Stimulation of Ca2+ influx in alpha T3-1 gonadotrophs via the cAMP/PKA signaling system

Marjan Hezareh, Werner Schlegel, and Stephen R. Rawlings

Fondation pour Recherches Médicales, University of Geneva, CH-1211 Geneva 4, Switzerland

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To investigate the regulation of free cytosolic calcium concentration ([Ca2+]i) by the adenosine 3',5'-cyclic monophosphate (cAMP) signaling system in clonal gonadotrophs, microfluorimetric recordings were made in single indo 1-loaded alpha T3-1 cells. Forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or a low concentration (100 pM) of the hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated Ca2+ step responses or repetitive Ca2+ transients, which were blocked by the removal of extracellular Ca2+ by the dihydropyridine (DHP) (+)PN 200-110 or by preincubation with the protein kinase A (PKA) antagonist H-89 (10 µM). Thus activation of the cAMP/PKA system in alpha T3-1 gonadotrophs stimulates Ca2+ influx through DHP-sensitive (L-type) Ca2+ channels. In contrast, high PACAP concentrations (100 nM) stimulated biphasic Ca2+ spike-plateau responses. The Ca2+ spike was independent of extracellular Ca2+, and similar responses were observed by microperfusion of individual cells with D-myo-inositol 1,4,5-trisphosphate, suggesting the involvement of the phospholipase C (PLC) signaling pathway. The Ca2+ plateau depended on Ca2+ influx, was blocked by (+)PN 200-110, but was only partially blocked by H-89 pretreatment. In conclusion, PACAP stimulates [Ca2+]i increases in alpha T3-1 gonadotrophs through both the PLC and adenylate cyclase signaling pathways. Furthermore, this is the first clear demonstration that the cAMP/PKA system can mediate changes in [Ca2+]i in gonadotroph-like cells.

pituitary adenylate cyclase-activating polypeptide; adenosine 3',5'-cyclic monophosphate; protein kinase A; vasoactive intestinal polypeptide; H-89; anterior pituitary cells; L-type calcium channels

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ANTERIOR PITUITARY GONADOTROPHS synthesize and secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) under the regulation of the hypothalamic factor LH-releasing hormone (LHRH or GnRH) and gonadal steroids. However, studies on the activity of this cell type have been hampered by the fact that the anterior pituitary gland contains at least six different cell types, and the gonadotrophs make up only 5-10% of the total population. Recently targeted oncogenesis was used to produce the alpha T3-1 cell line, which possesses gonadotroph-like characteristics (30). These cells express the LHRH receptor and the alpha -subunit common to LH and FSH, although they do not express the LH and FSH-specific beta -subunits (30). The alpha T3-1 cell line has proven very useful in biochemical studies of the regulation of gonadotroph-like cells (1, 4, 9, 12, 27, 31), and in particular on the regulation of alpha -subunit and early gene expression (27, 31). However, relatively little is known about the regulation of intracellular signaling in alpha T3-1 cells. In particular, although the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase-A (PKA) intracellular signaling pathway has been implicated in the regulation of alpha -subunit expression in both normal and clonal gonadotrophs (13, 31), the effects of this system on other intracellular signaling pathways, such as changes in free cytosolic Ca2+ concentration ([Ca2+]i) in alpha T3-1 cells, have not been widely studied.

The present article describes the effects of activation of the cAMP/PKA system on changes in [Ca2+]i in alpha T3-1 gonadotrophs. We tested the effects of forskolin, an activator of adenylyl cyclase (AC), the membrane- permeable cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and the hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) on Ca2+ changes in these cells. PACAP, which has been proposed to act as a hypophysiotropic factor regulating anterior pituitary cell activity (24), exists in both 38-amino acid (PACAP-38) and NH2-terminally shortened 27-amino acid (PACAP-27) forms and shares significant sequence homology with vasoactive intestinal polypeptide (VIP) (2). There are at least three subtypes of PACAP/VIP receptors (PVRs). The type 1 receptor PVR1 (also known as the PACAP-R) is selective for PACAP over VIP and couples to the activation of both AC and phospholipase C (PLC). In contrast, the PVR2 (VIP1R) and PVR3 (VIP2R) receptors do not distinguish between PACAP and VIP and couple to the activation of AC but not PLC (24). In the present study, PACAP was shown to be 100- to 1,000-fold more potent than VIP in stimulating Ca2+ changes in alpha T3-1 cells, suggesting the involvement of the PVR1. Low concentrations of PACAP, as well as forskolin and 8-BrcAMP, stimulated Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels in alpha T3-1 cells via the activation of the cAMP/PKA system. In contrast, higher PACAP concentrations stimulated both Ca2+ mobilization and Ca2+ influx responses, which were in part mediated by the action of the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]/diacylglycerol (DG) signaling system. Such results have important implications for understanding the ways in which the activation of the cAMP/PKA system may modulate gonadotroph activity, both alone and in concert with the Ins(1,4,5)P3/DG system (9, 24, 31).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The technique used for the alpha T3-1 cell culture has been previously reported (25). Briefly, the alpha T3-1 cells were grown as a monolayer culture in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose, 5% fetal bovine serum, and 5% horse serum at 37°C in a humidified atmosphere of 5% CO2-95% air. Medium was exchanged every 2-3 days, and cells were passaged at 7-day intervals. For the Ca2+ experiments, the cells were harvested by treatment with trypsin and then cultured for 3-4 days on round coverslips in six-well culture plates.

Measurement of [Ca2+]i. The technique used for the measurement of [Ca2+]i changes in pituitary cells has been described in detail elsewhere (25). Briefly, cells were loaded with 4.4 µM of the membrane-permeable acetyloxymethyl ester form of the Ca2+ fluorescent dye indo 1 (indo 1-AM) in standard medium [S medium: (in mM) 127 NaCl, 5 KCl, 2 MgCl2, 1.8 CaCl2, 5 NaHCO3, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose, pH 7.4] containing 0.04% pluronic F-127 for 30 min at room temperature. After the cells were washed three times with S medium, the coverslip containing cells was placed onto the stage of an inverted epifluorescence microscope (Diaphot, Nikon, Tokyo, Japan). The indo 1 within the cells was excited by ultraviolet light, and the resultant Ca2+-dependent fluorescence was collected by an oil immersion objective (Nikon Fluor) (40), split into two components by a dichroic mirror (lambda crit 455 nm), and detected by separate photomultipliers after passing through interference filters of either 405 or 480 nm wavelength. The outputs of the two photomultipliers were recorded at 33 Hz by use of an acquisition system (Acqui, SICMU, University of Geneva, Switzerland) run on an IBM-compatible computer. The fluorescence values obtained at the two wavelengths were ratioed (R = F405/F480) and then converted to Ca2+ values using the formula [Ca2+]i = Kd × beta  × (R - Rmin)/(Rmax - R), where Kd is the dissociation constant. The calibration constants were empirically determined from >= 50 individual cells with indo 1-loaded cells exposed to the calcium ionophore ionomycin (2 µM) in S medium containing either 10 mM [Ca2+] (Rmax) or essentially Ca2+ free with 10 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (Rmin). The value of beta  was calculated as the ratio of fluorescence at 480 nm for indo 1 in minimal and maximal Ca2+ concentrations. The Kd for indo 1/Ca2+ binding was taken as 250 nM.

Experiments were run at room temperature (20-22°C) using S medium or, where indicated, Ca2+-free S medium in which the CaCl2 was replaced by 1 mM EGTA.

Microperfusion of Ins(1,4,5)P3 into the cells. The technique used for the microperfusion of compounds into single anterior pituitary cells has been previously described (23). Briefly, the whole cell configuration of the patch-clamp technique was used to microperfuse Ins(1,4,5)P3 (20-40 µM) into individual alpha T3-1 cells. The standard pipette solution contained (in mM) 120 potassium aspartate, 20 KCl, 2 MgCl2, 20 HEPES-NaOH, 0.1 GTP, 2 ATP, and 0.04 indo 1 (pH 7.4), and it had a free [Ca2+] of 70-85 nM as measured with a Ca2+-sensitive electrode (23).

Data analysis. The data presented in this paper were gathered from a total of 42 experimental days. For any particular experimental manipulation, data were pooled from >= 3 separate days of experiments. Baseline [Ca2+]i values were calculated as an average over a 1-min period. [Ca2+]i values for the Ca2+-step, Ca2+-transient, and Ca2+-plateau responses were calculated as an average over the 60-s period between 1 and 2 min after the start of the Ca2+ response. [Ca2+]i values for the spike response were taken as the peak [Ca2+]i value in each case. The [Ca2+]i values are expressed in this paper as means ± SE. Student's t-test and Fisher's Exact Test were used to test for statistical differences between [Ca2+]i values and numbers of responding cells, respectively. For all analyses P <=  0.05 was taken as statistically significant.

Materials. The alpha T3-1 cells were kindly provided by Dr. P. L. Mellon (University of California, La Jolla, CA). PACAP-38, PACAP-27, VIP, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) were purchased from Calbiochem (Laufelfingen, Switzerland), and indo 1, indo 1-AM, and pluronic F-127 were obtained from Molecular Probes (Leiden, the Netherlands). Isradipine [(+)PN 200-110] and its inactive isoform [(-)PN 200-110] were a generous gift from Sandoz. All other reagents were obtained from Sigma Chemical (St. Louis, MO).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Concentration-dependent effects of PACAP and VIP on [Ca2+]i. We first tested the effects of the hypothalamic factor PACAP-38 on [Ca2+]i changes in alpha T3-1 gonadotrophs by use of microfluorimetric recordings in single indo 1-loaded cells. At low concentrations (<= 100 pM) PACAP-38 stimulated "step" or "repetitive transient" Ca2+ responses (Fig. 1, A and B), which will be described in more detail below. However, as the PACAP-38 concentration was increased, not only did more cells respond (Fig.1E), but an increasing proportion of cells exhibited biphasic "spike-plateau" Ca2+ responses (Fig. 1C). At the highest concentration tested (100 nM), PACAP increased [Ca2+]i in 23 of 28 cells (82%), and 21 of the 23 responding cells showed a Ca2+ spike-plateau response. The other two cells exhibited a Ca2+ step response typically observed at lower (<= 100 pM) PACAP-38 concentrations (Fig. 1A). Biphasic spike-plateau responses to PACAP-38 (100 nM) started from a mean [Ca2+]i value of 110 ± 10 nM and reached a Ca2+ peak of 450 ± 70 nM before decreasing to a Ca2+ plateau of 170 ± 20 nM measured between 1 and 2 min after the peak response (n = 21).


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Fig. 1.   Typical examples of pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38)- and vasoactive intestinal polypeptide (VIP)-stimulated Ca2+ responses in single alpha T3-1 cells. Changes in free cytosolic calcium concentration ([Ca2+]i) were measured in response to 100 pM PACAP-38 (A, B), 100 nM PACAP-38 (C), and 100 nM VIP (D) in single alpha T3-1 cells by microfluorimetry with the fluorescent Ca2+ indicator indo 1. PACAP-27 stimulated responses qualitatively similar to PACAP-38. Horizontal bars, PACAP-38 and VIP applications. E: proportion of alpha T3-1 cells exhibiting Ca2+ responses to a range of concentrations of PACAP-38, PACAP-27, and VIP. alpha T3-1 cells were exposed to indicated concentrations of PACAP-38 (bullet ), PACAP-27 (open circle ), and VIP (black-triangle), producing Ca2+ responses shown in plot as % of cells showing a Ca2+ response to indicated concentrations of peptides. Nos. of cells tested for data points (from lowest to highest concentration) were, for PACAP-38: 7, 7, 10, 26, 7, 8, 28; for PACAP-27: 7, 6, 6, 6, 7, 7, 10; and for VIP: 5, 5, 5, 7, 9, 12, 16.

Previous studies have demonstrated that alpha T3-1 cells express mRNA for both the PACAP-preferring PACAP/VIP type 1 receptor (PVR1) and the PVR3, which has similar affinity for PACAP and VIP (25). In an attempt to identify the dominant receptor contributing to the PACAP-stimulated Ca2+ responses in these cells, we recorded changes in [Ca2+]i at the single cell level in response to a range of PACAP-38, PACAP-27, and VIP concentrations (100 fM to 100 nM). To compare the sensitivity of the cells to PACAP-38, PACAP-27, and VIP, concentration-response curves were based on the number of cells responding to a particular peptide concentration (Fig. 1). Analysis of the curves so produced revealed that PACAP-27 and PACAP-38 had similar potencies, with ~25% of cells exhibiting Ca2+ responses at 100 fM PACAP, a proportion of responsive cells that increased to 80-90% at the top concentrations (10-100 nM) of PACAP tested (Fig. 1E). Both PACAP-27 and PACAP-38, however, were 100- to 1,000-fold more potent than VIP in stimulating changes in [Ca2+]i. The relative potency of PACAP-38, PACAP-27, and VIP suggests the action on the PVR1 in alpha T3-1 cells.

At all concentrations tested, PACAP-27 was equivalent to PACAP-38 in stimulating [Ca2+]i responses in alpha T3-1 cells (Fig. 1E). Furthermore, PACAP-27 caused similar changes in [Ca2+]i, i.e., predominantly spike-plateau response patterns at 100 nM and step responses or Ca2+ transients at concentrations <= 100 pM. In contrast, VIP even at the maximal dose of 100 nM stimulated either Ca2+ step or repetitive Ca2+ transients (Fig. 1D), and the biphasic Ca2+ spike-plateau response pattern was never observed in response to this peptide. Responses to VIP at 100 nM were observed in 10 of 16 cells (63%), producing a mean increase in [Ca2+]i over basal of 90 ± 10 nM (P <=  0.05). All VIP-stimulated Ca2+ responses were dependent on extracellular Ca2+ (25).

In summary, PACAP stimulates Ca2+ changes in alpha T3-1 cells through an action on the PVR1. High concentrations of PACAP-38 (100 nM) stimulate biphasic Ca2+ spike-plateau responses, whereas low concentrations of PACAP-38 (<= 100 pM) stimulate Ca2+ step or Ca2+ transient responses. Because low concentrations of PACAP-38 have previously been shown to preferentially stimulate cAMP production in alpha T3-1 cells (25, 26), the following series of experiments were designed to test the hypothesis that the Ca2+ responses stimulated by low (100 pM) PACAP-38 concentrations could be mediated through the cAMP signaling system.

Effect of 8-BrcAMP, forskolin, and low concentrations of PACAP on [Ca2+]i in alpha T3-1 cells. The effect of activation of the cAMP/PKA system on changes in [Ca2+]i was probed by raising intracellular cAMP concentration in alpha T3-1 cells by three independent mechanisms: 1) addition of the membrane-permeable cAMP analog 8-BrcAMP; 2) activation of endogenous AC by the addition of forskolin (12, 25); or 3) addition of a low concentration of PACAP-38, which activates an AC-coupled cell surface receptor expressed in these cells (25, 26).

The mean basal [Ca2+]i in alpha T3-1 cells was 110 ± 10 nM (n = 25). All three treatments, 8-BrcAMP (1 mM), forskolin (2 µM), and PACAP-38 (100 pM), stimulated increases in [Ca2+]i in single alpha T3-1 gonadotrophs, which were observed either as Ca2+ step responses (see Figs. 1A and 3A) or repetitive Ca2+ transients (Figs. 1B and 2, A and C). Eight of 16 cells (50%) responded to 1 mM 8-BrcAMP, producing a mean increase in [Ca2+]i over basal of 70 ± 10 nM (n = 8; P <=  0.05, Student's t-test). Eleven of 17 cells (65%) responded to 2 µM forskolin, showing a mean increase in [Ca2+]i of 170 ± 50 nM (n = 11; P <=  0.05). Eleven of 26 cells (42%) responded to 100 pM PACAP-38, producing a mean increase in [Ca2+]i of 100 ± 20 nM (n = 11; P <=  0.05).


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Fig. 2.   Membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) analog 8-bromo-cAMP (8-BrcAMP) and forskolin stimulate elevations of [Ca2+]i in single alpha T3-1 cells, which are dependent on extracellular Ca2+. Changes in [Ca2+]i were measured in single alpha T3-1 cells by microfluorimetry with the fluorescent Ca2+ indicator indo 1. 8-BrcAMP (1 mM) was added in normal S medium (see MATERIALS AND METHODS; A) and in Ca2+-free medium containing 1 mM EGTA (B). Under the latter condition, luteinizing hormone-releasing hormone (LHRH, 10 nM) was added to demonstrate continued responsiveness of cells. C: example of an alpha T3-1 cell in normal S medium stimulated by forskolin (2 µM). D: block of Ca2+ response by Ca2+ channel blocker NiCl2 (5 mM). Horizontal bars, times when drugs were added.

Involvement of Ca2+ influx through cell membrane Ca2+ channels. The Ca2+ responses stimulated by 8-BrcAMP, forskolin, and 100 pM PACAP-38 were absolutely dependent on extracellular Ca2+, because they were never observed in Ca2+-free medium (Fig. 2B; 8-BrcAMP: n = 8; forskolin: n = 9; 100 pM PACAP-38: n = 12; in all cases P <=  0.05, Fisher's Exact Test).

The Ca2+ responses stimulated by forskolin (2 µM) and PACAP-38 (100 pM) were blocked by the subsequent addition of the nonspecific Ca2+ channel blocker NiCl2 (Fig. 2D; forskolin: n = 11; 100 pM PACAP-38: n = 5; P <=  0.05). To more specifically identify the Ca2+ channel type involved, we tested the effect of (+)PN 200-110 (isradipine), a specific inhibitor of DHP-sensitive (L-type) voltage-activated Ca2+ channels expressed in alpha T3-1 cells (4). As shown in Fig. 3, the inactive PN 200-110 isoform [(-)PN 200-110; 100 nM] was without effect on the Ca2+ responses stimulated by forskolin (2 µM; n = 5; Fig. 3A) or PACAP-38 (100 pM; n = 5; Fig. 3B). In contrast, the Ca2+ responses stimulated by forskolin (2 µM) or PACAP-38 (100 pM) were completely blocked by addition of the active (+) isoform of PN 200-110 (100 nM) {forskolin: n = 5; PACAP-38: n = 5; P <= 0.05 compared with controls [(-)PN 200-110; Fig. 3, A and B]}. Similar effects were also observed with another L-type Ca2+ channel blocker, nifedipine (1-10 µM; data not shown).


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Fig. 3.   Effects of dihydropyridine-sensitive (L-type) Ca2+ channel blocker PN 200-110 (isradipine) on PACAP- and forskolin-stimulated Ca2+ responses. Single alpha T3-1 cells were stimulated by forskolin (2 mM) or PACAP-38 (100 pM) and were then exposed to (+)PN200-110 (100 nM; C and D) or to its inactive form (-)PN 200-110 (100 nM; "control"; A and B) for time indicated by horizontal bars.

Involvement of PKA in the Ca2+ responses. Taken together, the results detailed above suggest that a rise in intracellular cAMP concentration can lead to an increase in [Ca2+]i through the activation of Ca2+ influx via DHP-sensitive Ca2+ channels. To test whether this is a direct effect of cAMP, or an effect mediated by a cAMP-dependent protein kinase such as PKA, we performed a series of experiments with H-89, a competitive inhibitor of the ATP-binding site of PKA (5). This antagonist has been previously used in anterior pituitary cells to probe for the involvement of the cAMP/PKA system in corticotropin-releasing hormone (CRH)-stimulated ionic currents in adrenocorticotropic hormone (ACTH)-secreting pituitary cells (28), and PACAP-stimulated interleukin-6 release from rat folliculo-stellate cells (29). We also attempted to use the PKA antagonist RpcAMPS, the Rp diastereoma of adenosine 3',5'-cyclic monophosphothioate [1 mM; (22)] but found that preincubation with this compound had an appreciable effect to increase basal [Ca2+]i levels in alpha T3-1 cells (data not shown), possibly as a result of partial activation of the RII subunit of PKA (10) expressed in this cell type (9). For this reason we did not employ RpcAMPS in the present studies.

Cells were preincubated with 10 µM H-89 for 30 min before stimulation. Whereas basal [Ca2+]i was slightly, but not significantly (P > 0.05; Table 1), elevated by this pretreatment, the Ca2+ responses stimulated by 2 µM forskolin and 100 pM PACAP-38 were completely abolished (Fig. 4; Table 1). Such results strongly suggest that the effects of forskolin and low concentrations of PACAP on [Ca2+]i changes in alpha T3-1 cells are mediated through the cAMP/PKA signaling system.

                              
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Table 1.   Effects of the PKA inhibitor H-89 on PACAP- and forskolin-stimulated Ca2+ responses


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Fig. 4.   Effects of protein kinase A inhibitor H-89 on PACAP- and forskolin-stimulated Ca2+ responses. Cells were preincubated in S medium (control; A and B) or medium containing H-89 (10 µM; C and D) for 30 min at room temperature. Effects of forskolin (2 µM; A and C) and PACAP-38 (100 pM; B and D) on changes in [Ca2+]i in single alpha T3-1 cells were measured in the continual presence of H-89.

What is the mechanism of PACAP-stimulated Ca2+ spike-plateau response? The biphasic Ca2+ spike-plateau responses to high concentrations of PACAP-38 (Fig. 1C) were analyzed with regard to the role of Ca2+ influx. In the absence of extracellular Ca2+, only the Ca2+ plateau, but not the Ca2+ spike, was blocked (Fig. 5A). Furthermore, the Ca2+ plateau was blocked by the addition of the Ca2+ channel blockers (+)PN 200-110 (n = 5; Fig. 5B), nifedipine (1-10 µM; data not shown), or NiCl2 [5 mM; (25)]. These results suggest that the Ca2+ spike is mediated by the release of Ca2+ from an intracellular store, whereas the plateau phase of the response is dependent on Ca2+ influx (25). If PACAP-stimulated Ca2+ mobilization were through an Ins(1,4,5)P3-dependent mechanism, it would be expected that Ins(1,4,5)P3 should mimic the Ca2+ response pattern observed by PACAP. Intracellular application of Ins(1,4,5)P3 was achieved with the patch-clamp technique in its whole cell configuration (23). Ins(1,4,5)P3 at an intrapipette concentration of 20-40 µM caused a marked and rapid rise in [Ca2+]i starting a few seconds after break-in (n = 8) (Fig. 5C), effectively reproducing the pattern of the Ca2+ spike response to PACAP-38 (100 nM) observed in the absence of extracellular Ca2+ (Fig. 5A). Thus it is most likely that the spike response to PACAP in alpha T3-1 cells is mediated by Ins(1,4,5)P3, generated by the PACAP-stimulated activation of PLC (25, 26).


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Fig. 5.   PACAP-38 causes a Ca2+ spike response, which is independent of extracellular Ca2+ and which can be mimicked by intracellular application of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Changes in [Ca2+]i were measured in single alpha T3-1 cells by microfluorimetry with fluorescent dye indo 1. A: PACAP (100 nM; applied as indicated by bar) was added to a single alpha T3-1 in Ca2+-free medium containing 1 mM EGTA. Compare this response to that shown in B: an example of an alpha T3-1 cell stimulated by PACAP-38 (100 nM) in S medium, and block of Ca2+ plateau response by Ca2+ channel blocker (+)PN 200-110 (100 nM). C: [Ca2+]i measurement from a single alpha T3-1 cell in S medium during a patch-clamp recording in whole cell configuration with Ins(1,4,5)P3 (40 µM) in pipette. Arrow, time at which whole cell configuration was established and dialysis of cell cytosol with pipette solution started.

It is clear that the Ca2+ plateau response observed in response to high PACAP-38 concentrations has certain characteristics in common with the Ca2+ step response observed at low concentrations; both are sustained Ca2+ influx responses that are blocked by the removal of extracellular Ca2+ or addition of the Ca2+ channel blockers NiCl2 or (+)PN 200-110 (see Involvement of Ca2+ influx through cell membrane Ca2+ channels). To probe more directly for the involvement of PKA in the effects of high PACAP-38 concentrations on [Ca2+]i, we used the specific PKA inhibitor H-89. As described above, preincubation of alpha T3-1 cells with 10 µM H-89 for 30 min completely abolished the Ca2+ response to a low PACAP-38 concentration (100 pM) (Fig. 4; Table 1). In contrast, at high PACAP-38 concentrations (100 nM), H-89 attenuated (P <=  0.05, Student's t-test) but did not completely block the amplitude of the Ca2+ spike and the plateau [Ca2+]i levels (Table 1).

In conclusion, it appears that low concentrations (<= 100 pM) of PACAP stimulate Ca2+ influx through a cAMP/PKA-dependent mechanism. In contrast, at higher (>1 nM) concentrations, PACAP can stimulate both Ca2+ mobilization from an intracellular store and Ca2+ influx. The Ca2+ mobilization is probably mediated by an Ins(1,4,5)P3-dependent mechanism, whereas the Ca2+ influx (plateau) stimulated at high (100 nM) PACAP-38 concentrations is probably mediated by both PLC/Ins(1,4,5)P3/DG and AC/cAMP/PKA signaling pathways.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This is the first report, to our knowledge, to show that the cAMP/PKA system can mediate Ca2+ changes in gonadotroph-like cells. Such findings have important implications for the regulation of alpha T3-1 and gonadotroph cell activity by the cAMP/PKA system (13, 31). We have also demonstrated that PACAP can stimulate changes in [Ca2+]i in the alpha T3-1 gonadotrophs through both Ins(1,4,5)P3- and cAMP/PKA-dependent mechanisms.

Regulation of Ca2+ influx in alpha T3-1 cells through cAMP/PKA-dependent mechanisms. Three independent mechanisms of increasing cytosolic cAMP levels in alpha T3-1 gonadotrophs, forskolin, 8-BrcAMP, and a low concentration (100 pM) of PACAP-38 stimulated Ca2+ influx responses in such cells. The Ca2+ responses stimulated by forskolin and PACAP-38 were blocked by the PKA antagonist H-89 and by the DHP-sensitive Ca2+ channel blocker PN 200-110. These results suggest that an increase in cytosolic cAMP levels in alpha T3-1 gonadotrophs leads to an activation of PKA and the subsequent influx of Ca2+ through DHP-sensitive (L-type) Ca2+ channels in the cell membrane. However, what are the mechanisms mediating this effect? One possibility is that cAMP/PKA activates a voltage-independent Na+ influx, leading to membrane depolarization and the subsequent activation of voltage-activated Ca2+ currents. Such an effect occurs in rat somatotrophs in response to growth hormone-releasing hormone or the membrane-permeable cAMP analog SpcAMPS [the stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphorothioate (19)], and in ACTH-secreting human pituitary adenoma cells in response to CRH or forskolin (28). Significantly, in the latter study the CRH-stimulated inward Na+ current was blocked by H-89 pretreatment (28). An alternative possibility is that the activated PKA phosphorylates L-type Ca2+ channels, leading to an increase in their voltage sensitivity as has been shown in somatotroph-like GH3 cells (3). Such an action could theoretically lead to an increase in Ca2+ influx at the resting membrane potential (18) or increase the Ca2+ influx in response to membrane depolarization. The stimulation of Ca2+ influx by PACAP-38, 8-BrcAMP, and forskolin in rat somatotrophs may be mediated by similar mechanisms (21, 22).

In contrast to the present findings, PACAP, 8-BrcAMP, and forskolin (21, 22) do not stimulate [Ca2+]i changes in normal rat gonadotrophs, suggesting that there may be fundamental differences in the mechanisms by which an increase in cytoplasmic cAMP may modulate cellular activity in normal vs. clonal gonadotrophs.

PACAP stimulates Ca2+ changes through the PVR1. We have recently demonstrated the expression of mRNA for PVR1 and PVR3 in alpha T3-1 cells (25). However, the fact that PACAP is significantly more potent than VIP in binding (26), stimulation of cAMP and inositol phospholipid (PI) turnover (25, 26), and stimulation of Ca2+ changes (this study) suggests that the PVR1 is the dominant PACAP receptor expressed in the alpha T3-1 cell line. PACAP-38 and PACAP-27 were equally potent in stimulating Ca2+ changes and PI turnover in alpha T3-1 cells (25, 26), whereas in rat gonadotrophs, PACAP-38 is clearly more potent than PACAP-27 in stimulating PI turnover-dependent Ca2+ responses (11). One possible explanation for these differences could have been the preferential expression in alpha T3-1 cells of a recently described NH2-terminal splice variant of the PVR1 (PVR1vs for "very short"), which was shown to couple both PACAP-38 and PACAP-27 to PLC activation with similar potencies (20). However, reverse transcriptase-polymerase chain reaction studies in alpha T3-1 cells with primers common to the PVR1vs and the standard form of the PVR1 [kind gift of L. Journot (20)] revealed that PVR1vs mRNA was expressed at a very low level compared with PVR1 mRNA (D. Monnier and S. R. Rawlings, unpublished observations), and thus it is unlikely to play a major role in this cell type. It is interesting to note that a similar dichotomy on the relative potencies of PACAP-38 and PACAP-27 to stimulate PI turnover has been observed in PVR1-transfected Chinese hamster ovary cells (PACAP-38 congruent  PACAP-27) (7) or LLC-PK1 cells (PACAP-38 > PACAP-27) (14). It has been proposed that such differences in PVR1 binding and coupling may be due to differences in G protein expression and/or receptor numbers in different cell types (7, 14).

The PVR1 couples to the activation of both AC (leading to cAMP production) and PLC [leading to the production of Ins(1,4,5)P3 and DG] (24). In the present study, it was interesting to find that PACAP-stimulated Ca2+ responses are due to the activation of AC and cAMP at low (<= 100 pM) PACAP concentrations and the activation of both AC and PLC at higher (>= 1 nM) PACAP concentrations. Similar differences in the potencies of PACAP to stimulate cAMP production and PI turnover have also been observed in both alpha T3-1 cell populations (25, 26) and in cell lines transfected with the PVR1 (7, 14). These latter results suggest that these differences are probably due to the coupling characteristics of the PVR1 rather than to the expression of multiple receptor subtypes.

PACAP is a potent stimulator of alpha T3-1 cells. The Ca2+ responses observed in the present study are seen at PACAP concentrations (>= 100 fM) three orders of magnitude below those previously shown to stimulate cAMP production in alpha T3-1 cell populations (>= 100 pM) (25, 26). Although this may reflect gross differences between the standard cAMP assays on cell populations (25, 26) [cumulative measurement over a 40- to 45-min period at 37°C in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and the detection of cAMP-mediated effects on Ca2+ in single cells (dynamic responses to cAMP production over 0-5 min at room temperature in the absence of IBMX)], a more likely explanation is that the detection of cAMP-mediated Ca2+ influx responses may be a more sensitive assay for AC activation than measurement of total cAMP production in populations of these cells. Functional and ultrastructural studies have shown that ACs are often closely associated with sites of Ca2+ entry into a cell, and it has been proposed that activation of such ACs would produce local changes in cAMP concentration near to the cell membrane, where it could bind to PKA and lead to Ca2+ channel activation (6, 15). In such a case, a cAMP/PKA-sensitive Ca2+ channel would be a more sensitive detector of such local increases in cAMP than the standard cAMP assay, which measures total cellular cAMP production. Finally, it is interesting to note that similar differences in assay sensitivity have been observed between total cellular PI turnover measurements and recordings of PI turnover-dependent Ca2+ responses (11, 33).

Thus PACAP-38 is a very potent stimulator of Ca2+ influx responses in alpha T3-1 cells, with responses being observed at PACAP-38 concentrations as low as 100 fM. In individual pancreatic beta -cells, 1 fM PACAP stimulated Ca2+ responses in ~30% of cells tested, and 100 fM PACAP stimulated Ca2+ responses in 80% of cells and insulin release from beta -cell populations (32). As in alpha T3-1 cells, PACAP stimulated Ca2+ influx responses in pancreatic beta -cells through the activation of a PVR1-like receptor. The low concentrations of PACAP-38 that stimulate cAMP/PKA-dependent Ca2+ influx, and therefore presumably cAMP production, in alpha T3-1 gonadotrophs (100 fM to 100 pM) are within the range detected in rat hypophysial portal blood (50-100 pM) (8), thus suggesting that physiological concentrations of PACAP-38 may have effects on the cAMP/PKA system in gonadotroph cells in vivo.

PACAP stimulates the Ca2+ spike plateau response through an Ins(1,4,5)P3-dependent mechanism. Above 100 pM, PACAP also stimulated a spike-plateau Ca2+ response pattern, which was seen more frequently with increasing PACAP concentrations. Similar Ca2+ spike-plateau responses are observed in alpha T3-1 cells after activation of the PLC-coupled LHRH receptor (1, 17). The PACAP-stimulated Ca2+ spike was independent of extracellular Ca2+ and was mimicked by microperfusion of Ins(1,4,5)P3 into individual cells, suggesting that in alpha T3-1 cells, as in rat gonadotrophs (23), PACAP may stimulate Ca2+ release from an intracellular Ins(1,4,5)P3-sensitive store. In alpha T3-1 cells, microperfusion of Ins(1,4,5)P3 stimulated a Ca2+ spike-like response in all cases, and Ca2+ oscillations were never observed. In contrast, in rat gonadotrophs, the same procedure always produced repetitive Ca2+ oscillations (23). The reason for this difference is unclear but possibly relates to differences in the character of the Ins(1,4,5)P3-sensitive Ca2+ stores in these two cell types. The Ca2+ plateau stimulated by high PACAP concentrations is dependent on extracellular Ca2+ and is blocked by the Ca2+ channel antagonist PN 200-110, indicating the involvement of Ca2+ influx. This response pattern is only partly blocked by the PKA antagonist H-89 (Table 1), suggesting that other intracellular mechanisms may also be involved. One possibility is the DG/PKC system. PACAP-stimulated PLC would produce both Ins(1,4,5)P3 and DG, the latter leading to the activation of PKC. We have observed that the phorbol ester phorbol myristate acetate (PMA; 1 µM), which activates PKC, also stimulates small Ca2+ step responses in ~50% of alpha T3-1 cells tested (data not shown), and similar effects of PMA on [Ca2+]i and Ca2+ channel currents have been previously reported (1, 4). Thus the Ca2+ plateau observed in response to PACAP may be due to the activation of both the cAMP/PKA and DG/PKC systems.

Function of PACAP-stimulated cAMP production and Ca2+ influx in alpha T3-1 gonadotrophs. Activation of the cAMP/PKA signaling system has been previously shown to regulate a variety of cellular functions in both normal rat and clonal alpha T3-1 gonadotrophs, including the modulation of gene expression and alpha -subunit/LH release (31). However, the classical physiological regulators of gonadotroph cell function, including LHRH, do not stimulate cAMP production in such cells (12). The recent identification of the novel hypothalamic factor PACAP and the demonstration of its action on gonadotroph cells have led to the proposition that PACAP may be the physiological factor regulating the cAMP signaling system in gonadotrophs (24). In fact, PACAP has been shown to modulate both basal and LHRH-stimulated alpha -subunit/LH gene expression and secretion in both alpha T3-1 cells and normal rat gonadotrophs (24, 31), effects that are probably mediated through its activation of the cAMP signaling system (24, 31). However, there are clear differences in Ca2+ signaling between normal and clonal gonadotrophs; in rat gonadotrophs the cAMP/PKA signaling system has no apparent effect on [Ca2+]i changes (21-23), whereas, as shown in the present study, the same system stimulates Ca2+ influx in clonal alpha T3-1 gonadotrophs. Because it has been previously shown that Ca2+ influx is an important signal regulating immediate early gene transcription in clonal pituitary cells (16), further studies will need to examine whether the previously reported effects of the activation of the cAMP signaling system in alpha T3-1 cells are direct or whether they are mediated, at least in part, by the stimulation of Ca2+ influx.

    ACKNOWLEDGEMENTS

We thank Dr. P. L. Mellon for kindly providing the alpha T3-1 cells, Dr. Nicholas Demaurex for comments on an earlier draft of this paper, and the group of Dr. Karl-Heinz Krause for providing the use of their patch-clamp/microfluorimetry system.

    FOOTNOTES

This work was supported by Swiss National Science Foundation Grants 32-33514.92 and 31-45830.95.

Present address of M. Hezareh: Departments of Pathology and Medicine 0679, University of California at San Diego, La Jolla, CA 92093.

Address for reprint requests: S. R. Rawlings, Fondation pour Recherches Médicales, Univ. of Geneva, 64 Ave. de la Roseraie, CH-1211 Geneva 4, Switzerland.

Received 8 April 1997; accepted in final form 21 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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