of depolarization, which coincides with the second component of the response, was - 2 4 ___ 1 mV (n = 6). This value was identical to the mean V~ of responses to 50 gM ACh ( - 2 5 4- 1 mV, n = 6, results not shown) taken at the same time interval. The Vr for the AI I response corresponds with the equilibrium potential for CI - in Xenopus follicles, which is about - 1 9 mV to - 2 4 mV [1, 26]. The observation that AII- induced depolarization may be dependent on the gating of CI - ions was supported by pretreatment of follicles with 0.5 m M DIDS, an anionic channel blocker [29] for 5 min, which inhibited responses to 1 pM AII by 81% (P < 0.01, n = 4, results not shown). These observations suggest that AI I stimulates the same depolarizing Ca 2 +-

dependent C1 - current activated in the ACh response and in messenger-RNA-induced receptor responses [7, 31, 38].

Receptor mechanism of AII-evoked depolarization

Some indirect evidence has shown that AII-generated responses in follicles may be dependent on G proteins similar to those involved in ACh- and messenger-RNA- induced receptor stimulation [11, 27]. GTP[TS ] (563.0 Da), a nonhydrolysable analogue of GTP, binds irreversibly to and potently activates G proteins, pre- venting further G protein coupling with related receptors in the membrane [18]. We have assumed that injected substances with molecular masses below 1 kDa, such as GTP[TS], were able to pass through gap junctions into the surrounding follicular cells (see Materials and methods), where the AI I response is believed to originate [20, 36]. Xenopus ovarian follicles respond to GTP[TS ] injection by forming inositol phosphates [25]. Follicles microinjected with 50pmol and 100pmol GTP[TS] 15 min prior to stimulation by AI I exhibited significantly reduced sensi- tivity to I gM AII at both doses of GTP[TS ] tested (P < 0.01 for 50 pmol and 100 pmol/follicle, Fig. 2). Thus, AI I responses are likely to be dependent on G- protein-mediated activation pathways in the follicle.

More evidence for G protein involvement in the AII response was provided in experiments with pertussis toxin (PTX). PTX catalyses the ADP-ribosylat ion of the inhibi- tory G protein of adenylate cyclase (Gi) [45], and of Go, a GTP-binding protein of unknown function [43], leading to uncoupling of G proteins f rom their respective recep- tors.

130

PTX was freshly prepared before these experiments by adding sterile 0.1 M sodium phosphate buffer (pH 7.0), containing 0.05% bovine serum albumin, to lyophilized toxin. An aliquot of this mixture was added to modified Barths' solution containing follicles to produce a final concentration of 2 gg/ml PTX. PTX was continuously incubated with follicles at room temperature (17 - 2 1 ~ C). At approximately 2 4 - 2 6 h after the start of incubation, responses to 1 btM AII in PTX-treated follicles were found to be significantly reduced, with treated follicles averaging 6 + 2 mV (n -- 8) in amplitude, compared with control, untreated follicle responses of 28.8_ 0.9 mV (P < 0.01, n = 5, two toads; results not shown). The resting membrane potentials of toxin-treated cells were slightly elevated above control but averaged -4 1 4- 3 mV, which is well below the reversal potential of the AII response, and still allowed detection of AII- evoked depolarizations of up to approximately 20 mV in amplitude. In many cases, AII-induced CI- efflux was undetectable in toxin-treated cells. Treated follicles re- mained viable as they recovered their sensitivity to AII about 42 h later in spite of continued exposure to PTX. Therefore, as found in ACh- and messenger-RNA-in- duced receptor stimulation [11, 27, 29], AII-induced depolarization appears to be dependent on a pertussis- toxin-sensitive G protein.

The Ca2+-dependent CI- current evoked by AII closely resembles the two-component response induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [15], which is the putative second messenger in endogenous ACh receptor stimulation and exogenous messenger-RNA-in- duced receptor stimulation [22, 29]. Previously, we have been unable to detect significant inositol phosphate ac- cumulation during AII stimulation of follicles [22, 23]. However, the use of different experimental conditions has suggested that there is a small increase in inositol phosphates when follicles are stimulated by AII. Follicles were stimulated for short periods (1, 2, 10 and 20 min) in the absence of Li § in order to determine hormonally activated turnover in inositol phosphates. As shown in Fig. 3, we found that significant increases in both [aH]inositol bisphosphates (increase of41% over control, P < 0.05; two-tailed test) and [aH]inositol trisphosphates (increase of 17% over control, P < 0.05; two-tailed test) occurred after 1 min of stimulation by 2 I.tM AII in samples of 22 follicles (two experiments). The levels of [3H]inositol bisphosphates in AII-stimulated follicles remained significantly elevated above control values throughout the 20-min stimulation. In the same measure- ments, ACh-stimulated (1 mM) follicles had significantly higher levels of [aH]inositol trisphosphates (increase of 14% over control, P < 0.05; two-tailed test) after 2 rain of stimulation (results not shown). These results indicate a role for inositol phosphates in AII-evoked depolariza- tion in follicles.

As AII-stimulated increases in the levels of inositol phosphates were small, intraoocyte injections of inhibi- tors of the receptor-linked phosphoinositide pathway were also performed to determine whether the AII-in- duced CI- efflux was directly dependent on the pro- duction of inositol phosphates. Phytic acid (660.1 Da,

E

E O Z

A 400

300

200

Inositol bisphosphate

2 gM A I ! ~ _ _ _ ~

Control • S.E.M.

0 5 10 15 20

B 800 -

Inositol trisphosphate

600

400

0

~ \ ~ ' k \ \ \ \ \ \ \ \ \ \ \ \ \ \ 4 .L

Control • S.E.M.

5 10 15 20

Time of stimulation (min)

Fig. 3A, B. AII-activated mobilization of inositol phosphates in short-term stimulation (1 - 20 min). Follicle extracts were separated on Bio-Rad formate-form ion-exchange columns using a stepped salt gradient as described [3]. The line graphs show normalized count rates (cpm/50000 cpm) of (A) [aH]inositol bisphosphate and (B) [3H]inositol trisphosphate as detected in follicles after 1, 2, 10, and 20 min of stimulation by AII. Error bars represent SEM. The shaded regions beneath the line graphs indicate normalized count rates of respective inositol phosphates _+ SEM in control (unstimulated) samples of 22 follicles (n = 4). The values plotted in the line graphs represent the mean of two measurements. *, Values of P < 0.05 compared with control. The P values were calculated using Student's t-test (two-tailed analysis)

inositol hexakisphosphate) has been reported specifically to block the binding of Ins(1,4,5)P 3 to its intracellular receptor [32]. Follicles that were injected with 5 0 - 1000 pmol phytic acid 15 min prior to exposure to I gM AII or 50 gM ACh exhibited dose-dependent inhibition of both responses (Fig. 4). Microinjection of phytic acid was also found to evoke large fluctuations in the recorded current, especially at the maximum dose used in this experiment (1000 pmol). For the sake of statistical com- parison, it was necessary to take the average change in the current recording in response to a hormone between 1 min and 4 min after the start of hormone perfusion, rather than use the value corresponding to the maximal amplitude of a hormone-evoked current. Injection of 50 pmol phytic acid induced smaller fluctuations in the current (see traces in Fig. 4), which facilitated detection of responses to AII and ACh, and was found to inhibit depolarizing currents activated by AII and ACh substan- tially compared with vehicle-injected controls (P < 0.02, n = 3). Follicles injected with 1000 pmol phytic acid con- tinued to respond normally to 10 ~,M adenosine (n = 3, results not shown).

Low-molecular-mass heparin ( 4 - 6 kDa) has also been reported specifically to block the binding of Ins(1,4,5)P3 to its receptor, among other actions [14, 16]. Follicles were injected with low-molecular-mass heparin

"O

e~

- 400

- 200

u ~ nnn lmn la

- 600 500 nA 1 _ _

2 min

(3) (3 )

(3) (3) ** (4)

Vehicle- 50 250 1000 injected

Dose of phytic acid (pmol)

Fig. 4. Effect of intracellular injection of phytic acid on follicle responses to AII (1 gM; hatched bars) and acetylcholine (ACh) (50 gM; black bars). Follicles were injected with 50 nl vehicle (salt solution with equimolar concentrations of Mg 2 § and K + as phytic acid salt obtained from Sigma) or phytic acid 15 min prior to per- fusion of hormone solutions. The bar graph shows the mean change in current in response to agonist_+ SEM. Large doses of injected phytic acid activated current fluctuations in the follicle, as seen in the representative traces of responses to 1 pM AII above the bar graph. Instead of measuring the maximal amplitude of response evoked by hormone, the average difference in the current from rest to stimulation was taken 1 - 4 rain after the switch to hormone. Follicle responses to AII and ACh were significantly blocked by all three doses of phytic acid tested. Numbers in parentheses above the bars indicate the nmnber of follicles tested

131

"6

D..,

- 600 -

- 4 0 0

- 200

(5)

. . . . . (6)

Vehicle- injected

10 50 100

Dose of heparin ( g g / m l )

Fig. 5. Intraoocyte injections of low-molecular mass heparin 15- 25 min before application of hormones affect voltage-clamp re- sponses to AII (t BM; hatched bars) and ACh (50 gM; black bars). Responses to AII were significantly inhibited in follicles pre-injected with 100 gg/ml heparin, while responses to ACh were blocked in follicles injected with heparin at 50 gg/ml and i00 gg/ml. Numbers in parentheses above the bars indicate the number of follicles tested. Error bars represent SEM

(6)

(final dose 1 0 - 1 0 0 pg/ml) 15 min before stimulation by i gM AII or 50 laM ACh. Hepar in was observed to block the follicle responses to AII and ACh in a dose-dependent manner (Fig. 5), and the inhibition was significant in cells injected with 100 gg/ml heparin compared with water- injected controls (n = 13, P < 0.01). Responses to 10 gM adenosine were intact in follicles preinjected with 100 gg/ ml heparin (n = 3, results not shown).

Conditioning doses of HPLC-purif ied Ins(1,4,5)P3 were injected into oocytes 1 5 - 2 5 rain prior to hormone addition to determine the effect of this isomer on follicle responses to 1 BM AII and 50 gM ACh. Ins(1,4,5)P3 was observed to block responses to AI I and ACh potently in a concentrat ion-dependent manner (Fig. 6). Inhibition was significant at 0.1 pmol/follicle (P < 0.02) and maxi- mal at 0.5 pmol/follicle. Injections of HPLC-purif ied Ins(1,4)P2 at 0.5 pmol/follicle did not affect follicle re- sponses to the same doses of AII and ACh (n = 5 for AII , n = 6 for ACh).

Neomycin (908.9 Da), which has been reported to inhibit receptor-mediated hydrolysis of phosphatidyl- inositol 4,5-bisphosphate [4], was also injected into fol- licles to determine its effect on AI I and ACh responses. This antibiotic has also been shown to block inositol- phosphate-dependent responses in Xenopus oocytes at a dose of about 350 pmol [29]. In our hands, follicles in- jected with 350 pmol neomycin continued to respond at control values to 1 gM AII (n = 9, P = 0.18) while re- sponses to 100 gM ACh were diminished by 57% in these cells (n = 9, P < 0.01, results not shown). Higher doses of neomycin not only inhibited responses to AII [n = 5;

- 600

c O e~

- 400 "6

e~

- 200

0 .

(4)

Water- injected

(s)

0.05 0.1 0.5 5

Dose Ins(1,4,5)P 3 injected/oocyte (pmol)

Fig. 6. Effects of conditioning doses of HPLC-purified inositol 1,4,5- trisphosphate 15-25 min before stimulation of follicles by AII (1 gM; hatched bars) and ACh (50 gM; black bars). Responses to AII and ACh were significantly inhibited in follicles pre-injected with at least 0.1 pmol Ins(1,4,5)P3. Numbers in parentheses above the bars indicate the number of follicles tested. Error bars represent SEM

control = - 4 5 2 4- 7 4 n A vs, treated (700-- 1750 pmol) = - 3 0 _+ 18 nA], but also to adenosine (10 BM, n = 5; control = - 3 0 _ + 13mV vs, treated ( 5 0 0 - 700 pmol) = - 5 4- 2 mV]. These levels of neo- mycin were often found to be lethal, as injected follicles exhibited elevated resting membrane potentials of about - 1 5 mV [9 out of 13 (69%) injected follicles], indicating cell death. We concluded that the results f rom neomycin injections were invalidated by the apparent ly nonspecific, and sometimes fatal, effects of this antibiotic.

132

A

B

- 2 O

>

I = - 4 0

- 6 0

C - 2 0

- 4 O

>

E - 6 0

- 8 0

50 gM ACh 1 I~M All i

1 rain

1 gM All 10 pM adenosine

5 rain

10 p.M epinephrine 1 gM All

Fig. 7 A - C . Cross-reactivity of the AII response with other en- dogenous follicle responses. (A) A representative trace of an appli- cation of AII at a dose causing maximal depolarization (1 gM) during a response to a maximally stimulating dose of ACh (50 taM), which still evoked C1- efflux, superimposed over that induced by ACh (n = 4). This was also observed in follicles stimulated by the reverse order of addition of hormones (n = 5). (B) The adenosine (10 gM) or epinephrine (10 ~tM, not shown) response was not blocked by prior stimulation of the follicle with AII (1 ~tM, n = 5 for adenosine, n = 3 for epinephrine). Likewise, in (C), stimulation with epinephrine (10 gM) or adenosine (10 ~tM, not shown) did not cross-desensitize the follicle to 1 pM AII (n = 3 for both hormones)

Self- and cross-desensitization characteristics of AH and ACh responses

Follicle responses to AII and ACh generally do not exhibit heterologous desensitization with each other, although AII is desensitized homologously, as previously described [20]. Even when ACh was simultaneously ap- plied during a follicle response to AII, the cholinergic response was superimposed over that of AII (n = 5), and a similar effect was observed when ACh was added before AII (n = 4, Fig. 7 A). ACh did not significantly alter the amplitudes of currents evoked by AII, since the mean amplitudes in response to AII (1 gM) applied to follicles immediately following recovery of depolarization caused by 50 gM ACh were between 79% and 128% of those to AII in controls (each measurement repeated on least three follicles from each of four toads). Similarly, stimulation of follicles with 1 gM AII failed to affect amplitudes of responses to 50 gM ACh significantly, which were 81% - 143% of those in control cells (each measurement re- peated on least three follicles from each of four toads; results not shown). As an exception to this, follicles from one toad were found to have larger responses to 50 gM ACh (estimated mean peak amplitude of response = - 524 + 22 nA, n = 11, compared with an overall average from six other toads of -438 _ 30 nA, n = 46; P < 0.05), which significantly blunted responses to AII by 42% (n =

7, P < 0.02) 10-20 min after application of ACh. The long duration of C1- current oscillations evoked by ACh (averaging 7.4 + 0.4 min, n = 35) made it difficult to test AII responses less than 10 rain after the start of ACh stimulation. Larger amplitudes of depolarization have been correlated with more potent desensitization effects, presumably because of increased levels of second messen- ger produced [40].

Application of a maximally stimulating dose of AII (1 ~tM) failed to cross-desensitize follicle responses to 10 gM adenosine (n = 3) and 10 gM epinephrine (n = 3, Fig. 7 B), both of which activate a cyclic-AMP-mediated hyperpolarizing K + efflux [19, 46]. Full responses to adenosine could still be observed without a washout period after stimulation by 1 gM AII. In contrast, ACh potently blocked follicle responses to the same doses of adenosine and epinephrine (n = 4) [9, 44, 46]. Prior ex- posure of follicles to 10 gM adenosine (n = 5) or 10 laM epinephrine (n = 3, Fig. 7 C) failed to block responses to 1 gM AII. We concluded from these observations that ACh and AII may utilize independent receptor signalling mechanisms.

Discussion

The results in this paper suggest that stimulation of intra- cellular Ca 2 + release and gating of Ca 2 +-dependent C1- channels by AII in Xenopus ovarian follicles is dependent upon the production of inositol phosphates. The increase in inositol trisphosphate is small in response to AII and does not accumulate during prolonged stimulation in the presence of Li + [22, 23]. In contrast, ACh potently stimulates the production of inositol trisphosphate for up to 90 rain in similar conditions [22, 23]. These obser- vations show diverse patterns of inositol trisphosphate release during stimulation by AII and ACh.

The strikingly different ways in which AII and ACh evoke the release of inositol phosphates, as well as their dissimilar desensitization characteristics, suggest that at least two separate mechanisms linking receptors to the production of inositol phosphates may exist in ovarian follicles. This proposal is supported by electro- physiological results demonstrating that AII and ACh responses do not potently desensitize each other (also shown in [20]). The endogenous ACh response is known to cross-desensitize depolarizations evoked by ligands binding to receptors expressed in messenger-RNA-in- jected oocytes (for review, see [41]). It has been proposed that heterologous desensitization by ACh arises from convergence of different types of receptors onto a com- mon pathway mediated by inositol trisphosphate [41]. Inositol 1,4,5-trisphosphate desensitizes Xenopus oocytes to further applications of itself when injected into the same region of the cell [2], and also desensitizes mRNA- injected oocytes to stimulation of serotonin receptors [40].

Further indications that separate pathways exist for AI1 and ACh are apparent in the different effects of these hormones on responses in follicles that involve pro- duction of cAMP, leading to activation of a hyper-

polarizing K + efflux. ACh is known to desensitize follicles to hormones generating cAMP, such as adenosine and epinephrine (an effect that has been attributed to acti- vation of protein kinase C by ACh [9]), while AII at maximally depolarizing doses was unable to affect electrophysiological responses to these hormones.

There are several possibilities that could explain why AII and ACh mobilize inositol trisphosphate and activate release ofintracellular Ca 2 + in common without potently cross-desensitizing each other. The two responses may be compartmentalized, so that AII and ACh receptors and their associated effector systems are localized within sep- arate cell structures. For example, the two responses may be polarized at different points around the oocyte, or two populations of follicle cells could exist on ovarian follicles, each separately expressing ACh and AII recep- tors. Mammalian ovarian follicles have recently been re- ported to contain at least two populations of granulosa cells, which separately produce receptors to follicle- stimulating hormone and AII [13]. Alternatively, AII may activate the production of inositol phosphates only in the follicle cells surrounding the oocyte, while ACh may activate inositol phosphate production mainly within the oocyte. The follicle cells are known to be electrically coupled to the oocyte by means of gap junctions [6, 47]. To address this possibility, enzymic and manual de- folliculation techniques have been employed in our lab- oratory combined with electrophysiological screening of hormone responses and scanning electron microscopy of follicle surfaces during defolliculation (P. Lacy, unpub- lished observations). Both ACh and AII responses were eradicated or greatly reduced upon removal of the follicle cells from the oocyte using either collagenase treatment (n = 8) or manual defolliculation (n = 11), provided that defolliculation was complete according to scanning elec- tron micrographs. Collagenase treatment did not destroy receptor function, as responses to AII and ACh remained in follicles that had been treated with collagenase for the same period of time as stripped oocytes but, unlike stripped oocytes, did not have their follicular layers re- moved. In support of these observations, octanol, an inhibitor of gap junctions [20, 36], was found to eradicate responses to both ACh and AII. Hexanol, used as a negative control at an equivalent dose to octanol, did not affect follicle responses to AII. Similar conclusions have been reached in experiments conducted by Sandberg and coworkers [36]. Thus, from our findings, it seems likely that both ACh and AII receptors were present in the follicle cells of ovarian follicles used in our experiments.

Other reasons for the observed differences between ACh and AII responses may be related to the receptor stimulation pathways. The homologous desensitization seen in responses to ACh and AII suggests that de- sensitization within these responses occurs at the level of the receptors. However, Ins(1,4,5)P3 and protein kinase C have been shown to mediate desensitization of expressed serotonin receptors both at the level of the receptor and downstream of receptor stimulation in Xenopus oocytes [40], which would be expected in stimulation by ACh and AII. To account for this, receptor occupancy may not have reached high enough proportions to evoke cross-

133

desensitization by exhaustion of Ins(l,4,5)P3-sensitive Ca z + stores, although both hormones were employed at maximally stimulating doses. Another plausible expla- nation for these findings is that AII and ACh may stimu- late two inositol phosphate pathways linked to distinct intracellular Ca z + pools. Recently, two different recep- tors were shown to be linked to distinct Ca 2 + stores in Xenopus oocytes [39]. In addition, Lechleiter et al. [21] detected responses in Xenopus oocytes expressing cloned m2 and m3 muscarinic receptors, which could be dis- tinguished by their patterns of Ca z + release and their sensitivities to PTX, suggesting that the two receptors were linked to distinct G proteins native to the oocyte. Although endogenous responses to AII and ACh are both inhibited by PTX (inhibition of the follicle cholinergic response by PTX has been demonstrated by Moriarty et al. [27]), their different effects on adenylate cyclase responses would lend weight to the idea that distinct G proteins may be involved in the signalling pathways for AII and ACh. Alternatively, the ACh receptor may re- cruit larger numbers of the same G protein as the one employed in AII stimulation. In either case, the G pro- teins are likely to couple to a common receptor signalling pathway involving phospholipase-C-induced generation of CaZ+-releasing Ins(1,4,5)P3 (for reviews, see [6, 41]), with activation by ACh being somewhat greater in po- tency than AII.

A second important issue to consider is the interpre- tation of results from heparin injections. The high mo- lecular mass of heparin ( 4 - 6 kDa) should prevent its movement into the follicle cells following injection into the oocyte [6]. This implies that inositol phosphates mobilized by ACh and AII may pass from the follicle cells to the oocyte and release Ca 2 + from pools situated within the oocyte. In support of this view, Ins(1,4,5)P3 and Ca 2 + have been shown to pass freely through gap junctions in isolated hepatocytes [35]. Thus an intercellu- lar communication pathway using second messengers may exist between receptors in the follicle cells and effec- tor systems in the oocyte. The possibility of such a path- way existing in Xenopus follicles has been proposed by Sandberg et al. [36]. However, results from heparin exper- iments must be interpreted with caution in view of its nonspecific actions on other cytoplasmic proteins [5].

A recent report has shown that phytic acid evokes a biphasic current when injected into Aplysia neurones [37], suggesting a direct cytoplasmic function for phytic acid. It is possible but not probable that injection of phytic acid into follicles forms small quantities of Ins(1,4,5)P3 capable of desensitizing the cell to stimulation by hor- mones [40]. Very slow hydrolysis of inositol pentakis- phosphate to an inactive tetrakisphosphate has been ob- served in homogenates of J(enopus ovarian follicles [25]. Phytic acid is also a known chelator of Ca 2+, although it is not likely to bind avidly to Ca 2 + at the comparatively low intracellular levels of Ca 2 + [48]. For these reasons, the results from phytic acid injection have not been as conclusive as observations from heparin and Ins(l,4, 5)P3 and quantification of inositol phosphates. However, taking together all the results, these experiments have demonstrated the involvement of inositol phosphates in

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the second-messenger pa thway st imulated by A I I in Xenopus follicles.

The funct ional role o f A I I in Xenopus follicles appears to be associated with matura t ion , as incubat ion o f A I I and progesterone with some batches o f follicles resulted in enhanced ma tu ra t ion rates over those obta ined f rom follicles exposed to proges terone alone (supported by observat ions o f Sandberg et al. [36]); in follicles f rom other toads, an effect by A I I could no t be detected (P. Lacy, unpubl ished observations). ACh was observed to accelerate the rate o f ma tu ra t ion induced by progesterone more potent ly (P. Lacy, unpubl ished observations) [10], which is interesting in view o f its ability to stimulate inositol phospha te p roduc t ion more vigorously than AI I . Fur ther work in this area will help to contr ibute to our unders tanding o f the control o f oocyte funct ion and matu ra t ion mediated by hormones act ing on follicle cells.

In conclusion, we have described a depolar izing re- sponse to A I I in Xenopus follicles tha t closely resembles, and yet does no t directly interact with ACh responses in this tissue. These responses differentially produce inositol phospha te dur ing agonist applicat ion and they differ in their desensitization patterns. Fur thermore , we have suggested the presence o f an intercellular communica t ion pa thway in these responses, which appears to involve passage o f receptor-mobil ized inositol phosphates f rom somatic cells (the follicle cells) to a germ cell (the oocyte). The possible existence o f an intercellular communica t ion pa thway provides new possibilities for second-messenger modula t ion o f meiotic ma tu ra t ion in oocytes. It also holds very interesting implications for h o r m o n a l re- sponses in other tissues conta in ing mixed cell types, in which differential receptor and response sitings m a y com- bine to mediate complex physiological functions.

Acknowledgements. We acknowledge support from the Medical Re- search Council of New Zealand in the form of a Postgraduate Scholarship (M.P.L.) and a Senior Fellowship (R.P.M.) We thank Dr. Robin Irvine for kindly supplying us with Ins(1,4,5)P3 and Ins(l,4)P2. The Dagan 8500 voltage clamp was funded by the New Zealand Lotteries Board of Control, and some working expenses were provided by the Wellington Medical Research Foundation.

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