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Molecular Endocrinology, doi:10.1210/me.2005-0363
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Molecular Endocrinology 20 (9): 2231-2246
Copyright © 2006 by The Endocrine Society

Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

Arturo E. Gonzalez-Iglesias, Yonghua Jiang, Melanija Tomic, Karla Kretschmannova, Silvana A. Andric, Hana Zemkova and Stanko S. Stojilkovic

Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland 20892-4510

Address all correspondence and requests for reprints to: Dr. Stanko Stojilkovic, Endocrinology and Reproduction Research Branch/National Institute of Child Health and Human Development/National Institutes of Health, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenyltio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN MANY CELL types, Ca2+ and cyclic nucleotide signaling pathways are tightly interconnected not only at the level of intracellular messenger generation but also at the level of their intracellular effectors (1, 2). Changes in intracellular calcium concentration ([Ca2+]i) can influence cAMP production and/or degradation; Ca2+ activates some isoforms of adenylyl cyclase (AC) but inhibits others, and selectively stimulates some phosphodiesterase (PDE) subtypes (3, 4). Calcium also affects cGMP levels through the activation of Ca2+-sensitive PDEs (5), as well as by activating nitric oxide synthases (6) and modulating the nitric oxide-dependent soluble guanylyl cyclase (sGC) activity (7). In turn, changes in the cyclic nucleotide intracellular levels and activity of their kinases can influence membrane potential (Vm) and Ca2+ signaling. These include the effects on Ca2+ influx through the plasma membrane channels, Ca2+ clearance from cytosol, and conductivity of inositol 1,4,5-triphosphate and ryanodine receptor channels (2, 8, 9, 10, 11, 12). The reciprocal modulation of Ca2+ and cyclic nucleotide signaling also represents one of the mechanisms by which cells control their secretion (13).

Both normal (14, 15) and immortalized pituitary cells (16) express Ca2+-inhibitable ACs. These cells also express other subtypes of ACs, as well as numerous PDEs (17, 18). Pituitary cells also express {alpha}1ß1 sGC dimer, and the nitric oxide-dependent activity of this enzyme is modulated by protein kinase A (PKA)-mediated phosphorylation of {alpha}1-subunit (19). Normal and immortalized pituitary cells generate action potentials spontaneously (20, 21); in pituitary lactotrophs and somatotrophs, such electrical activity and the associated voltage-gated calcium influx (VGCI) are sufficient to maintain high hormone release in vitro (22). The mRNA transcripts for cyclic nucleotide-gated channels were also identified in pituitary cells (23). These cells also express PKA-regulated L-type voltage-gated Ca2+ and Na+ channels (24), as well as an unidentified PKA-regulated depolarizing current (25). These findings could indicate the possible role of cyclic nucleotides and their kinases in control of pacemaking and hormone secretion, a hypothesis that has not been tested experimentally in any of the secretory anterior pituitary cells.

Here we studied the effects of basal and stimulated AC and sGC activity on spontaneous generation of action potentials, VGCI-dependent Ca2+ transients, and Ca2+-controlled hormone release using pituitary lactotrophs as a cell model. We initially examined the reciprocal modulation of cyclic nucleotides and VGCI in spontaneously firing pituitary lactotrophs, a mechanism that, in other excitable cell types, accounts for the control of pacemaking and VGCI (13, 26). We also studied the AC subtype(s) contributing to the basal cAMP accumulation in pituitary cells, as well as the effects of up- and down-regulation of AC and sGC activity on the pattern of cAMP and cGMP release, pacemaking, VGCI, and prolactin (PRL) secretion. Finally, we examined the messenger roles of cAMP and cGMP in control of Ca2+ influx and PRL release. The results suggest that multiple AC subtypes, including Ca2+-inhibitable forms, contribute to the basal cAMP production. Cyclic nucleotides do not seem to be critical for spontaneous firing of action potentials but provide an effective mechanism for facilitation of pacemaking, VGCI, and PRL release in pituitary lactotrophs. It also appears that cAMP, but not cGMP, is essential in up-regulation of electrical activity and Ca2+ influx-controlled PRL release, which predominantly operates through a PKA-independent pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Inhibition of PDEs on Cyclic Nucleotide and PRL Release
The addition of 3-isobutyl-1-methylxanthine (IBMX), a common inhibitor of PDE subtypes, to pituitary cells in static cultures (1 x 106/well) increased cAMP (Fig. 1AGo) and cGMP (Fig. 1CGo) intracellular concentrations and their release in a dose-dependent manner. Application of IBMX also caused a dose-dependent increase in basal PRL release. Furthermore, no changes in stored PRL were observed in IBMX-treated cells compared with controls (data not shown). There was a linear relationship between intracellular and extracellular cyclic nucleotide concentrations in IBMX-treated cells (Fig. 1Go, B and D), indicating that the released cAMP and cGMP reflect well the status of AC and sGC activities in pituitary cells. Moreover, there was an obvious difference in the extent of cAMP and cGMP release. As shown in Fig. 1Go, A and C, most of cGMP and only a fraction of cAMP were released, suggesting a preference of cyclic nucleotide export pump for cGMP, which in turn could favor cAMP-dependent intracellular signaling functions in pituitary cells. Finally, Fig. 1Go, E and F, illustrates that small changes in intracellular content of cyclic nucleotides have a profound effect on PRL release.


Figure 1
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  		Fig. 1. Effects of IBMX, a Common Inhibitor of PDEs, on Cyclic Nucleotide Accumulation and PRL Release in Pituitary Cells in Static Culture

A and C, Dose-dependent effects of IBMX on cAMP (A) and cGMP (C) release and cell content. B and D, Correlation between released and cell content of cyclic nucleotides in IBMX-treated cells. E and F, The relationship between cAMP intracellular content and PRL release (E) and cGMP intracellular content and PRL release (F). At the end of 3-h incubation, medium was collected, cells were scraped, and cAMP, cGMP, and PRL levels were determined in medium (released) and cell extracts (cell content). Data shown are means ± SEM from sextuplicate incubations in one of three similar experiments. R, Coefficient of correlation.

 
In general, stimulation of PRL release by an increase in basal cyclic nucleotide levels could reflect VGCI-independent or VGCI-dependent actions of cyclic nucleotides on hormone secretion, changes in the de novo PRL synthesis, and/or changes in the rate of priming of secretory vesicles. Measurement of cyclic nucleotides and PRL in cells cultured for 1–3 h certainly does not provide an adequate system with which to address the first hypothesis, because Vm and Ca2+ recordings are commonly done at a minute time scale. To overcome this limitation, pituitary cells attached on beads and loaded in chambers (1.5 x 107 cells per chamber) were perifused at flow rate of about 0.8 ml/min at 37 C, samples were collected every minute, and cyclic nucleotides and PRL contents were measured. In unstimulated cells, there were measurable levels of cAMP (Fig. 2AGo) and cGMP (Fig. 2BGo) in perifusion medium. During perifusion with IBMX-containing medium, the release of both cyclic nucleotides increased in a dose-dependent manner, reaching the steady-state levels between 3 and 15 min (Fig. 2Go, A and B). The averaged data from four experiments, shown in Fig. 2Go, D and E, indicate dose-dependent profiles comparable to those observed in cells in static cultures. Based on this comparison and the strong correlation between intracellular and extracellular cyclic nucleotide levels in cells in static cultures (Fig. 1Go, B and D), we may conclude that release of cAMP and cGMP provides a valuable indicator of the status of AC and sGC activities in pituitary cells.


Figure 2
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  		Fig. 2. Stimulation of Cyclic Nucleotide and PRL Release by IBMX in Perifused Pituitary Cells

A–C, Concentration-dependent effects of IBMX on cAMP (A), cGMP (B), and PRL (C) release. Cells were perifused for 2.5 h to establish stable baselines and samples were then collected every minute and analyzed for their cAMP, cGMP, and PRL contents. The profiles of cAMP, cGMP, and PRL levels are representative of four independent experiments. Gray areas indicate the duration of IBMX application. Numbers above gray areas indicate IBMX concentration. D–F, Mean ± SEM values derived from four experiments at the steady-state level.

 
Basal PRL release in perifused pituitary cells was high, and IBMX further increased PRL release, whereas removal of IBMX led to a progressive decrease in PRL release toward the basal levels (Fig. 2CGo). It is interesting that there was a consistent delay in decline of PRL and cyclic nucleotide release of 3–5 min after the removal of IBMX. This delay was not observed in other treatments (see for example Fig. 3Go) and probably reflected a slow dissociation of IBMX from PDEs. The stimulatory effects of low IBMX concentrations on PRL release in perifused pituitary cells (Fig. 2FGo) and those in static cultures (Fig. 1Go, E and F) further support the hypothesis that the status of basal ACs, sGC, and PDEs influences PRL secretion.


Figure 3
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  		Fig. 3. Extracellular Ca2+ Dependence of Spontaneous Electrical Activity, Ca2+ Transients, and PRL Release

A, Identification of lactotrophs by activation of Ca2+-mobilizing TRH receptors. TRH was applied only during 2-sec period. B–D, Effects of depletion of extracellular Ca2+ by perfusion with Ca2+-deficient medium containing 0.5 mM EGTA and inhibition (1 µM nifedipine) and activation (1 µM Bay K 8644) of voltage-gated Ca2+ channels on the pattern of electrical activity (B) and Ca2+ transients (C) in single lactotrophs, and PRL release (D) in perifused pituitary cells. In experiments with Ca2+ measurements, 100 nM TRH was added at the end of experiments to identify lactotrophs. Horizontal bars and gray areas indicate the duration of EGTA, nifedipine, and Bay K 8644 applications.

 
Extracellular Ca2+ Dependence of Vm Activity, VGCI, and Cyclic Nucleotide and PRL Release
It is well established that ACs are not only effectors of specific G protein-coupled receptors, but are also regulated by convergent signaling pathways, including cytosolic Ca2+ and kinases (27). The activity of sGC in pituitary cells is also modulated in a PKA- and Ca2+-dependent manner (7, 19). Furthermore, phosphorylation and intracellular Ca2+ contribute to the control of PDE activities (5). As shown in Fig. 3Go, spontaneous electrical activity, calcium signaling, and basal PRL release are also dependent on extracellular Ca2+ concentration.

In electrophysiological studies, we identified single lactotrophs by their Vm responses to 100 nM TRH applied for 2–5 sec at the beginning of the experiments. In these cells, TRH acts on its Ca2+-mobilizing receptors, leading to a transient membrane hyperpolarization followed by a sustained depolarization (28). Figure 3AGo illustrates the hyperpolarizing effects of TRH in spontaneously active (top) and quiescent lactotrophs (middle and bottom). In general, TRH-responsive cells fired single action potentials (top and middle) or showed bursting type of firing (bottom). Removal of extracellular Ca2+ and the addition of 1 µM nifedipine in Ca2+-containing medium silenced Vm activity of the cells (Fig. 3BGo, top and middle). Conversely, 1 µM Bay K 8644, an L-type Ca2+ channel activator, increased the frequency of spiking (Fig. 3BGo, bottom) and initiated the firing of action potentials in quiescent cells (data not shown). These treatments had similar effects on spontaneous Ca2+ transients in single lactotrophs (Fig. 3CGo) and basal PRL secretion in perifused pituitary cells (Fig. 3DGo).

We also used perifused pituitary cells to examine the dependence of AC activity on the status of VGCI. EGTA treatment led to a rapid increase in cAMP release, whereas BayK 8644 treatment attenuated basal AC activity, both in a reversible manner. As shown in Fig. 4AGo, these effects were observed in IBMX-treated and untreated cells, confirming that Ca2+-inhibitable ACs contribute to the basal cAMP production. It is also important to stress that residual Ca2+-independent basal cAMP production was substantial, indicating the relevance of other AC subtypes in cAMP signaling in cells under resting conditions. Earlier studies clarified that EGTA and Bay K 8644 treatments had similar effects on basal sGC activity in pituitary cells (7).


Figure 4
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  		Fig. 4. Extracellular Ca2+ Dependence of Cyclic Nucleotide Production

A, Effects of depletion of extracellular Ca2+ by 0.5 mM EGTA (left) and facilitation of VGCI by 1 µM Bay K 8644 (right) on cAMP release in perifused pituitary cells in the presence (closed circles) or absence (open circles) of 1 mM IBMX. Gray areas indicate the duration of EGTA and Bay K 8644 application. B, RT-PCR identification of mRNA transcripts for Ca2+-inhibitable ACs in anterior pituitary (bottom) and GH3 cells (top). C, Western blot analysis of expression of AC3 (left), AC5/6 (center), and AC9 (right) in GH3 cells and anterior pituitary. Arrows indicate band of interest. RT, Reverse transcriptase.

 
The calcium dependence of basal AC activity in intact cells shown here is in agreement with previous studies with cell homogenates (14, 15). In general, there are three known subtypes of Ca2+-inhibitable AC enzymes: AC5, AC6, and AC9 (29). Intracellular Ca2+ also inhibits AC3 subtype upon its phosphorylation by Ca2+-calmodulin kinase II (30). Our RT-PCR analysis revealed the presence of mRNA transcripts for all four subtypes of these enzymes in a mixed population of pituitary cells but only AC3, AC6, and AC9 subtypes in GH3 immortalized pituitary cells (Fig. 4BGo). Western blot analysis was consistent with RT-PCR analysis (Fig. 4CGo); the expression of AC3 and AC9 proteins was detected in both cell types, expression of AC6 was observed in GH3 cells, and AC5 and/or AC6 proteins could be demonstrated in pituitary cells (the antibody used does not distinguish between these two isoforms).

The potential participation of these enzymes in Ca2+-dependent inhibition of cAMP production was analyzed using the protocol with EGTA treatment in the presence and absence of specific inhibitors of these enzymes. The contribution of AC9 in basal cAMP production was characterized by inhibiting calcineurin, which serves as a Ca2+-sensor that mediates the negative feedback effects of Ca2+ on the activity of this enzyme (31). We used three different blockers of calcineurin: FK-506 (100 nM), cyclosporin A (10 µM), and (R,S)-{alpha}-cyano-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cypermethrin; 10 nM). All three blockers slightly inhibited basal cAMP levels, and removal of extracellular Ca2+ still increased cAMP production, indicating that participation of this enzyme in VGCI-controlled cAMP production is minor (10–15%). To address the participation of AC3 subtype, we evaluated the effects of two blockers of Ca2+-calmodulin kinase II, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera-zine (KN62; 50 µM) and 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93; 10 µM), which should inhibit this AC isoform (30). In such treated cells, the stimulatory effect of removal of extracellular Ca2+ on cAMP production was only slightly (~5%) affected. We also tested cells with 2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80) (100 µM), an inhibitor of AC5 (32), which attenuated the EGTA-induced rise in cAMP release by about 15%. Finally, the EGTA-induced rise in cAMP production was reduced by 50% when cells were treated with 500 µM 2',5'-dideoxyadenosine (2'5'-ddAdo), an AC inhibitor with high preference for AC6 (33). These results indicate that the stimulatory effect of VGCI blockade on cAMP production was mediated by multiple AC subtypes.

Dependence of Basal PRL Release on Cyclic Nucleotide Signaling
Because the Ca2+-inhibitable ACs account only partially for basal cAMP production, in further studies we used several common blockers of Ca2+-inhibitable and -nonihibitable enzymes to study the relevance of basal cyclic nucleotide production on PRL release (33, 34, 35). Figure 5Go illustrates the effects of cis-N-(2-phenylcyclopenthyl)azacyclotridec-1-en-2-amine HCl (MDL12330A) (panel A), 2'5'-ddAdo (panel B), and 2',5'-dideoxyadenosine-3'-O-bis(S-pivaloyl-2-thioethyl)-phosphate (2'5'-ddAdo-SATE) (panel C) on cAMP and cGMP release in cells with inhibited PDEs with 1 mM IBMX. It is interesting that MDL12330A inhibited both cAMP and cGMP release (Fig. 5AGo), whereas 2'5'-ddAdo (Fig. 5BGo), 2'5'-ddAdo-SATE (Fig. 5CGo), and 9-(tetrahydro-2'-furyl)adenine (SQ22536) (data not shown) were specific for ACs.


Figure 5
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  		Fig. 5. Effects of Common AC Inhibitors on cAMP (left panels) and cGMP (right panels) Release in Perifused Pituitary Cells

Gray areas indicate the duration of cell perifusion with MDL12330A (A), 2'5'-ddAdo (B), and 2'5'-ddAdo-SATE (C). To amplify cyclic nucleotide release, cells were perifused with medium containing 1 mM IBMX.

 
In addition to blocking cAMP production, SQ22536 exhibited AC-independent stimulatory effects on VGCI in lactotrophs (data not shown) and thus could not be used for secretory studies. All other inhibitors decreased basal PRL release in perifused pituitary cells, but the percentage of inhibition varied among blockers in the presence and absence of IBMX (Table 1Go). None of these compounds completely blocked PRL release, clearly indicating the presence of cyclic nucleotide-dependent and -independent components in Ca2+-controlled exocytosis. As shown in Fig. 6AGo, application of 0.1 and 1 µM 2'5'-ddAdo-SATE required about 25 min to reach the steady PRL release. In contrast to 2'5'-ddAdo-SATE, 1H-(1,2,4)oxadioazolo[4,3-a]quinozalin-1-one (also known as ODQ) inhibited sGC without affecting basal AC activity and PRL release (Fig. 6BGo). These results suggest that basal AC activity, rather than basal sGC, contributes to the control of PRL release.


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  		Table 1. Effects of AC Inhibitors on Basal PRL Release (ng/min) in Perifused Pituitary Cells

 

Figure 6
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  		Fig. 6. Dependence of Basal PRL Release on AC but Not sGC Signaling Pathway in Perifused Pituitary Cells

A, Dose-dependent effects of 2'5'-ddAdo-SATE, a potent AC inhibitor, on basal PRL release. For effects of 2'5'-ddAdo-SATE on cyclic nucleotide release, see Fig. 5CGo. B, The lack of effects of ODQ, a specific inhibitor of sGC (bottom trace) on cAMP (middle trace), and PRL (top trace) release. Cells were perifused with IBMX-free medium. ODQ, 1H-(1,2,4)oxadiazolo[4,3-a]quinoalin-1-one.

 
Single-cell Ca2+ measurements revealed that none of these inhibitors abolished spontaneous Ca2+ transients when applied for 10–20 min. The extent to which inhibition of basal AC activity affected the amplitude or frequency of Ca2+ signals was difficult to determine with these semiquantitative measurements. Electrophysiological measurements revealed that a transient exposure of pituitary cells to MDL12330A led to abolition of spontaneous firing of action potentials only in a small fraction of cells (five of 27 cells). In 10 of 27 cells, we also observed a reduction in the frequency or amplitude of action potentials, whereas in the residual spontaneously active cells no obvious changes in the pattern of electrical activity was noticed. Application of MDL12330A initiated firing of action potentials in none of the quiescent cells. These results suggest that spontaneous pacemaking in lactotrophs is independent of the status of basal cyclic nucleotide production, but that modulation of electrical activity by basal cAMP production may contribute to the control of PRL release.

Effects of Cell-Permeable Cyclic Nucleotides and Forskolin on VGCI and PRL Release
To further study the relevance of cyclic nucleotides on pacemaking and VGCI-controlled PRL release and to dissociate which signaling molecule, cAMP and/or cGMP, is critical in those processes, we used two cell-permeable cyclic nucleotide analogs: 8-Br-cAMP and 8-Br-cGMP. Application of 8-Br-cAMP initiated firing of action potentials in quiescent lactotrophs (Fig. 7AGo), whereas application of 8-Br-cGMP was ineffective (data not shown). 8-Br-cAMP also increased PRL release in perifused pituitary cells (Fig. 7BGo), whereas 8-Br-cGMP was ineffective (data not shown), as well as 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP, Fig. 7BGo), an agonist of Epac cAMP receptor. The stimulatory effect of 8-Br-cAMP was dependent on the presence of physiological Ca2+ concentrations in medium (Fig. 7CGo), indicating that cAMP influences secretion through a Ca2+-dependent step in exocytosis. In cells with 2'5'-ddAdo-SATE-induced inhibition of AC activity and PRL release, application of 8-Br-cGMP was ineffective (Fig. 7DGo), but secretion was rescued in cells treated with 8-Br-cAMP (Fig. 7EGo). In the same experiment, the combined effect of 8-Br-cAMP and 8-Br-cGMP on PRL release was less effective compared with treatment with 8-Br-cAMP alone (Fig. 7Go, panel F vs. panel E).


Figure 7
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  		Fig. 7. Effects of Cell-Permeable Cyclic Nucleotide Analogs on Electrical Activity in Single Lactotrophs and PRL Release in Perifused Pituitary Cells

A, Initiation of firing of action potentials with 8-Br-cAMP in an identified lactotroph. B, Stimulation of basal PRL release with 8-Br-cAMP. In the same experiment but different column, 8-pCPT-2Me-cAMP, an Epac cAMP receptor agonist, was ineffective. C, The lack of effects of 8-Br-cAMP on PRL release in cells with blocked Ca2+ influx by 0.5 mM EGTA treatment. Notice the increased PRL release in 8-Br-cAMP-treated cells before and after application of Ca2+-deficient medium. D–F, Effects of 8-Br-cGMP (D), 8-Br-cAMP (E), and both analogs applied together (F) on recovery of PRL release in cells with inhibited AC activity with 2'5'-ddAdo-SATE. All three treatments were performed in the same experiment with different columns, and the control column is shown in panel D.

 
In further experiments, pituitary cells were treated with forskolin, a common activator of AC1 to AC8 subtypes (29). Figure 8AGo illustrates stimulatory effects of forskolin on cAMP (left) and cGMP (right) release in perifused pituitary cells without inhibited PDEs. Forskolin also initiated firing of action potentials in the majority of quiescent lactotrophs (Fig. 8BGo, left) and increased the frequency of spiking in spontaneously firing cells (Fig. 8BGo, right). The stimulatory effects of forskolin on Vm oscillations occurred within 30 sec of application and persisted for several minutes after its removal. In parallel to Vm measurements, forskolin increased [Ca2+]i in the majority of quiescent lactotrophs (58 of 73 cells). As shown in Fig. 8CGo (left), the stimulatory effect of forskolin was not instantaneous, but developed gradually. In spontaneously active cells, forskolin increased the frequency or amplitude of [Ca2+]i transients within 1 min (Fig. 8CGo, right). Finally, there was a gradual increase in PRL release during forskolin treatment in perifused pituitary cells, followed by a slow decrease toward the basal levels after its washout (Fig. 8DGo).


Figure 8
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  		Fig. 8. Effects of Forskolin on Cyclic Nucleotide and PRL Release in Perifused Pituitary Cells and on Electrical Activity and Ca2+ Signaling in Single Lactotrophs

A, Stimulatory effects of forskolin on cAMP (left) and cGMP (right) release. B, Forskolin-induced initiation of firing of action potentials in quiescent lactotrophs (left traces) and increase in the firing frequency in spontaneously active cells (right traces). C, Forskolin-induced Ca2+ transients in quiescent cells (left traces) and increase in the frequency and/or amplitude of spiking in spontaneously active cells (right traces). D, Stimulatory effects of forskolin on PRL release. Gray areas in A, C, and D and horizontal bars in B indicate the duration of forskolin application.

 
Application of 2'5'-ddAdo-SATE attenuated the forskolin-induced elevation in cAMP (Fig. 9AGo) and PRL release (Fig. 9BGo), clearly indicating the importance of cAMP in VGCI-controlled exocytosis. Forskolin-induced cAMP production was also inhibited by 1 µM 2'5'-ddAdo-SATE, 75 µM MDL12330A, 1 mM SQ22536, and 100 µM NKY80 by 66%, 33%, 71%, and 74%, respectively. Forskolin-induced Ca2+ signaling and PRL release were also significantly inhibited by these three blockers. Figure 9CGo illustrates effects of MDL12330A on forskolin-induced Ca2+ transients, and Fig. 9DGo illustrates effects of MDL12330A on forskolin-induced PRL release. The MDL12330A-induced inhibition of PRL release was rescued by application of 8-Br-cAMP (Fig. 9EGo) but not by 8-Br-cGMP (data not shown). These results clearly show an Epac cAMP receptor-independent stimulatory action of cAMP on excitability, VGCI, and Ca2+-controlled PRL release.


Figure 9
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  		Fig. 9. Effects of AC Inhibitors on Forskolin-Induced Ca2+ Signaling in Single Lactotrophs and PRL Release in Perifused Pituitary Cells

A and B, Inhibition of forskolin-induced cAMP production (A) and PRL release (B) by 2'5'-ddAdo-SATE. C and D, Abolition of forskolin-induced intracellular Ca2+ transients (C) and PRL release (D) by MDL12330A. E, Recovery of PRL release in MDL12330A-treated cells with 8-Br-cAMP. In the same experiment, cGMP was ineffective. For comparison with 2'5'-ddAdo-SATE-treated cells, see Fig. 7EGo. Gray areas indicate the duration of AC inhibitor application, and horizontal bars indicate the duration of forskolin and 8-Br-cAMP treatments. MDL, MDL12330A.

 
Role of PKA in cAMP-Induced Pacemaking, VGCI, and PRL Release
The cAMP-dependent protein kinase (PKA) family of enzymes is assembled from the products of four regulatory and two catalytic subunit genes (36). To study the potential role of PKA in cAMP effects in lactotrophs, we used four specific inhibitors of this enzyme: 4-cyano-3-methylisoquinoline (CMI) (37), N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, 2HCl (H89) (38), 8-(4-chlorophenylthio)cAMP thioate Rp-isomer (Rp-8-CPT-cAMPS) (39), and the myristoylated version of PKI amide 14–22 peptide (38). In the concentration range of 0.1 to 10 µM, CMI was ineffective in stopping spontaneous (Fig. 10AGo, top and middle) and forskolin-induced (Fig. 10AGo, bottom) electrical activity. Moreover, in CMI-treated cells, forskolin was able to increase the firing frequency (Fig. 10AGo, middle). The pattern of spontaneous firing in pituitary cells was also not affected by application of 1 mM Rp-8-CPT-cAMPS and 1 µM H89 (data not shown).


Figure 10
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  		Fig. 10. Independence of Spontaneous and Forskolin-Induced Electrical Activity and Ca2+ Signaling of the Status of PKA Signaling Pathway

A, The lack of effects of CMI treatment on electrical activity in spontaneously active lactotrophs exhibiting plateau bursting (top trace) and single spiking type of action potential (middle trace) and in a cell with forskolin-induced electrical activity (bottom trace). B and C, The lack of effects of CMI (B) and H89 (C) on spontaneous and forskolin-induced Ca2+ transients. Arrows indicate the moment of drug applications. In Ca2+ experiments with two treatments, the first compound was present during the application of the second compound.

 
Single-cell Ca2+ measurements revealed the lack of effects of 0.5 and 1 µM CMI on basal [Ca2+]i in spontaneously active and quiescent cells. Forskolin initiated Ca2+ transients in the presence of CMI in quiescent cells and increased the amplitude in spontaneously active cells (Fig. 10BGo, top three traces). Forskolin-induced oscillatory and nonoscillatory Ca2+ responses were also not affected by sustained application of 0.5 and 1 µM CMI (Fig. 10BGo, bottom two traces). When applied in 5 and 10 µM concentrations, CMI did not abolish spontaneous Ca2+ transients but initiated spiking in a fraction of lactotrophs. In such treated cells, 1 µM forskolin increased the frequency/amplitude of Ca2+ transients (data not shown). Spontaneous Ca2+ transients were also not abolished by H89 and PKI 14–22 in 1, 5, and 10 µM concentrations (data not shown), further indicating the independence of VGCI of the status of PKA signaling pathway. H89 (Fig. 10CGo) and PKI 14–22 (data not shown) were also ineffective in blocking the stimulatory effects of forskolin on Ca2+ signals. Like CMI, both compounds, in 5 and 10 µM concentrations, initiated Ca2+ spiking in a fraction of lactotrophs.

In parallel to Ca2+ signaling, basal PRL release was not affected by application of 1 mM Rp-8-CPT-cAMPS, 0.5 and 1 µM CMI (Fig. 11AGo), and 1 µM H89 (data not shown), whereas 10 µM CMI slightly elevated basal PRL release (Fig. 11CGo). In both 1 and 10 µM CMI-treated cells and 10 µM PKI 14–22-treated cells, forskolin increased PRL release in a manner comparable to that observed in controls (Fig. 11Go, B and C), confirming the independence of cAMP-induced PRL release of the status of PKA family of enzymes.


Figure 11
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  		Fig. 11. Independence of Basal and Forskolin-Induced PRL Release of PKA Signaling Pathway in Perifused Pituitary Cells

A, The lack of effects of Rp-8-CPT-cAMPS and CMI on basal PRL release. B and C, Stimulatory effect of forskolin on PRL release in cells treated with 1 µM CMI (B), 10 µM CMI, and 10 µM PKI 14–22 (C). Gray areas indicate duration of forskolin application and horizontal bars indicate duration of PKA inhibitor treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Dependence of Spontaneous Pacemaking and Basal PRL Release on AC Signaling Pathway
Normal and immortalized pituitary cells generate action potentials spontaneously (20, 21), and the firing occurs either as a baseline spiking or a periodic plateau-bursting activity (22). Such spontaneous VGCI is critical for basal PRL release, and cell-permeable cyclic nucleotides cannot rescue PRL secretion in cultures bathed in Ca2+-deficient medium. Lactotrophs and PRL-secreting GH3 cells express numerous plasma membrane channels, including inactivating and noninactivating voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, multiple calcium-sensitive K+ and Cl– channels, M channels, and inwardly rectifying K+ channels (24, 41, 42, 43, 44, 45). The primary roles of plasma membrane channels involved in action potential spiking and VGCI are relatively well characterized in these cells, in contrast to channels and intracellular signaling pathways controlling their pacemaking activity (20, 21).

The finding that IBMX, a common inhibitor of PDEs, and forskolin, a common activator of ACs, elevate cAMP and cGMP levels and stimulate PRL release is consistent with the role of cyclic nucleotides in modulation of electrical activity, VGCI, and PRL release. This hypothesis was directly confirmed in experiments with forskolin and 8-Br-cAMP, which initiated the firing of action potentials in quiescent cells and increased the frequency of spiking in spontaneously active cells. Furthermore, the finding that pacemaking activity was preserved in the majority of cells with blocked cAMP production indicates that this signaling pathway is not critical for spontaneous generation of action potentials. Earlier studies excluded the role of cGMP signaling pathway in spontaneous electrical activity, VGCI, and PRL release (46). Finally, electrophysiological experiments suggested that basal cyclic nucleotides could contribute to the modulation of firing activity in lactotrophs.

PRL release was reduced, but not abolished, in cells treated with different AC inhibitors, further supporting the conclusion about the cyclic nucleotide-dependent and -independent pathways controlling spontaneous VGCI and exocytosis. In PC12 cell types, cAMP, through PKA, affects the size of slowly and rapidly releasable secretory vesicle pools without affecting the kinetics of vesicle fusion (47). In lactotrophs, inhibitors of PKA pathway did not have rapid effects on basal PRL release, whereas 8-Br-cAMP facilitated pacemaking activity and secretion, arguing against the role of this pathway in priming of secretory pool during the short-term treatment. IBMX, in concentrations that only slightly elevated cyclic nucleotides, also stimulated PRL release, further suggesting that small changes in cyclic nucleotide levels are sufficient to fully activate cells and PRL release. Taken together, these results are consistent with the hypothesis that basal cAMP production contributes to the control of VGCI and PRL release by modulating electrical activity and that the inhibitory effect of VGCI on Ca2+-inhibitable ACs may serve as a negative feedback mechanism.

Reciprocal Modulation of Cyclic Nucleotides and VGCI in Pituitary Lactotrophs
The reciprocal modulation of VGCI and cyclic nucleotides represents one of the mechanisms by which cells control their secretion. For example, in pancreatic ß-cells cAMP potentiates Ca2+-dependent exocytosis and mediates the stimulation of insulin release by glucagon. In these cells, Ca2+ oscillations are coupled to cAMP oscillations, indicating that these two messengers are interlinked and reinforce each other (13). In embryonic Xenopus spinal neurons, there are dynamic interactions between cAMP transients and spontaneous Ca2+ spikes driven by transient plasma membrane depolarization and Ca2+ influx through voltage-gated Ca2+ channels. The authors (26) developed a mathematical model of Ca2+ and cAMP oscillations in which these signaling molecules are strictly interdependent. In GT1 immortalized hypothalamic neurons, agonist-induced Ca2+ mobilization was associated with sustained VGCI (48) and stimulation of AC1 (49). These neurons express functional cyclic nucleotide-gated channels (50, 51), and cAMP stimulates pacemaking activity (52), findings consistent with the operation of a positive feedback mechanism between VGCI and cAMP generation.

In contrast, Ca2+ inhibition of basal AC activity was observed in normal and immortalized rat pituitary cell membranes and homogenates (14, 15, 16), and concentrations of extracellular Ca2+ required for inhibition of AC activity were in the range observed in intact spontaneously active lactotrophs (22). Consistent with these results, we also observed that VGCI attenuated intrinsic AC activity in intact cells independently of the status of PDEs. In GH4C1 pituitary cells, there is an intimate colocalization of ACs with capacitative calcium entry channels, which are activated by depletion of intracellular Ca2+ stores (53). In lactotrophs, the recruitment of Ca2+ from intracellular stores by calcium-induced calcium release mechanism does not occur in spontaneously firing cells (54), and thus VGCI should solely account for inhibition of intrinsic AC activity.

Our results further indicate that anterior pituitary cells express mRNA transcripts and proteins for all known forms of Ca2+-inhibitable AC subtypes: AC3, AC5, AC6, and AC9. Others previously identified AC9 and characterized its role in AtT20 immortalized mouse pituitary cells (31). In these cells, agonist-induced stimulation of AC9 facilitates dihydropyridine-sensitive Ca2+ influx, which in turn inhibits the enzyme. The authors also identified calcineurin as a Ca2+ sensor that mediates the negative feedback effects of VGCI on receptor-stimulated cAMP production. Our observations are also in accordance with earlier published data concerning the expression of AC6 in several immortalized pituitary cell types (55) and its inhibition by submicromolar [Ca2+]i (56, 57). We also show that none of the Ca2+-inhibitable AC inhibitors completely blocks EGTA-induced elevation in cAMP production, suggesting that multiple forms of Ca2+-inhibitable ACs contribute to the negative regulation of cAMP production by VGCI. It is also important to point out that other subtypes of these enzymes contribute to the basal cAMP production. Partial dependence of basal PRL release on cyclic nucleotides and partial inhibition of AC activity by spontaneous VGCI are findings consistent with reciprocal modulation of cyclic nucleotides and VGCI in spontaneously firing pituitary lactotrophs. In contrast to other cell types, such a mode of regulation contributes only to the control of spontaneous electrical activity and basal PRL release, whereas the AC-independent action potential secretion coupling accounts for the majority of basal PRL release.

Messenger(s) Involved in Cyclic Nucleotide-Dependent Vm Activity, VGCI, and PRL Release
Whereas experiments with IBMX and forskolin demonstrated unquestionably the dependence of electrical activity, VGCI, and PRL release on cyclic nucleotide production, they did not dissociate which signaling molecule cAMP, cGMP, their dependent kinases, or Epac cAMP receptor accounts for these effects. Furthermore, in our experimental conditions, changes in AC activity in pituitary cells frequently paralleled with changes in cGMP levels, raising the possibility that both messengers could contribute to the control of electrical activity and PRL release. In experiments with IBMX, the increase in both nucleotides reflected the attenuation in their degradation by PDEs. On the other hand, parallelism in the actions of forskolin on cAMP and cGMP levels in cells with inhibited PDEs resulted indirectly from the combined role of PKA stimulation of cGMP synthesis by increasing nitric oxide production (58) and directly by phosphorylating the {alpha}-sGC subunit, which enhanced the nitric oxide-dependent sGC activity (19). Finally, MDL12330A, the well-established inhibitor of AC (59), also decreased cGMP release, presumably by inhibiting sGC activity.

However, the potential role of cGMP and its kinase in control of pacemaking activity and PRL secretion was excluded. First, selective inhibition of basal cGMP production did not affect basal PRL release in contrast to selective inhibition of AC activity by 2'5'-ddAdo, 2'5'-ddAdo-SATE, and NKY80. Second, application of 8-Br-cGMP was ineffective in initiation of electrical activity and PRL release in control cells and did not rescue secretion in cells treated with AC inhibitors. In contrast, 8-Br-cAMP stimulated pacemaking activity and PRL release and rescued secretion in cells with blocked AC activity. Third, stimulation of sGC activity did not affect VGCI and PRL secretion (46). Consistent with the role of cAMP rather than cGMP in these intracellular processes, the present data and our ongoing experiments indicate that the majority of cGMP is rapidly released from cells by MRP5 export pump (60).

In general, the stimulatory action of cAMP on electrical activity and secretion could be mediated by Epac cAMP receptor or PKA. Regarding the former effector molecule, our results with 8-pCPT-2'-O-Me-cAMP, the specific Epac cAMP receptor agonist (61), argue against this pathway. We also used numerous PKA inhibitors in a wide range of concentrations, and none of them abolished spontaneous and forskolin-induced VGCI and PRL release. We observed initiation of Ca2+ transients only in a fraction of cells with inhibited PKA, an action consistent with expression of voltage-gated Na+ channels in these cells (24) and their modulation by this enzyme (62). These observations strongly support a PKA-independent role of cAMP in control of pacemaking, VGCI, and PRL release.

Channel(s) Involved in Cyclic Nucleotide-Dependent Modulation of Vm Activity
In several neuronal cell types, AC signaling pathway controls pacemaking through hyperpolarization-activated cation channels. These channels conduct both K+ and Na+, and their activation leads to slow depolarization of cell membrane and initiation of firing (63). Normal and immortalized pituitary cells (64, 65) also express functional hyperpolarization-activated cation channels, which may contribute to the control of pacemaking. Cyclic nucleotide-gated channels also participate in pacemaking. For example, normal and immortalized GnRH-secreting neurons express cyclic nucleotide-gated channels, which in these cells operate as the pacemaking channels (50, 51, 66). They predominantly conduct Na+, leading to depolarization of cells and activation of VGCI and are regulated by both cAMP and cGMP (9). Pituitary cells also express mRNA transcripts for three subtypes of cyclic nucleotide-gated channels (23). The ability of cAMP but not cGMP to initiate firing in quiescent lactotrophs is more consistent with the role of hyperpolarization-activated cation channels, because these channels are more sensitive to cAMP than cGMP regulation (67). However, the lack of effects of cGMP in lactotrophs does not necessarily argue against the functional role of cyclic nucleotide-gated channels, because our results indicate that the majority of de novo produced cGMP is released. In that scenario, cGMP may not accumulate intracellularly to the levels needed to modulate these channels. Certainly, the possibility that another channel or ion transporter is affected by cAMP should not be excluded.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
8-pCPT-2'-O-Me-cAMP and Rp-8-CPT-cAMPS were purchased from BIOLOG (Bremen, Germany), 2',5'-ddAdo and MDL12330A were obtained from Alexis (Lausen, Switzerland), and FK-506 was obtained from A.G. Scientific, Inc. (San Diego, CA); SQ22536, NKY80, CMI, H89, and PKI 14–22 amide, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, (R,S)-{alpha}-cyano-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, and cyclosporine were obtained from EMD Biosciences-Calbiochem (La Jolla, CA). All other compounds, including 2',5'-ddAdo-SATE, were from Sigma Chemical Co. (St. Louis, MO) if not otherwise specified.

Cell Cultures and Treatments
Experiments were performed on anterior pituitary cells from normal postpubertal female Sprague Dawley rats obtained from Taconic Farms (Germantown, NY) and GH3 immortalized pituitary cells [American Type Culture Collection (ATCC), Manassas, VA]. Pituitary cells were dispersed by trypsin procedure and cultured in medium 199 containing Earle’s salts, sodium bicarbonate, 10% heat-inactivated horse serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). A two-stage Percoll discontinuous density gradient procedure was used to obtain enriched lactotrophs, and further identification of lactotrophs in single-cell measurements was done by the addition of TRH (Peninsula Laboratories, Inc., Belmont, CA) at the beginning (in electrophysiological studies) or at the end of recording (Ca2+ imaging). GH3 immortalized pituitary cells were cultured in Ham’s F12K medium supplemented with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 15% heat-inactivating horse serum, 2.5% fetal bovine serum, and gentamicin (100 µg/ml).

PRL, cGMP, and cAMP Measurements
Anterior pituitary cells (1 million per well) were plated in 24-well plates in M199 containing serum and incubated overnight at 37 C under 5% CO2-air and saturated humidity. If not otherwise stated, medium was removed on the following day, after which cells were washed and then stimulated at 37 C under 5% CO2-air and saturated humidity for 3 h. PRL and cyclic nucleotides were measured in incubation medium and, in some experiments, in cell extracts. Hormone secretion and cyclic nucleotide release were also monitored using cell column perifusion experiments. Briefly, 1.5 x 107 cells were incubated with preswollen cytodex-1 beads in 60-mm Petri dishes for 20 h. The beads were then transferred to 0.5-ml chambers and perifused with Hanks’ M199 containing 25 mm HEPES, 0.1% BSA, and penicillin (100 U/ml)/streptomycin (100 µg/ml) for 2.5 h at a flow rate of 0.8 ml/min and at 37 C to establish stable basal secretion. Fractions were collected in 1-min intervals, stored at –20 C, and later assayed for PRL, cAMP, and cGMP content using RIA. Primary antibody and standard for PRL assay were purchased from the National Pituitary Agency and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA), and secondary antibody was purchased from Sigma. Cyclic nucleotides were determined using specific antisera provided by Albert Baukal (NICHD, Bethesda, MD). [125I]PRL, [125I]cAMP, and [125I]cGMP tracers were purchased from PerkinElmer Life Sciences (Boston, MA).

Single-Cell [Ca2+]i Measurements
For measurements of [Ca2+]i, cells were incubated in Hanks’ M199 and loaded with 2 µM fura-2 AM (Molecular Probes, Inc., Eugene, OR) at 37 C for 60 min. Coverslips with cells were then washed with Krebs-Ringer buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. The ratio of light intensities, F340/F380, which reflects changes in Ca2+ concentration, was followed in several single cells at the rate of one point per second.

Electrophysiological Recordings
Current-clamp recordings were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) and were low-pass filtered at 2 kHz. Membrane potential (Vm) was measured using the perforated patch recording technique. Briefly, nystatin (Sigma) stock solution (50 mg/ml) was prepared in dimethylsulfoxide and stored for up to 1 wk at –20 C. Just before use, nystatin and dispersing agent pluronic F-127 (Molecular Probes) were added to the intracellular solution from stock solution to obtain a final concentration of 250 µg/ml each. Patch electrodes were pulled from borosilicate glass tubes (1.5 mm outer diameter; World Precision Instruments, Sarasota, FL) using a Flaming Brown Horizontal Puller (P-87; Sutter Instruments, Novato, CA). Electrodes were heat polished to a final tip resistance of 3–6 M{Omega}. A series resistance of less than 25 M{Omega} was reached 10 min after the formation of a gigaohm seal (seal resistance > 5 G{Omega}) and remained stable for up to 1 h. Data acquisition and analysis were done with a PC equipped with a Digidata 1200 A/D interface in conjunction with Clampex 8 (Molecular Devices). The extracellular medium contained (in mM): 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.3 with NaOH. The pipette solution contained (in mM): 70 KCl, 70 K+-aspartate, 1 MgCl2, and 10 HEPES (pH adjusted to 7.2 with KOH). The bath contained less than 500 µl of saline and was continuously perifused at a rate of 2 ml/min. Applications of TRH, forskolin, and drugs, lasting for 2–60 sec, were performed using fast gravity-driven microperfusion system (BPS-8; ALA Scientific Instruments, Westbury, NY) containing eight glass tubes with a common outlet of approximately 400 µm in diameter. The application tip was routinely positioned at a distance of about 500 µm from the recorded cell and approximately 50 µm above the surface of the cultured cells.

RT-PCR Analysis of Adenylyl Cyclase Expression
Total RNA was extracted from rat pituitary or GH3 cell using TRIzol reagent (Invitrogen, Carlsbad, CA). Then, 5 µg RNA was digested with DNase I to eliminate genomic DNA contamination. The first-strand cDNA was synthesized with oligo(dT) primer using SuperScript III reverse-transcriptase (Invitrogen) and was used as the template for PCR. Primer sets were designed according to the nucleotide sequence of adenylyl cyclase deposited in GenBank. Sequences for sense and antisense primers, respectively, were as follows: AC3 (NM-130779), 5'-CCGCCCAGGTGGTAAAGAAGAGAA-3' and 5'-ATCCCGGTTGTCACGTTGTAAGGT-3'; AC5 (NM-022600), 5'-GCGGCGCAATGATGAACTGTACTA-3' and 5'-CATACGGCTGGCCACATTTACTG-3'; AC6 (NM-012821), 5'-CTCTTGGCCAGCTCCGTCTTCCTC-3' and 5'-CCGCTGTCATGGTTCTTGGAGTA-3'; AC9 (XM-220178), 5'-GCTGCTTCTGCTCTACATCTCGCTA-3' and 5'-CCGCTGTCATGGTTCTTGGAGTA-3'. The amplification was conducted in GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA) in a 25-µl reaction volume containing 0.5 µl of the first-strand cDNA as template, 0.5 µl of AccuPrime Taq DNA polymerase (Invitrogen), 0.5 µM of each primer, and 1x AccuPrime buffer I. Amplification of cDNA template was initiated by a denaturation step at 94 C for 3 min, followed by 30 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 58 C, and extension for 1 min at 68 C. To confirm the lack of genomic DNA contamination, RNA sample was also amplified under the same condition as the negative control. After PCR, a 10-µl aliquot of PCR products was size fractionated by electrophoresis in a 1.5% agarose gel and visualized by ethidium bromide staining.

Immunoprecipitation and Western Blot
The presence of AC3, AC5/6, and AC9 was confirmed by immunoprecipitation coupled with Western blot. Briefly, protein extracted from GH3 cells (2 x 107) or rat anterior pituitaries was incubated with 5 µl of rabbit polyclonal antibody against AC3 (ab14778, Abcam, Cambridge, MA), AC5/6 (SC-590; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or goat polyclonal antibody against AC9 (SC-8578, Santa Cruz Biotechnology) for 1 h at 4 C, followed by incubation with 50 µl of recombinant protein G agarose (Invitrogen) for 1 h at 4 C. The beads were then washed five times with RIPA buffer (1% Triton X-100, 50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, and 0.02% sodium azide) and boiled with 25 µl 2x protein sample buffer for 5 min. Eluted protein was fractionated in 6% SDS-PAGE and electrophoretically transferred to a polyvinylidine difluoride membrane (0.45-µm Immobilon transfer membrane; Millipore Corp., Bedford, MA) in XCell II Blot Module (Invitrogen). Immunoblotting was performed using the corresponding antibody as the primary antibody and horseradish peroxidase-conjugated rabbit antigoat IgG or goat antirabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) as the secondary antibody. Signals were detected using SuperSignal West Pico Luminol kit (Pierce Chemical Co., Rockford, IL) and exposed using x-ray film.

Calculations
Perifusion data, [Ca2+]i, and Vm oscillations are shown as representative traces of at least three independent experiments. Unless otherwise indicated, the static culture results shown are means ± SEM values of sextuplicate incubations in one of at least three similar experiments, each giving the same statistical conclusions. Significant differences with P < 0.05 were determined by one-way ANOVA with Neuman-Keuls multiple comparison test.


   ACKNOWLEDGMENTS
 
We thank Dr. Takayo Murano for help in PRL measurements.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

Present address for H.Z.: Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.

A.E.G.-I., Y.H., M.T., K.K., S.A.A., H.Z., and S.S.S. have nothing to declare.

First Published Online April 27, 2006

Abbreviations: AC, Adenylyl cyclase; 8-Br-cAMP, 8-bromo-cAMP; [Ca2+]i, intracellular calcium concentration; CMI, 4-cyano-3-methylisoquinoline; 2'5'-ddAdo, 2',5'-dideoxyadenosine; 2'5'-ddAdo-SATE, 2',5'-dideoxyadenosine-3'-O-bis(S-pivaloyl-2-thioethyl)-phosphate; H89, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, 2HCl; IBMX, 3-isobutyl-1-methylxanthine; MDL12330A, cis-N-(2-phenylcyclopenthyl)azacyclotridec-1-en-2-amine HCl; NKY80, 2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone; 8-pCPT-2'-O-Me-cAMP, 8-(4-chlorophenyltio)-2'-O-methyl-cAMP; PDE, phosphodiesterase; PKA, protein kinase A; PKI 14–22; protein kinase A inhibitor 14–22 amide myristoylated; PRL, prolactin; Rp-8-CPT-cAMPS, 8-(4-chlorophenylthio)cAMP thioate Rp-isomer; sGC, soluble guanylyl cyclase; SQ22536, 9-(tetrahydro-2'-furyl)adenine; VGCI, voltage-gated calcium influx; Vm, membrane potential.

Received for publication September 7, 2005. Accepted for publication April 18, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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