Article
Reference
Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol trisphosphate-dependent
mechanism
RAWLINGS, Stephen Roy, DEMAUREX, Nicolas, SCHLEGEL, Werner
Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP) increases cAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) that appear to be independent of cAMP. To study the mechanisms involved in this response, we used the patch-clamp technique to microperfuse various agents into single rat gonadotrophs while monitoring [Ca2+]i with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (> 1 microM) oscillations in [Ca2+]i, which were blocked by intracellular application of GDP beta S (guanosine 5'-O-2-thiodiphosphate), indicating the involvement of a G-protein. To identify the intracellular messenger(s) involved, we microperfused gonadotrophs with cAMP, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of cAMP (100 microM) had no significant effect on basal [Ca2+]i and did not alter the PACAP-stimulated Ca2+ response. Heparin, but not its inactive [...]
RAWLINGS, Stephen Roy, DEMAUREX, Nicolas, SCHLEGEL, Werner. Pituitary adenylate cyclase-activating polypeptide increases [Ca2]i in rat gonadotrophs through an inositol
trisphosphate-dependent mechanism. Journal of Biological Chemistry , 1994, vol. 269, no. 8, p. 5680-6
PMID : 7907085
Available at:
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Pituitary Adenylate Cyclase-activating Polypeptide Increases [Ca2+Ji in Rat Gonadotrophs through an Inositol
Trisphosphate-dependent Mechanism*
(Received for publication, June 22, 1993, and in revised form, September 14, 1993) Stephen R. RawlingsS, Nicolas Demaurexlin, and Werner Schlegel
From the Fondation pour Recherches Mkdicales, University of Geneva, CH-1211 Geneva 4, Switzerland, and §Division of Infectious Diseases, University Hospital Geneva
Pituitary adenylate cyclase-activating polypeptide (PACAP) increases CAMP production and stimulates hormone release from a variety of anterior pituitary cells. However, in anterior pituitary gonadotrophs PACAP stimulates oscillations in cytosolic free Ca2+ con- centration ([Ca2+li) that appear to be independent of CAMP. To study the mechanisms involved in this re- sponse, we used the patch-clamp technique to microper- fuse various agents into single rat gonadotrophs while monitoring [Ca2+li with microfluorometry. Extracellular application of PACAP to single gonadotrophs stimulated high amplitude (>1 p ~ ) oscillations in [Ca2+Ii, which were blocked by intracellular application of GDPpS (guanosine 5’-0-2-thiodiphosphate), indicating the in- volvement of a G-protein. To identify the intracellular messengerb) involved, we microperfused gonadotrophs with CAMP, inositol 1,4,5-trisphosphate tIns(l,4,5)P3), or heparin, an antagonist of the Ins(1,4,5)P3 receptor. A high concentration of CAMP (100 PM) had no significant effect on basal [Ca2+Ii and did not alter the PACAP- stimulated Ca2+ response. Heparin, but not its inactive isoform, completely blocked the PACAP-stimulated in- crease in [Ca2+li, while Ins(1,4,5)Ps stimulated oscilla- tions in [Ca2+Ii very similar to those observed in re- sponse to PACAP. These results strongly suggest that PACAP mobilizes Ca2+ through an Ins(l,4,5)P3-depend- ent mechanism. The fact that PACAP stimulates two sig- naling pathways in pituitary cells could substantially enhance the signaling potential of this hypothalamic peptide.
Pituitary adenylate cyclase-activating polypeptide (PACAPl1 is the most recently discovered member of the secretid glucagodvasoactive intestinal polypeptide (VIP) family of pep- tides. It is a 38-amino acid, C terminus-amidated, peptide (PACAPSS), which also exists in a 27-amino acid form
* This work was supported by the Swiss National Science Foundation Grants 32-33514.92 and 32-30161.90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. Tel.: 022-382-3841;
Fax: 022-347-5979.
1 Supported by the Janggen-Pohn Foundation (St. Gallen) and the Ciba-Geigy Foundation (Basel).
activating polypeptide; PACAP38, the 38-amino acid form of PACAP;
The abbreviations used are: PACAP, pituitary adenylate cyclase- PACAP27, the 27-amino acid form of PACAP; VIP, vasoactive intestinal polypeptide; LH, luteinizing hormone; LHRH, LH-releasing hormone;
[Ca2+Ii, cytosolic free Ca2+ concentration; Ins(1,4,5)P3, inositol 1,4,5- trisphosphate; G-protein, GTP-binding protein; GDPpS, guanosine 5’- 0-2-thiodiphosphate; indo-UAM, the membrane-permeable acetoxy- methyl ester form of indo-1.
(PACAP271 (Miyata et al., 1989,1990). The first 28 amino acids of PACAP38 share 68% sequence homology with porcine VIP, although the remaining sequence is unique (Miyata e t a l . , 1989). However, PACAP38 is approximately 1000 times more potent at stimulating CAMP production in anterior pituitary cells than is VIP (Miyata et a l . , 1989). PACAP-like immunore- activity is found in the median eminence of the hypothalamus (Koves et al., 1990) and specific PACAP binding sites, which do not significantly bind VIP, are present on anterior pituitary cells (Gottschall et al., 1990). In addition, PACAP can modulate the in vitro and in vivo release of a variety of pituitary hor- mones including luteinizing hormone (LH) (Culler and Pas- chall, 1991; Hart et al., 1992; Osuga e t a l . , 1992), and growth hormone (Goth e t a l . , 1992; Hart e t a l . , 1992; Jarry et al., 1992).
Thus it has been proposed that PACAP may act as a hypotha- lamic regulator of anterior pituitary cell function.
The intracellular mechanisms regulated by PACAP have yet to be fully determined. I n addition to its effect on CAMP levels, PACAP has been shown to increase [Ca2+li in a number of anterior pituitary cell types (Canny et al., 1992; Vigh e t a l . , 1992; Gracia-Navarro et al., 1992). In somatotrophs, which se- crete growth hormone, PACAP stimulates Ca2+ influx through a CAMP-dependent mechanism (Canny e t a l . , 1992; Rawlings et a l . , 1993). In contrast, in gonadotrophs PACAP stimulates Ca2+
release from a n intracellular store, a n effect that is not blocked by a n antagonist to CAMP-dependent protein kinase (Canny et al., 1992; Rawlings et al., 1993). It thus appears that PACAP can stimulate two different intracellular pathways in closely related cell types. This study addresses the mechanisms by which PACAP stimulates the mobilization of Ca2+ in rat go- nadotrophs.
A number of intracellular messengers are capable of stimu- lating the release of Ca2+ from intracellular stores including inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) (Berridge and Irvine, 1989), CAMP (Capiod e t a l . , 19911, cADP ribose (Galione, 1993) and Ca2+ itself (Fabiato, 1983). The present study has investi- gated the mechanisms underlying the PACAP-stimulated Ca2+
release in single identified gonadotrophs. The whole-cell patch clamp technique was used to apply various compounds intra- cellularly while [Ca2+li was monitored with microfluorometry.
It was found that the action of this peptide in the gonadotroph is G-protein-mediated, and stimulates the release of Ca2+ from a n intracellular store through an Ins(1,4,5)P3-dependent, CAMP-independent, mechanism.
EXPERIMENTAL PROCEDURES
Anterior Pituitary Cell Dissociation-Male Sprague-Dawley rats (200-300 g, IFFA Credo, l’kbresle, France) were kept in a controlled environment before use (lights on, 0700-1900,25 “C). For each experi- mental day, a single rat was decapitated, the anterior pituitary gland removed, finely diced, and the cells dissociated in Spinner’s minimum essential medium (Life Technologies, Inc.) containing 0.1% trypsin 5680
PACAP38 Regulation
of[Ca2+li in Rat Gonadotrophs
5681(Difco), 0.2% penicillidstreptomycin (Life Technologies, Inc.), and 0.1%
bovine serum albumin at 37 "C for 1 h. The cells were resuspended in RPMI medium (Life Technologies, Inc.), containing 0.1% bovine serum albumin and 0.2% penicillidstreptomycin, at 400,000 cells/ml and plated onto poly-L-lysine-coated glass coverslips (Rawlings et al., 1991).
The cells were incubated a t 37 "C in a humidified atmosphere contain- ing 5% COz for at least 1 h before use.
[Ca'+], Measurements-Anterior pituitary cells were loaded with indo-1/AM (2.5 PM) in RPMI for 30 min a t room temperature, washed with RPMI to remove any remaining ester, and then kept at room temperature until use. The coverslip holding the indo-1-loaded cells was fixed into a chamber on the stage of a n inverted microscope (Nikon Diaphot) equipped for dual emission recordings (Demaurex et al., 19931, and changes in [Ca2+], were monitored by recording the fluorescent emission of the indo-1 in response to UV light. The excitation light was provided by a mercury lamp and was attenuated by neutral density filters (x 256), passed through a 355-nm interference filter, and re- flected to the stage of the microscope by a dichroic mirror (critical wavelength (Acrit) = 400 nm). The emitted fluorescence light was col- lected by a n objective (Nikon Fluor x 40, oil immersion) and split into two by a second dichroic mirror (Acrit = 455 nm), the high wavelength light then passing through a 480-nm interference filter to be detected by one photomultiplier, while the low wavelength light passed through a 405-nm interference filter to be detected by a second photomultiplier.
Emitted indo-1 fluorescence a t t h e two wavelengths was recorded si- multaneously a t 50 Hz using a 12-bit analogue-digital interface (Acqui, SICMU, Geneva) and stored on a n IBM computer. The output of the two photomultipliers was also recorded directly onto a digital tape recorder (DTR-1201 Biologic, 38640 Claix, France) at 12 kHz to provide a backup of the recording.
Whole Cell Patch Clamp Electrophysiology-The whole-cell configu- ration of the patch clamp technique (Hamill et al., 1981) was used to voltage-clamp and microperfuse substances into the cells. Borosilicate glass pipettes were made using a BB-CH-PC puller (Mecanex, Nyon, Switzerland) and had resistances of between 6 and 12 megohms when using the standard solutions described below. Pipette-cell membrane seal resistances were between 20 and 50 gigohms, and access resis- tances upon establishing the whole cell configuration ranged from 15 to 40 megohms. Recordings were made with a n Axopatch 200A amplifier (Axon Instruments Inc., Foster City, CA) in the voltage-clamp mode.
Recording of current and holding voltage were made by the same A/D interface used for the [Ca2+l, measurements, allowing for concurrent recordings of electrophysiological and [Ca2+li data. The data were also backed u p onto the digital tape recorder at a sampling rate of 24 kHz.
Whole-cell recordings were made with cells voltage-clamped to a potential close to the normal resting membrane potential. To determine this value, recordings of membrane potential at zero applied current were made. The resting membrane potential of identified gonadotrophs was found to range between -59 and -43 mV, with a mean value of -50
f 2 mV (mean 2 S.E.; n = 10). Thus whole-cell voltage-clamp recordings were made at a holding potential of -50 mV.
Calculation of[Ca"+l, Values-Values for [Ca2+li were calculated us- ing the formula (Grynkiewicz et al., 1985): [Ca2+l, = K,.p.(R - RmiJ (R,,, - R ) , where R = F405/F480. F405 and F4,, are the recorded fluores- cence intensities at 405 nm and 480 nm wavelengths, respectively. The calibration constants (R,,,, R,,, K d . p ) were determined in whole-cell recordings under the same experimental conditions (Almers and Neher, 1985). R,,, (0.47 f 0.02, mean f S.E.; n = 8 ) was determined after
perfusing cells with the standard pipette solution buffered to free [Ca2+]
< 10 nM with 10 mM EGTA. R,,, (1.61 ? 0.05; n = 10) was measured with a pipette solution containing 10 mM Ca2+. Kd.P (1217 2 30; n = 9) was calculated by solving the above equation using the R value measured following break-in with a pipette solution containing 9.2 mM EGTA and 5.4 m CaC12, which produced a fixed Ca2+ concentration of 291 nM, as measured with a Ca2+-sensitive electrode.
Solutions-Experiments were conducted at 22 "C in experimental medium containing (in m): 127 NaCl, 5 KCl, 2 MgCl,, 1.8 CaCI,, 5 NaHCO,, 10 HEPES-NaOH, and 10 glucose, pH 7.4. For the nominally Ca2+-free medium, the CaCl, was replaced with the Ca2+ buffer EGTA (1 mM). The standard pipette solution contained (in mM), 120 potassium aspartate, 20 KCl, 2 MgC12, 20 HEPES-NaOH, 0.1 GTP, 2 ATP, 0.04 indo-1, pH 7.4. This pipette solution had a free Ca2+ concentration of between 70 and 85 nM, measured with a Ca2+-sensitive electrode.
Microperfusion of Compounds into the Cell-The use of the whole-cell configuration of the patch clamp technique allowed compounds to be microperfused into the inside of single cells during recording. Where appropriate, heparin, de-N-sulfated heparin, Ins(1,4,5)P3, or CAMP
volving GDPPS, this nucleotide replaced GTP in the pipette solution.
Achievement of the whole-cell configuration generally resulted in little change in [Ca2+],. However, when Ins(1,4,5)P3 was included in the pi- pette a rapid (within 2 s) rise in [Ca2+li was observed (see Fig. 5). This indicates that the diffusion of low molecular weight compounds from the pipette into the cell is very rapid. A high access resistance between the pipette and the inside of the cell significantly reduces the ability of the pipette contents to diffuse into the cell (Marty and Neher, 1983), a n effect that is particularly profound for large molecular weight com- pounds like heparin. For this reason, when blockers were introduced into the cell, only recordings where the access resistance was less than 40 megohms were considered. In general PACAP was applied to the cell 2 min after establishing the whole-cell configuration to ensure t h a t even the large molecular weight compounds such as heparin had sufficient time to diffuse into the cell.
Cell Identification-Only 3-5% of anterior pituitary cells secrete LH in response t o LHRH and thus can be identified as gonadotrophs (Raw- lings et al., 1991). LHRH is a specific stimulator of gonadotrophs, and thus can be used as a tool to identify such cells (Naor, 1990). In this study lobed cells of 10-12-pm diameter were selected for recordings, since a high proportion of such cells have been shown to be LH-secreting gonadotrophs (Rawlings et al., 1991). In our hands, >95% of the cells selected on these morphological criteria showed a clear Ca2+ response to LHRH. Only cells exhibiting a Ca2+ response to LHRH (Leong and Thorner, 1991), added a t t h e end of the experiment, were included in the statistics for this study. The maximal concentration of LHRH (IO nM) used was capable of overcoming the potential blocking actions of intra- cellularly applied heparin and GDPPS (Tse and Hille, 1992).
Materials-PACAP (1-38) was obtained from either Peninsula Labo- ratories Europe Ltd. (St. Helens, UK), or Calbiochem (Laufelfingen, Switzerland). PACAP (1-27) was obtained from Calbiochem. Indo-1/AM and indo-1-free acid were purchased from Molecular Probes, Inc. (Eu- gene, OR). Ins(1,4,5)P3 was purchased from DuPont NEN. The heparin used had a n M , of 6,000 (product no. H-2149; Sigma). All other peptides and reagents, unless otherwise stated, were obtained from Sigma.
RESULTS
This study is the first to employ whole-cell patch clamp re- cordings to study the action of PACAP at the single cell level. As such, it was necessary to test whether this technique signifi- cantly alters the responsiveness of gonadotrophs to PACAP.
Thus the peptide was added to intact cells and cells in whole- cell recording configuration to compare the stimulated changes in [Ca2+li under otherwise identical experimental conditions.
In intact gonadotrophs, a maximum concentration of PACAP (100 m; Rawlings et al., 1993) stimulated an increase in [Ca2+Ii (Fig. IA). Of 18 gonadotrophs tested in this way, 15 cells (83%) showed an increase in [Ca2+Ii, and 12 of these 15 cells (80%) showed high amplitude (>1 p ~ ) oscillations in [Ca2+Ii as shown in Fig. lA. The other three cells exhibited a low amplitude (0.2-0.4 p ~ ) , steplike increase in [Ca2+li, as previously de- scribed (Canny et al., 1992; Rawlings et al., 1993). In gonado- trophs subjected to whole-cell voltage-clamp recordings, PACAP (100 nM) stimulated an increase in [Ca2+Ii (Fig. 1B, top).
Of 23 cells tested in this way, 21 cells (91%) showed increases in [Ca2+li, and of these 21 cells, 19 (90%) showed the Ca2+
oscillations response (Fig. 1 B ) . Hence, the responsiveness of the gonadotrophs to PACAP in the whole-cell recording configu- ration was not significantly altered compared to intact cells.
Significantly, in both intact and patch clamped cells, the pres- ence of PACAP in the medium did not preclude the Ca2+ re- sponse to a high concentration (10 nM) of LHRH (Fig. 1).
The whole-cell voltage-clamp technique also allowed us to monitor changes in cell membrane conductance. With cells volt- age-clamped a t -50 mV, close to the resting membrane poten- tial of these cells (see "Experimental Procedures"), both PACAP and LHRH stimulated changes in membrane conductance, which appeared to closely follow their effects on changes in [Ca2+li (Fig. 1B, bottom). Similar LHRH-stimulated currents have been previously reported (Kukuljan et al., 1992; Tse and Hille, 1992) and have been used instead of indo-1 to monitor
PACAP38 Regulation of [Ca2+li in Rat Gonadotrophs
A. lntarr Cell
PACAP LHRH I
0 1 2 3 4 5 6 7
Time (mid
B. Whole-Cell Recording
' 1 PACAP ,LHRH
Current (PA) Whole-Cell 0
-40
0 1 2 3 4 5 6 7
Time (mid
C . Inrocr Cell
'I
LHRH (1 pM)0 1 2 3 4 5 6
Time (mid
phs. A, PACAF' (100 n M ) was applied extracellularly (solid bar) to an FIG. 1. PACAP-stimulated Ca2+ responses in single gonadotro- intact rat gonadotroph in which [Ca2+1- was monitored by microfluo- rometry with indo-I. The cell was identified by the subsequent addition of LHRH (10 n M ; open bar). In this, and all subsequent figures, a given under "Results." B , a whole-cell patch-clamp recording from a representative recording is shown, and the number of experiments is gonadotroph voltage-clamped at -50 mV, close to its resting membrane potential (see "Experimental Procedures"). The upper trace shows brane current. PACAF' (100 nM; solid bar) and LHRH (10 nM; open bar) [Ca2+l,, and the lower trace shows the concurrent recording of cell mem- were applied extracellularly. As with all recordings, LHRH was added to identify the cell type. C , microfluorometric measurement in an intact gonadotroph of the Ca2+ response t o 1 PM LHRH.
the measurements of membrane conductance were used simply as a monitor of the cell stability during microperfusion.
A maximal concentration of LHRH (10 nM) generally stimu- lates a characteristic "spike-plateau'' Ca2+ response pattern in gonadotrophs, as most clearly observed in Fig. lA (Shangold et al., 1988; Iida et al., 1991; Leong and Thorner, 1991). In con- trast, lower concentrations of LHRH (around the picomolar level) stimulate repetitive Ca2+ oscillations, as shown in Fig. 1C (Shangold et al., 1988; Iida et al., 1991; Leong and Thorner, 1991). Similarities in the Ca2+ response patterns for PACAP (Fig. 1, A and B ) and LHRH (Fig. 1C) suggest the possibility of a common mechanism of action. The present study has inves- tigated the intracellular mechanisms regulating the PACAP- stimulated Ca2+ oscillation response in gonadotrophs. The in- frequently observed steplike Ca2+ response pattern (Rawlings et al., 1993) has not been specifically addressed in the present study.
GTP-binding Proteins (G-proteins)
The cell membrane receptor-mediated stimulation of the Ca2+ mobilization may occur through G-protein-dependent (Taylor et al., 1991) and G-protein-independent (Kim et al., 1991; Gusovsky et al., 1993) mechanisms. To test for the in- volvement of a G-protein in the PACAP stimulation of Ca2+
CDPgS
'1
PACAP LHRH
i
0 1 2 3 4 5 6 7
Time (mid
mediated through a G-protein. Measurement of [Ca2+], during a FIG. 2. Ca2+ mobilizing response to PACAP in gonadotrophs is whole-cell recording of a gonadotroph voltage-clamped at -50 mV. The whole-cell configuration, established at time = 0, allowed for the mi- croperfusion of the cell with 2 mM GDPpS included in the pipette solu- tion. PACAP and LHRH were applied as in Fig. 1.
mobilization, the competitive inhibitor of GTP-binding to G- proteins GDPpS replaced GTP in the pipette solution. Mi- croperfusion of GDPpS (intrapipette concentration = 2 mM) into the cell blocked the Ca2+ response t o the subsequent addition of PACAP in all cells tested (Fig. 2, n = 6). This concentration of GDPpS also blocked the Ca2+ mobilizing action of low concen- trations of LHRH (data not shown), which is known to involve a G-protein (Conn et al., 1987; Naor, 1990). However, as previ- ously suggested (Tse and Hille, 1992), the use of a maximal concentration of LHRH (10 nM) was capable of overcoming this block, thus allowing the identification of the gonadotrophs (Fig.
2). In conclusion, the effect of PACAP on Ca2+ mobilization in gonadotrophs appears to be mediated by the activation of a G-protein.
Cyclic AMP
A previous report using a competitive antagonist to CAMP- binding of CAMP-dependent protein kinase has demonstrated that the PACAP-stimulation of Ca2+ mobilization in rat go- nadotrophs is not through the activation of CAMP-dependent protein kinase (Rawlings et al., 1993). However, CAMP is also known to have CAMP-dependent protein kinase-independent actions (Nakamura and Gold, 1987; DiFrancesco and Tortora, 1991). To test for the effect of CAMP on [Ca2+Ii, and the subse- quent response to PACAP, a high concentration of CAMP (100 PM) was microperfused into identified gonadotrophs. This con- centration of CAMP significantly stimulates Ca2+ mobilization in guinea pig hepatocytes (Capiod et al., 1991). However, it had no effect on [Ca2+Ii in four gonadotrophs, and produced a small decrease in basal [Ca2+li in one cell (Fig. 3). Microperfusion of CAMP had no noticeable effect on the subsequent Ca2+ response to the addition of PACAP (Fig. 3, n = 5). These results strongly suggest that the PACAP stimulation of Ca2+ mobilization is not through a CAMP-dependent mechanism.
Ins(1,4,5)P3
Effect of the Ins(1,4,5)P3 Receptor Antagonist Heparin- Previous reports have used the competitive Ins(1,4,5)P3 recep- tor blocker heparin (Ghosh et al., 1988) to distinguish between Ins(l,4,5)P3-dependent and -independent stimulation of Ca2+
mobilization in a variety of cell types including gonadotrophs (Pape et al., 1988; Capiod et al., 1991; Tse and Hille, 1992;
Takasawa et al., 1993). Heparin (300 J ~ M ) microperfused into gonadotrophs completely blocked the PACAP-stimulated Ca2+
response in all cells tested (Fig. 4 A , n = 7). In contrast, inclusion of 300 p~ of the inactive form of heparin (de-N-sulfated hepa- rin), had no effect on the PACAP-stimulated Ca2+ mobilization response (Fig.
a,
6 of 7 cells). In agreement with an earlier study (Tse and Hille, 1992), we have found that the Ca2+ re- sponse to low concentrations of LHRH were blocked by this concentration of heparin (data not shown). However, addi-PACAP38 Regulation
of[Ca2+li in Rat Gonadotrophs
5683CAMP
PACAP ,LHRH I
0 1 2 3 4 5 6 7
Time (min)
FIG. 3. PACAP-stimulated Cas+ mobilization response is not mediated through C A M P . Measurement of [Ca2+li during a whole-cell recording of a gonadotroph voltage-clamped at -50 mV. The whole-cell allowed for the microperfusion of the cell with 100 p~ CAMP contained configuration was established at the time indicated by the arrow and in the pipette solution. PACAP and LHRH were applied as in Fig. 1.
A.
Heparin
PACAP LHRH
I 2 1
0 1 2 3 4 5 6 7
Time (mid
B.
De-N-Sulphated Heparin
'1
PACAP ," I
0 1 2 3 4 5 6 7
Time ( m i d
FIG. 4. PACAP-stimulated Cas+ response is mediated through the activation of the CaB+store Ins(l,4,6)Ps receptor. Measure- ment of [Caz+li during a whole-cell recording of gonadotrophs voltage- clamped at -50 mV. Whole-cell configuration was established at the time = 0, and allowed for the microperfusion of the cell with the isoform of de-N-sulfated heparin (300 p ~ ; B ) contained in the pipette Ins(1,4,5)P3 receptor antagonist heparin (300 ~ M ; A ) , or with its inactive solution. PACAP and LHRH were applied as in Fig. 1.
tion of the maximal concentration of LHRH (10 m) to identify the cell type was able to elicit a Ca2+ response. Such effects of high concentrations of LHRH to overcome the heparin block have been previously reported (Tse and Hille, 1992).
Reproduction of the PACAP-stimulated Ca2+ Response by Zns(1,4,5)P3 -If the effect of PACAP to mobilize Ca2+ was through the production and action of Ins(1,4,5)P3, it would be expected that the microperfusion of Ins(1,4,5)P3 into the cell should reproduce the Ca2+ response patterns observed for PACAP. Four concentrations of Ins(1,4,5)P3 were tested. Mi- croperfusion of 5 p~ Ins(1,4,5)P3 into gonadotrophs resulted in either no response (3 cells; Fig. 5 A ) , or the production of a single Ca2+ spike after a delay of 40 s (1 cell). Microperfusion of 10 p~ Ins(1,4,5)P3 resulted in either no effect (4 cells), or re- petitive Ca2+ oscillations (3 cells; Fig. 5 B ) . Microperfusion of 20
all of the cells tested (Fig. 5C; n = 7). The top concentration of Ins(1,4,5)P3 tested with this protocol (50 p ~ ) produced a series of rapid Ca2+ oscillations (Fig. 5 0 ; n = 6). Comparison of the Ca2+ oscillation response pattern stimulated by PACAP (Figs.
1, 3, and 4 B ) and the response to the microperfusion of Ins(1,4,5)P3 (Fig. 5) clearly demonstrate that physiological con- centrations of Ins(1,4,5)P3 can mimic the Ca2+ mobilizing ac- tion of PACAP.
Block of the PACAP Response by a High Concentration of Zntracellular Ins(1,4,5)P3 -The intracellular messenger Ins(1,4,5)P3 is known to address specific intracellular Ca2+
stores. If PACAP acts through a n Ins(l,4,5)P3-dependent mechanism, the emptying of these Ca2+ stores should preclude further responses to PACAP. To empty the Ins(l,4,5)P3-sensi- tive stores we microperfused a pharmacologically high concen- tration of Ins(1,4,5)P3 (200 PM) into gonadotrophs bathed in a nominally Ca2+-free medium containing 1 mM EGTA. In five of six cells tested in this way, Ins(1,4,5)P3 stimulated a rapid, large amplitude rise in [Ca2+Ii, which was followed by a number of high amplitude Ca2+ oscillations before the [Ca2+lj returned to basal levels, generally within 3 min (Fig. 6 A ) . In the other cell, Ins( 1,4,5)P3 microperfusion stimulated a single Ca2+
spike, which was not accompanied by any repetitive Ca2+ os- cillations. Clamping the intracellular Ins(1,4,5)P3 level in this way completely blocked the Ca2+ response to a subsequent addition of PACAP ( n = 5) (Fig. 6A). In control cells (no Ins(1,4,5)P3 microperfusion, in Ca2+-free medium), 4 of 6 cells tested exhibited a Ca2+ response to PACAP (Fig. 6B). The ad- dition of a high concentration of LHRH at the end of the ex- periment was generally capable overcoming the block by Ins(1,4,5)P3 and eliciting a small response (Fig. 6A). Only cells identified in this way were included in the results detailed above.
In conclusion, these data demonstrate that PACAP can stimulate the release of Ca2+ from intracellular Ca2+ stores through a n Ins(l,4,5)P3-dependent mechanism in the rat go- nadotroph.
DISCUSSION
PACAP was first isolated, and in fact was named, by its action to stimulate the production of CAMP in anterior pituitary cells (Miyata et al., 1989). A number of its actions in the regu- lation of pituitary cell function appear to be through the CAMP system, including the stimulation of changes in Ca2+ influx in somatotrophs (Rawlings et al., 1993), the release of interleu- kin-6 from folliculo-stellate cells (Tatsuno et al., 19911, and the release of a-melanocyte-stimulating hormone from mela- notrophs (Koch and Lutz-Bucher, 1992). The present study has directly demonstrated at the single cell level that PACAP can have a specific cellular action through the activation of the Ins(1,4,5)P3 system.
PACAP Stimulation of Ca2+ Mobilization-The effect of PACAP to mobilize Ca2+ from intracellular Ca2+ stores was blocked by the Ins(1,4,5)P3 receptor antagonist heparin. This results argues against the involvement of such Ca2+ mobilizing agents as cADP ribose and Ca2+ itself, which act through a heparin-insensitive ryanodine receptor (Galione, 1993; Palade et al., 1989). The mechanism of cAMP-stimulated Ca2+ mobili- zation in hepatocytes is unclear, however, it is not blocked by heparin (Capiod et al., 1991). Furthermore, in the present study, 100 p~ CAMP could not stimulate Ca2+ mobilization in the rat gonadotroph, nor could it inhibit the response to PACAP. Thus it appears that the PACAP-stimulated Ca2+ rise is mediated through the Ins(1,4,5)P3 receptor on the intracel- lular Ca2+ store. The fact that microperfusion of physiological concentrations of Ins(1,4,5)P3 can mimic, and that pharmaco-
A.
LHRH
=
20 p M lns(1,4,5)P3 LHRH
I
B.
lop M Ins(1,4,5)P3
2 l
J-
LHRH1 -
'3 -
LHRH0'
4
i ~ ~ " l ~ " ' l " " i ' " ' l " ' ~ l " ' ~ l
0 1 2 3 4 5 6
Time (min)
FIG. 5. Microperfusion of Ins(1,4,5)P3 into gonadotrophs can mimic the PACAP-stimulated Ca2+ response. Measurement of [Ca2+l, during a whole-cell recording of gonadotrophs voltage-clamped at -50 mV. The whole-cell configuration was established at the times indicated by the arrows and allowed for the microperfusion of the cells with Ins(1,4,5)P3 (5-50
v,
as indicated in panels A-D). LHRH was applied as in Fig.1, to identify the cells as gonadotrophs.
PACAP supports this conclusion.
PACAP Stimulation of Phospholipase C-But how is PACAP causing this effect? The most logical explanation is that the stimulation of the PACAP receptor leads to the activation of phospholipase C (PLC) and the subsequent hydrolysis of phos- phatidylinositol 4,5-bisphosphate into the intracellular mes- sengers Ins(1,4,5)P3, and 1,2-diacylglycerol. A similar mecha- nism exists in gonadotrophs for the activation of the LHRH receptor (Naor et al., 1986; Morgan et al., 1987; Chang et al., 19881, and the endothelin receptor (Stojilkovic et al., 1992). In support of this idea, PACAP has been recently shown to stimu- late the production of inositol phosphates in clonal adrenal medullary cells (Deutsch and Sun, 1992; Isobe et al., 1993). In addition, PACAP-stimulated LH release from the gonadotroph appears to be dependent, at least in part, on the activation of protein k i n a s e 4 ( H a r t et al., 1992). Thus PACAP action in the gonadotroph may involve, in addition to Ins(1,4,5)P3-mediated Ca2+ mobilization, the activation of protein kinase C by 1,2- diacylglycerol.
There are a number of possible mechanisms for the activa- tion of phospholipase C isoforms. Activation of many growth factor receptors is through a direct (non-G-protein-mediated) tyrosine phosphorylation of the y-isozyme of phospholipase C (phospholipase C-y) (e.g. see Kim et al. (1991)). In a variation of this system, the muscarinic receptor-mediated tyrosine phos- phorylation of phospholipase C-y is also dependent upon Ca2+
influx (Gusovsky et al., 1993). The present study has demon- strated that the PACAP action is mediated by a G-protein con- sistent with a recent binding study demonstrating the exist-
ence of a G-protein-complexed PACAP receptor in rat brain (Schafer et al., 1991). In addition, the PACAP-stimulation of the Ca2+ oscillations response pattern is independent of extra- cellular Ca2+ (Fig. 6B) (Canny et al., 1992). Thus the PACAP stimulation of Ins(1,4,5)P3 in the gonadotroph is most likely to be through the G-protein-mediated activation of the P-isozyme of phospholipase C (Taylor et al., 1991).
! h o PACAP-stimulated Intracellular Pathways in Pituitary Cells--The present study has demonstrated that PACAP action in gonadotrophs can be mediated by Ins(1,4,5)P3, whereas pre- vious data has shown that in somatotrophs PACAP action is mediated by CAMP (Rawlings et al., 1993). Thus PACAP can apparently stimulate both the CAMP and the phosphoinositol signaling pathways in anterior pituitary cells. How could PACAP influence these two systems? Recent cloning data may provide an answer to this question. The PACAP type I receptor has been very recently cloned (Pisegna and Wank, 1993; Hashi- mot0 et al., 1993; Spengler et al., 1993), and shown to exist in at least five splice variants (Spengler et al., 1993). One of the splice variants is linked solely to the stimulation of CAMP pro- duction, while the other four splice variants of the PACAP receptor can be linked to the activation of both adenylate cy- clase and phospholipase C (Spengler et al., 1993). Although PACAP38 and PACAP27 stimulate CAMP production with ap- proximately equal potency for these four splice variants of the receptor, PACAP38 stimulates inositol phosphate production with a potency 2-3 logs greater than for PACAP27. These re- sults are consistent with an earlier study showing a differential action of PACAP38 and PACAP27 on the stimulation of CAMP
PACAP38 Regulation
ofI(
A.
200pM I n s ~ l , l R P , (Cd*-free Medium)2 1
i PACAP ,LHRH
,
0 2 4 6 8 IO
Time (min)
B.
Control (Caz+free Medium)PACAP ,LHRH 1
t
I ~ . ' . ~ . , ,
0 2 4 6 8 I O
Time (mid
FIG. 6. Microperfusion of a high Ins(1,4,5)P3 concentration into gonadotrophs can block the PACAP-stimulated Ca2+ re- sponse. Measurement of [Ca2+l, during whole-cell recordings from go- nadotrophs voltage-clamped at -50 mV. These experiments were per- formed in a nominally Ca2+-free medium containing 1 m~ EGTA. The whole-cell configuration was established at the time indicated by the arrow and allowed for the microperfusion of the cell with the standard pipette solution ( B ) or with the standard pipette solution containing 200 PM Ins(1,4,5)P3 (A). PACAP (100 nM) and LHRH (10 n M ) were applied a s indicated by the bars.
production and inositol phospholipid turnover in the sympa- thoadrenal PC12 cell line (Deutsch and Sun, 1992). Thus, if the action of PACAP in the rat gonadotroph is through the activa- tion of one of these receptor subtypes we may expect to see a differential action of PACAP38 and PACAP27 on Ins(1,4,5)P3- stimulated Ca2+ oscillations in these cells.
In initial experiments we tested three concentrations of PACAP38 and PACAP27 (10 nM, 100 nM, and 1 p ~on their ) ability to stimulate Ca2+ oscillations in the rat gonadotroph.
Remarkably, given the data described above, we found no strik- ing difference in the potency of these two PACAP isoforms. For both PACAP38 and PACAP27 the threshold concentration for the stimulation of Ca2+ oscillations was between 10 nM and 100 nM ( n = 12). How can we reconcile such data? Perhaps the simplest explanation lies in the differences in the methods of detecting inositol phosphate production between our study and the studies described above (Deutsch and Sun, 1992; Spengler et al., 1993). Our study, by measuring the release of Ca2+ from Ins(l,4,5)P3-sensitive stores, detects the production of an effec- tive concentration of Ins(1,4,5)P3 over a time period of seconds at the single cell level, although it can give only an approximate idea of the levels of Ins(1,4,5)P3 formed. In contrast, the other studies measured the cumulative production of all inositol phosphates (InsP,,) over a time period of 60 min (Deutsch and Sun, 1992) and 20 min (Spengler et al., 1993).
Whatever the reasons for the differences between these re- sults, the new found complexity in the PACAP receptor subtype field and our initial results given above show that a simple comparison of the effects of PACAP38 and PACAP27 will not
7
,a'
L+li in Rat Gonadotrophs
5685pressed in the rat gonadotroph. Further studies, probably in- volving molecular biology techniques, will be necessary to iden- tifiy the PACAP receptor subtype(s) involved in Ins(1,4,5)P3 production, and hence Ca2+ mobilization, in this cell type.
Possible Physiological Role of PACAP-In the gonadotroph LHRH can produce similar oscillations in [Ca2+li to those ob- served by PACAP stimulation in the present study (Fig. 1C) (Shangold et al., 1988; Iida et al., 1991; Leong and Thorner, 1991; Tse and Hille, 1992). Such Ca2+ oscillations are closely coupled with exocytosis (and thus probably LH release) (Tse et al., 1993) and have also been proposed to be a method of regu- lating LHRH receptor numbers and LH synthesis (Leong and Thorner, 1991; Leong, 1991). Thus the action of PACAP to stimulate Ca2+ oscillations may play an important role in the regulation of gonadotroph function.
The fact that PACAP can act through two different intracel- lular signaling systems provides the possibility of an enhanced signaling potential for this peptide's effects on anterior pitui- tary cell function. On the one hand, the same peptide could selectively address different pituitary cell types for which one or the other pathway appears to be dominant (phospholipase C for gonadotrophs; CAMP for somatotrophs). Furthermore, if the two pathways coexist in the same cells, PACAP could produce a more complex signal, dependent upon the interactions or
"cross-talk" (Houslay, 1991) between these two signaling path- ways. There is some evidence that other hypothalamic factors may stimulate more than one signaling pathway, for example the action of thyrotropin-releasing hormone on clonal pituitary cells (Paulssen et al., 1992). However, this is the first clear demonstration that a hypothalamic factor can significantly stimulate both of these systems in normal identified pituitary cells, and, furthermore, it may address different systems in different cell types (see above). These results should thus en- courage further studies on the action of PACAP on anterior pituitary cell physiology.
Acknowledgments-We thank Dr. Karl-Heinz Krause and Dr. Rose- mary Murray-Whelan for helpful comments on an earlier draft of the manuscript. We also thank the group of Joel Bockaert for providing a preprint of the paper on the cloning of the PACAP receptor (Spengler et al., 1993).
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