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• W2079085209 abstract "It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca2+ and activating Ca2+-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca2+signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that Gs-coupled receptors stimulatedde novo cGMP production. Furthermore, cholera toxin (an activator of Gs), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of Gs-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells. It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca2+ and activating Ca2+-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca2+signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that Gs-coupled receptors stimulatedde novo cGMP production. Furthermore, cholera toxin (an activator of Gs), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of Gs-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells. Soluble guanylyl cyclases (sGC) 1The abbreviations used are: sGCsoluble guanylyl cyclaseGPCRsG protein-coupled receptorsGHRHgrowth hormone-releasing hormoneCRFcorticotropin-releasing hormoneTRHthyrotropin-releasing hormoneETendothelinNOSnitric-oxide synthaseeNOSendothelial NOSnNOSneuronal NOSACadenylyl cyclasePDEsphosphodiesterasesIBMX3-isobutyl-1-methylxanthineCTXcholera toxinODQ1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-oneNS 20284H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one8-Br-cAMP8-bromo-cAMP catalyze the formation of cGMP in response to a wide variety of agents including hormones and neurotransmitters acting through G protein-coupled receptors (GPCRs). Stimulation of sGC is especially well established for GPCRs that signal through phospholipase C- and adenylyl cyclase (AC)-dependent pathways. It is generally accepted that calcium mediates the coupling of these receptors to sGC (1.Christopoulos A. El-Fakahany E.E. Life Sci. 1999; 65: 1-15Crossref PubMed Scopus (57) Google Scholar). Activation of the phospholipase C signaling pathway leads to inositol trisphosphate-induced Ca2+ mobilization accompanied by facilitation of voltage-sensitive and/or -insensitive Ca2+influx (2.Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6174) Google Scholar), whereas activation of the AC signaling pathway usually facilitates Ca2+ influx without affecting Ca2+mobilization (3.Stojilkovic S.S. Tomic M. Koshimizu T. Van Goor F. Conn P.M. Means A.R. Principles of Molecular Regulation. Humana Press Inc., Totowa, NJ2000: 149-185Google Scholar). Both cAMP and protein kinase A stimulate voltage-gated Ca2+ influx, the former through activation of cyclic nucleotide-gated channels (4.Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (422) Google Scholar) and the latter by activating nonselective cationic channels (5.Takano K. Takei T. Teramoto A. Yamashita N. Am. J. Physiol. 1996; 270: E1014-E1057Google Scholar, 6.Naumov A.P. Herrington J. Hille B. Pflugers Arch. 1994; 427: 414-421Crossref PubMed Scopus (52) Google Scholar). The rise in intracellular calcium concentration ([Ca2+]i) induced by these receptors is sufficient to stimulate two nitric-oxide (NO) synthase (NOS) enzymes, endothelial (eNOS) and neuronal (nNOS) (7.Xu X. Zeng W. Diaz J. Lau K.S. Gukovskaya A.C. Brown R.J. Pandol S.J. Muallem S. Cell Calcium. 1997; 22: 217-228Crossref PubMed Scopus (47) Google Scholar). Because sGC is activated by NO, either as an intracellular or plasma membrane permeable agonist (8.Lucas K.A. Pitary G.M. Kazerounian S. Ruiz-Stewart I. Park J. Schultz S. Chepenik K.P. Waldman S.A. Pharmacol. Rev. 2000; 52: 375-413PubMed Google Scholar), calcium-dependent NO production should result in stimulation of sGC. soluble guanylyl cyclase G protein-coupled receptors growth hormone-releasing hormone corticotropin-releasing hormone thyrotropin-releasing hormone endothelin nitric-oxide synthase endothelial NOS neuronal NOS adenylyl cyclase phosphodiesterases 3-isobutyl-1-methylxanthine cholera toxin 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one 8-bromo-cAMP Several lines of evidence support the operation of the NOS/sGC signaling system in the anterior pituitary. In normal and immortalized pituitary cells, calcium-sensitive nNOS and eNOS were detected (9.Xu X. Miller K.J. Biochem. Pharmacol. 2000; 59: 509-516Crossref PubMed Scopus (6) Google Scholar, 10.Lozach A. Garrel G. Lerrant Y. Berault A. Counis R. Mol. Cell. Endocrinol. 1998; 143: 43-51Crossref PubMed Scopus (45) Google Scholar, 11.Vancelecom H. Matthys P. Denef C. J. Histochem. Cytochem. 1997; 45: 847-857Crossref PubMed Scopus (55) Google Scholar, 12.Wolff D. Datto G.A. Biochem. J. 1992; 128: 201-206Crossref Scopus (59) Google Scholar, 13.Garrel G. Lerrant Y. Siriostis C. Berault A. Marge S. Bouchaud C. Counis R. Endocrinology. 1998; 139: 2163-2170Crossref PubMed Google Scholar, 14.Imai T. Hirata Y. Marumo F. Biomed. Res. 1992; 13: 371-374Crossref Scopus (14) Google Scholar, 15.Ceccatelli S. Hulting A.L. Zhang X. Gustafsson L. Villar M. Hokfelt T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11292-11296Crossref PubMed Scopus (272) Google Scholar). The activity of these enzymes was confirmed by measurements of NO, NO2, and NO3 under different experimental paradigms (16.Ohita K. Hirata Y. Imai T. Marumo F. J. Endocrinol. 1993; 138: 429-435Crossref PubMed Scopus (17) Google Scholar, 17.Tsumori M. Murakami Y. Koshimura K. Kato Y. J. Neuroendocrinol. 1999; 11: 451-456Crossref PubMed Scopus (11) Google Scholar). sGC is also expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH3-immortalized cells. This enzyme is exclusively responsible for cGMP production in unstimulated cells (18.Kostic T.S. Andric S.A. Stojilkovic S. Mol. Endocrinol. 2001; 15: 1010-1022Crossref PubMed Scopus (53) Google Scholar). Consistent with the Ca2+-calmodulin sensitivity of nNOS (19.Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar), agonists that increase [Ca2+]i, including the Ca2+-mobilizing thyrotropin-releasing hormone (TRH) and Ca2+ influx-dependent growth hormone-releasing hormone (GHRH), were found to modulate NO levels and hormone secretion (10.Lozach A. Garrel G. Lerrant Y. Berault A. Counis R. Mol. Cell. Endocrinol. 1998; 143: 43-51Crossref PubMed Scopus (45) Google Scholar, 17.Tsumori M. Murakami Y. Koshimura K. Kato Y. J. Neuroendocrinol. 1999; 11: 451-456Crossref PubMed Scopus (11) Google Scholar, 20.Kato M. Endocrinology. 1992; 131: 2133-2138Crossref PubMed Scopus (112) Google Scholar, 21.Vesely D.L. Mol. Cell. Biochem. 1985; 66: 145-149Crossref PubMed Scopus (6) Google Scholar, 22.Bugajski J. Borycz J. Gadek-Michalska A. Glod R. J. Physiol. Pharmacol. 1998; 49: 607-616PubMed Google Scholar, 23.Antoni F.A. Dayanithi G. Biochem. Biophys. Res. Commun. 1989; 158: 824-830Crossref PubMed Scopus (26) Google Scholar). Furthermore, basal sGC activity is partially dependent on spontaneous voltage-gated calcium influx (18.Kostic T.S. Andric S.A. Stojilkovic S. Mol. Endocrinol. 2001; 15: 1010-1022Crossref PubMed Scopus (53) Google Scholar). These findings support a generally accepted view about the relevance of calcium in activation of the NOS/sGC signaling pathway by GPCRs. Recent studies have also indicated that high [Ca2+]i inhibits cGMP accumulation, an action presumably mediated by direct inhibitory effects of [Ca2+]i on sGC activity (24.Andric S.A. Kostic T.S. Tomic M. Koshimizu T. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 844-849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 25.Parkinson S.J. Jovanovic A. Jovanovic S. Wagner F. Terzic A. Waldman S.A. Biochemistry. 1999; 38: 6441-6448Crossref PubMed Scopus (35) Google Scholar). Here we addressed the hypothesis that cAMP, in addition to [Ca2+]i, mediates the action of GPCRs on sGC-controlled cGMP production. Experiments were done in cultured pituitary cells and immortalized GH3 cells. We focused investigations on two receptors signaling through a Gs pathway, GHRH and corticotropin-releasing factor (CRF) receptors; two receptors signaling through a Gq/G11 pathway, TRH receptor (that also couples to a Gs signaling pathway) (26.Allgeier A. Offermanns V.S.J. Spicher K. Schultz G. Dumont J.E. J. Biol. Chem. 1994; 269: 13733-13735Abstract Full Text PDF PubMed Google Scholar) and the endothelin (ET)-A receptor that couples to a Gi/Go signaling pathway (27.Tomic M. Zivadinovic D. Van Goor F. Yuan D. Koshimizu T. Stojilkovic S.S. J. Neurosci. 1999; 19: 7721-7731Crossref PubMed Google Scholar, 28.Burris T.P. Kanyicska B. Freeman M.E. Eur. J. Pharmacol. 1991; 198: 223-225Crossref PubMed Scopus (19) Google Scholar); and the dopamine D2 receptor that signals through a Gi/Go pathway without activating phospholipase C (29.Freeman M.E. Kanyicska B. Lernat A. Nagy G. Physiol. Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1858) Google Scholar). GHRH receptors are expressed in somatotrophs and immortalized GH3 pituitary cells (30.Bluet-Pajot M.-T. Epelbaum J. Gourdji D. Hammond C. Kordon C. Cell. Mol. Neurobiol. 1998; 18: 101-123Crossref PubMed Scopus (113) Google Scholar). CRF receptors are expressed in corticotrophs (31.Abou-Samra A.-B. Harwood J.P. Manganiello V.C. Catt K.J. Aguilera G. J. Biol. Chem. 1987; 262: 1129-1136Abstract Full Text PDF PubMed Google Scholar). TRH receptors are expressed in thyrotrophs, lactotrophs, and GH3-immortalized cells (32.Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). D2 receptors are expressed in lactotrophs and somatotrophs (29.Freeman M.E. Kanyicska B. Lernat A. Nagy G. Physiol. Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1858) Google Scholar), whereas ETA receptors are expressed in all secretory cell types (33.Stojilkovic S.S. Balla T. Fukuda S. Cesnjaj M. Merelli F. Krsmanovic L.Z. Catt K.J. Endocrinology. 1992; 130: 465-474Crossref PubMed Google Scholar, 34.Kanyicska B. Freeman M.E. Am. J. Physiol. 1993; 265: E601-E608PubMed Google Scholar, 35.Samson W.K. Biochem. Biophys. Res. Commun. 1992; 187: 590-595Crossref PubMed Scopus (34) Google Scholar). Experiments were performed on anterior pituitary cells from normal female Sprague-Dawley rats obtained from Taconic Farms (Germantown, NY) and immortalized GH3 cells. Pituitary cells were dispersed as described previously (36.Koshimizu T. Tomic M. Wong A.O.L. Zivadinovic D. Stojilkovic S.S. Endocrinology. 2000; 141: 4091-4099Crossref PubMed Scopus (42) Google Scholar). Cells were cultured in medium 199 containing Earle's salts, sodium bicarbonate, 10% horse serum, and antibiotics. GH3 cells were cultured in F12K nutrient mixture containing 1.5 g/liter NaHCO3, 2.5% fetal bovine serum, and 15% horse serum. Cells were stimulated by GHRH, CRF, TRH (from Bachem, Torrance, CA), forskolin, and 8-Br-cAMP (from RBI, Natick, MA). Phosphodiesterases (PDEs) were inhibited by 3-isobutyl-1-methylxanthine (IBMX), dipyridamole, and zaprinast from Sigma-RBI. Two compounds, 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) from Sigma-RBI and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) from Sigma were used to inhibit sGC. Cells (1 × 106per well) were plated in 24-well plates in serum-containing M199 and incubated overnight at 37 °C under 5% CO2/air and saturated humidity. Experiments were done in cells 16 h after dispersion. Prior to the experiments, medium was removed, and cells were washed with serum-free M199 and stimulated at 37 °C under 5% CO2/air and saturated humidity. Cells were stimulated with GHRH, CRF, and TRH in serum-free medium and in the absence or presence of PDE inhibitors. At the end of the incubation period cells were dialyzed in an IBMX-containing medium, and cyclic nucleotide concentrations were determined by radioimmunoassay in medium and in dialyzed cells as previously described (24.Andric S.A. Kostic T.S. Tomic M. Koshimizu T. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 844-849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) using specific antisera provided by Albert Baukal, (NICHD, National Institutes of Health, Bethesda, MD). For [Ca2+]i measurements, cells were incubated in Krebs-Ringer buffer supplemented with 2 μmfura-2/AM (Molecular Probes, Eugene OR) at 37 °C for 60 min. Coverslips with cells were washed with this 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 ×40 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 simultaneously. cAMP and cGMP data are shown as total (cell content + medium) nucleotide levels. The results shown are means ± S.E. from sextuplicate determination in one of at least three similar experiments. Asterisks indicate a significant difference among means, estimated by Student's t test. In a mixed population of pituitary cells bathed in medium containing no PDE inhibitors, basal cGMP was about 1–2 pmol/well (Fig. 1 A, right panel). Addition of 1 mm IBMX, a nonselective inhibitor of PDEs, increased cGMP levels to about 10 pmol/well (Fig. 1 B,right panel) suggesting that PDEs participate in the control of basal cGMP levels in pituitary cells. As shown in Fig. 1 C, right panel, no further increase in cGMP levels was observed in cells bathed in medium containing 1 mm IBMX together with two cGMP-specific PDE inhibitors, dipyridamole (50 μm) and zaprinast (50 μm). Basal cAMP levels also increased in cells bathed in 1 mmIBMX-containing medium (Fig. 1, A versus B, left panels), whereas the addition of all three PDE inhibitors did not further elevate cAMP levels (Fig. 1 C, left panel). These results suggest that 1 mm IBMX is sufficient to silence PDEs in anterior pituitary cells. GHRH, CRF, and TRH induced significant elevations in cGMP levels comparable to basal levels. As shown in Fig. 1, right panels, all three agonists stimulated cGMP accumulation when PDEs were not silenced (A), but also in cells bathed in IBMX-containing medium (B), and medium containing a mixture of three inhibitors (C). Agonist-induced cGMP accumulation was also observed in cells treated with 50 μm zaprinast and 50 μm dipyridamole alone (Table I). GHRH-induced cGMP accumulation was observed in cells bathed in medium containing 10 μmvinpocetine, a specific PDE1 inhibitor (basal, 3.44 ± 0.04; 1 nm GHRH-treated, 5.04 ± 0.235 pmol/well;p < 0.05). GHRH, CRF, and TRH also induced a significant and PDE-independent increase in cAMP accumulation (Fig. 1,left panels and Table I). These data indicate that an agonist-induced rise in cGMP levels does not result from modulation of PDE activity but represents de novo cGMP production.Table IEffects of dipyridamole (50 μm) and zaprinast (50 μm) on agonist (100 nm)-induced cyclic nucleotide accumulation in anterior pituitary cells during a 60-min incubationAgonistsDipyridamoleZaprinastcAMPcGMPcAMPcGMPpmol/wellBasal3.28 ± 0.163.62 ± 0.380.98 ± 0.173.51 ± 0.49TRH8.28 ± 0.841-ap < 0.05 vs. basal.4.46 ± 0.131-ap < 0.05 vs. basal.2.18 ± 0.291-ap < 0.05 vs. basal.5.74 ± 0.051-ap < 0.05 vs. basal.CRF24.59 ± 3.961-ap < 0.05 vs. basal.6.23 ± 0.341-ap < 0.05 vs. basal.16.37 ± 2.841-ap < 0.05 vs. basal.6.73 ± 0.311-ap < 0.05 vs. basal.GHRH157 ± 6.491-ap < 0.05 vs. basal.8.36 ± 0.221-ap < 0.05 vs. basal.97.89 ± 4.291-ap < 0.05 vs. basal.8.63 ± 0.411-ap < 0.05 vs. basal.1-a p < 0.05 vs. basal. Open table in a new tab To identify which guanylyl cyclase, particulate or soluble, is responsible for agonist-induced cGMP production, cells were incubated in the presence of ODQ and NS 2028, two inhibitors of sGC (37.Garthwaite J. Southam E. Boulton C.L. Nielsen E.B. Schmidt K. Mayer B. Mol. Pharmacol. 1995; 48: 184-188PubMed Google Scholar, 38.Olesen S.-P. Drejer J. Axelsson O. Moldt P. Bang L. Nielsen-Kudsk J.E. Busse R. Mulsch A. Br. J. Pharmacol. 1998; 123: 299-309Crossref PubMed Scopus (101) Google Scholar). As shown in Fig. 2 A, left panel, ODQ significantly inhibited basal and GHRH-induced cGMP production in two doses, 0.1 μm and 1 μm, in cells bathed in medium without PDE inhibitors. NS 2028 also inhibited basal and GHRH-induced cGMP accumulation in a concentration-dependent manner with an estimated EC50 of about 100 nm when cells were bathed in 1 mm IBMX-containing medium (Fig. 2 B, left panel). None of these inhibitors affected basal and GHRH-induced cAMP production (right panels). NS 2028 also inhibited CRF and TRH-induced cGMP production when added in 1 μmconcentration (not shown). These results indicate that basal and agonist-induced cGMP production in pituitary cells is mediated by sGC. GHRH and CRF increased [Ca2+]i in cells bathed in 2 mm Ca2+-containing medium (Fig. 3, A and B,left traces) but not in 10 μmCa2+-containing medium (right traces), indicating that [Ca2+]i transients induced by these agonists were dependent on Ca2+ influx. TRH-induced Ca2+ response (Fig. 3 C, left trace) was not abolished by depletion of extracellular Ca2+ (not shown). However, when cells were bathed in Ca2+-deficient medium in the presence of 1 μmthapsigargin for 30 min prior to addition of TRH, no response in [Ca2+]i was observed (right trace) indicating that the Ca2+-signaling action of this agonist was abolished when the intracellular Ca2+ pool was depleted. In cells bathed in Ca2+-deficient medium, GHRH and CRF induced a significant increase in cGMP accumulation (Fig. 3,D and E, right panels). TRH was also able to stimulate cGMP production when bathed in Ca2+-deficient and thapsigargin-containing medium (Fig. 3 F, right panel). Agonist-induced cAMP accumulation was also not blocked by abolition of the Ca2+-signaling function of these receptors (Fig. 3, D–F, left panel). These results indicate that all three GPCRs can stimulate cGMP production and cAMP production in a [Ca2+]i-independent manner. When stimulated with 100 nm agonist concentrations, the amplitudes of cGMP responses were highest in GHRH-stimulated cells followed by CRF and TRH. The same order was observed in cells bathed in Ca2+-containing medium with and without PDE inhibitors (Fig. 1) and in cells bathed in Ca2+-deficient medium (Fig. 3). In all experimental conditions cGMP production paralleled cAMP production raising the possibility that cAMP, in addition to [Ca2+]i, mediates the action of these receptors on cGMP production. To further compare cAMP and cGMP production, cells bathed in 1 mm IBMX-containing medium were stimulated with increasing concentrations of GHRH, CRF, and TRH, and both nucleotides were measured from the same samples. Fig. 4 A illustrates the concentration-dependent effects of these agonists on cAMP production (left panel) and cGMP production (right panel). In all concentrations tested the Ca2+-mobilizing TRH was less potent stimulating cGMP production and cAMP production compared with CRF and GHRH. Correlation analysis of these data combined revealed the existence of a significant linear relationship between cAMP and cGMP production (Fig. 4 B). Further experiments were done with GH3-immortalized cells expressing both GHRH and TRH receptors. As in pituitary cultured cells, GHRH and TRH stimulated cAMP and cGMP production in a concentration-dependent manner (Fig. 4 C). There was also a parallel in cAMP and cGMP accumulation, as illustrated by linear regression in Fig. 4 D, derived from these two experiments. In contrast to primary culture, in GH3 cells GHRH- and TRH-induced cAMP and cGMP responses were almost comparable, suggesting that the cross-coupling of TRH receptors to Gsis more effective in these cells than in pituitary cells. Dissociation between Ca2+signaling and cGMP production and parallelism between cAMP and cGMP production suggest that coupling of GHRH, CRF, and TRH receptors to the Gs signaling pathway accounts for their actions on cGMP production. To test this hypothesis more directly, cells were treated with 10 ng/ml CTX, an activator of Gs, for variable times. During a 2-h incubation in the presence of 1 mm IBMX this treatment significantly increased cAMP production (Fig. 5 A, left panel) as well as cGMP production (Fig. 5 A, right panel). As in agonist-stimulated cells (Fig. 4), there was a linear relationship between cAMP and cGMP levels in untreated (Fig. 5 B, left panel) and CTX-treated cells (right panel). In cells treated with CTX for 12 h and 24 h without IBMX, then washed, and incubated for an additional 60 min in the presence of 1 mm IBMX, cAMP production was several fold higher than in controls (Fig. 5 C, left panel). In these cells cGMP production was also significantly elevated (right panel). The addition of 1 μmforskolin, an AC activator (Fig. 5 D, left panel), and 8-Br-cAMP, a permeable cAMP analog, also induced the time-dependent rise in cGMP production in cells bathed in 1 mm IBMX-contained medium (Fig. 5 D, right panel). Thus, the nonreceptor-mediated activation of the Gs-AC signaling pathway also leads to stimulation of sGC activity. To test the specificity of AC-dependent signaling in GPCR-controlled cGMP production, in further experiments we stimulated cells with ET-1, an agonist for ETA receptors and apomorphine, a specific agonist for D2 receptors. Both agonists abolished spontaneous [Ca2+]i transients in pituitary cells (Fig. 6, A andB). ET-1, but not apomorphine, also stimulated Ca2+ mobilization resulting in a rapid and transient [Ca2+]i response. In accord with literature data (27.Tomic M. Zivadinovic D. Van Goor F. Yuan D. Koshimizu T. Stojilkovic S.S. J. Neurosci. 1999; 19: 7721-7731Crossref PubMed Google Scholar, 39.Missale C. Nash R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2750) Google Scholar), ET-1 and apomorphine inhibited cAMP production in a concentration-dependent manner (Fig. 6 C), and this inhibition was accompanied by a decrease in cGMP production (Fig. 6 D). Consistent with the role of Ca2+ mobilization in activation of NOS/sGC (7.Xu X. Zeng W. Diaz J. Lau K.S. Gukovskaya A.C. Brown R.J. Pandol S.J. Muallem S. Cell Calcium. 1997; 22: 217-228Crossref PubMed Scopus (47) Google Scholar), inhibition of cGMP production was less prominent in ET-1 than apomorphine-stimulated cells. To test the hypothesis that protein kinase A mediates the action of cAMP on stimulation of sGC activity, cells were treated with 1 μm H89, a concentration that predominantly inhibits protein kinase A. Addition of H89 for 15 min prior to stimulation with agonists resulted in a significant reduction of receptor-induced cGMP production (Fig. 7 A). Forskolin and CTX-induced cGMP production was also reduced in the presence of 1 μm H89 (Fig. 7 B). On the other hand, cAMP levels measured in the same samples were not affected by H89 (Table II).Table IIcAMP levels in anterior pituitary cells treated with agonists (100 nm) for 60 min in 1 mm IBMX-containing medium in the absence and presence of 1 μm H89Agonists (100 nm)−H89+H89pmol/wellGHRH65.45 ± 1.3263.83 ± 2.81CRF54.55 ± 1.4559.18 ± 0.73TRH13.55 ± 0.8513.66 ± 0.29 Open table in a new tab It is generally accepted that GPCRs stimulate NO-sensitive sGC by increasing [Ca2+]i and activating Ca2+-dependent nNOS and/or eNOS (1.Christopoulos A. El-Fakahany E.E. Life Sci. 1999; 65: 1-15Crossref PubMed Scopus (57) Google Scholar, 18.Kostic T.S. Andric S.A. Stojilkovic S. Mol. Endocrinol. 2001; 15: 1010-1022Crossref PubMed Scopus (53) Google Scholar). In accordance with this hypothesis, earlier studies have indicated that sGC is expressed in pituitary cells as well as nNOS and eNOS, and that spontaneous electrical activity and calcium signaling provide an effective mechanism for activation of NOS and subsequent stimulation of sGC (18.Kostic T.S. Andric S.A. Stojilkovic S. Mol. Endocrinol. 2001; 15: 1010-1022Crossref PubMed Scopus (53) Google Scholar, 24.Andric S.A. Kostic T.S. Tomic M. Koshimizu T. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 844-849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Here we extended these investigations by studying the actions of several GPCRs on cGMP production. The focus in investigations was on receptors that stimulate and inhibit spontaneous, voltage-gated calcium influx-dependent [Ca2+]i transients, as well as on receptors that facilitate calcium release from intracellular Ca2+ pools. In cultured pituitary cells, ETA and D2receptors inhibited spontaneous [Ca2+]i transients and decreased cGMP production, whereas GHRH, CRF, and TRH elevated [Ca2+]i and stimulated cGMP production. Both ETA and D2 receptors signal through Gi/o pathways leading to inhibition of AC and stimulation of inward rectifier potassium current that hyperpolarizes cells, terminates spontaneous firing of action potentials, and abolishes voltage-gated Ca2+ influx (27.Tomic M. Zivadinovic D. Van Goor F. Yuan D. Koshimizu T. Stojilkovic S.S. J. Neurosci. 1999; 19: 7721-7731Crossref PubMed Google Scholar, 39.Missale C. Nash R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2750) Google Scholar). On the other hand, GHRH and CRF receptors stimulate AC activity through Gs (30.Bluet-Pajot M.-T. Epelbaum J. Gourdji D. Hammond C. Kordon C. Cell. Mol. Neurobiol. 1998; 18: 101-123Crossref PubMed Scopus (113) Google Scholar, 31.Abou-Samra A.-B. Harwood J.P. Manganiello V.C. Catt K.J. Aguilera G. J. Biol. Chem. 1987; 262: 1129-1136Abstract Full Text PDF PubMed Google Scholar). Somatotrophs respond to GHRH with a robust increase in cAMP production and stimulation of nonselective cationic channels, presumably through protein kinase A, depolarization of cells, and facilitation of voltage-gated Ca2+ influx (5.Takano K. Takei T. Teramoto A. Yamashita N. Am. J. Physiol. 1996; 270: E1014-E1057Google Scholar, 6.Naumov A.P. Herrington J. Hille B. Pflugers Arch. 1994; 427: 414-421Crossref PubMed Scopus (52) Google Scholar,40.Lussier B.T. French M.B. Moor B.C. Kraicer J. Endocrinology. 1991; 128: 592-603Crossref PubMed Scopus (79) Google Scholar, 41.Lussier B.T. French M.B. Moor B.C. Kraicer J. Endocrinology. 1991; 128: 570-582Crossref PubMed Scopus (74) Google Scholar, 42.Kwiecien R. Tseeb V. Kurchikov A. Kordon C. Hammond C. J. Physiol. 1997; 499: 613-623Crossref PubMed Scopus (32) Google Scholar). CRF receptors also increase excitability of corticotrophs and facilitate voltage-gated calcium influx (43.Guerineau N. Corcuff J.-B. Tabarin A. Mollard P. Endocrinology. 1991; 129: 409-420Crossref PubMed Scopus (89) Google Scholar, 44.Kuryshev Y.A. Childs G.V. Ritchie A.K. Endocrinology. 1995; 136: 3925-3935Crossref PubMed Scopus (51) Google Scholar). Finally, TRH receptor is a member of the Gq/11-coupled calcium-mobilizing receptors whose activation leads to inositol trisphosphate-induced Ca2+ release followed by facilitated calcium influx (32.Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). This receptor is also cross-coupled to the Gs-AC signaling pathway (26.Allgeier A. Offermanns V.S.J. Spicher K. Schultz G. Dumont J.E. J. Biol. Chem. 1994; 269: 13733-13735Abstract Full Text PDF PubMed Google Scholar), and protein kinases A and C pathways may play an important role in controlling the sustained voltage-gated Ca2+ influx by inhibiting spontaneously active inward-rectified potassium channels (3.Stojilkovic S.S. Tomic M. Koshimizu T. Van Goor F. Conn P.M. Means A.R. Principles of Molecular Regulation. Humana Press Inc., Totowa, NJ2000: 149-185Google Scholar). Thus, Ca2+signaling by these receptors is consistent with the [Ca2+]i dependence of sGC-derived cGMP production. Several lines of evidence presented here, however, indicate that Ca2+ is not an exclusive signaling pathway for stimulation of cGMP production by GPCRs. GHRH and CRF were found to stimulate sGC in cells bathed in Ca2+-deficient medium, a treatment that abolished agonist-induced Ca2+ influx. The Ca2+-mobilizing action of TRH, but not the agonist-induced cGMP accumulation, was abolished in cells with intracellular Ca2+ pools depleted by thapsigargin. In contrast to [Ca2+]i signals, cGMP production was found to correlate well with cAMP levels, suggesting that GPCRs stimulate the NOS/sGC signaling pathway through the cAMP-dependent mechanism. A decrease in cGMP production observed in cells stimulated with ET-1 and apomorphine also paralleled a decrease in cAMP production. The dependence of cGMP accumulation on cAMP levels was further documented in experiments with CTX, a treatment that elevated the levels of both nucleotides. To directly activate AC, we treated cells with forskolin. Furthermore, to exclude AC-independent actions of forskolin, cells were treated with 8-Br-cAMP, a permeable cAMP analog. In both experiments, a significant increase in cGMP production was observed confirming a hypothesis that cAMP mediates the action of GHRH, CRF, and TRH on cGMP production. Finally, receptor-, CTX-, and forskolin-induced stimulation of cGMP production, but not cAMP production, was reduced in the presence of H89. These results indicate that GPCRs can modulate sGC activity through the AC signaling pathway. The dependence of sGC activity on the protein kinase A signaling pathway is consistent with several reports indicating that phosphorylation of eNOS makes this enzyme active in the absence of [Ca2+]i signaling. The serine/threonine protein kinase Ark/PKB-induced phosphorylation of eNOS is well established (45.Fulton D. Gratton J.-P. McCabe T.J. Fontana J. Fujio Y. Wlsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 65-70Crossref Scopus (2232) Google Scholar, 46.Dimmeler S. Fleming I. Fissithaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar). One report also suggests that eNOS from endothelial cells is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases (47.Butt E. Bernhardt M. Smolenski A. Kotsonis P. Frohlich L.G. Sickmann A. Meyer H.E. Lohmann S.M. Schmidt H.H.H.W. J. Biol. Chem. 2000; 275: 5179-5187Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Earlier studies have also indicated that protein kinase C and protein kinase A can phosphorylate sGC in vitro, leading to the facilitation of enzyme activity (48.Zwiller J. Revel M.O. Malvia A.N. J. Biol. Chem. 1985; 260: 1350-1353Abstract Full Text PDF PubMed Google Scholar, 49.Zwiller J. Revel M.O. Basse P. Biochem. Biophys. Res. Commun. 1981; 101: 1383-1387Crossref Scopus (56) Google Scholar). In intact PC12 cells, phorbol ester-activated protein kinase C also phosphorylates sGC and increases the enzyme activity (50.Louis J.C. Revel M.O. Zwiller J. Biochim. Biophys. Acta. 1993; 1177: 299-306Crossref PubMed Scopus (51) Google Scholar). At the present time, our experiments cannot distinguish between the possible direct actions of protein kinase A on sGC from that mediated by NOS. However, it is unlikely that phosphorylation of eNOS accounts for GHRH-induced cGMP production in somatotrophs because these cells do not express eNOS, but do express nNOS (18.Kostic T.S. Andric S.A. Stojilkovic S. Mol. Endocrinol. 2001; 15: 1010-1022Crossref PubMed Scopus (53) Google Scholar), phosphorylation of which leads to a decrease in NO production (51.Bredt D.S. Ferris C.D. Snyder S.H. J. Biol. Chem. 1992; 267: 10976-10981Abstract Full Text PDF PubMed Google Scholar). A calcium-independent increase in cGMP levels observed in agonist-stimulated cells is also compatible with findings that this ion plays an important role in the control of PDE1 activity in a calmodulin-dependent manner. Phosphorylation of PDEs by protein kinases A and G also modulates the activity of these enzymes (52.Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1643) Google Scholar, 53.Conti M. Mol. Endocrinol. 2000; 14: 1317-1327Crossref PubMed Google Scholar, 54.Rybalkin S.D. Rybalkina I.G. Feil R. Hofmann F. Beavo J.A. J. Biol. Chem. 2002; 271: 3310-3317Abstract Full Text Full Text PDF Scopus (193) Google Scholar). Thus, calcium depletion/repletion in culture media in our experiments could influence the rate of cGMP degradation. However, that does not provide a rationale for agonist-induced cGMP accumulation, because it was also observed in cells bathed in medium containing 1 mm IBMX, an inhibitor that blocks PDE1 with an IC50 of 4 μm, as well as PDEs 2, 3, 4, 5, 6, 7, 10, and 11, with IC50 values of 2–100 μm(52.Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1643) Google Scholar). Agonist-induced cGMP accumulation was also observed in cells bathed in the presence of vinpocetine, a specific PDE1 inhibitor, as well as in the presence of specific inhibitors of cGMP-dependent PDEs, dipyridamole, and zaprinast (52.Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1643) Google Scholar). The ability of GHRH, CRF, and TRH to stimulate cGMP accumulation in cells bathed in medium containing a mixture of three PDE inhibitors in high concentrations further argues against the hypothesis that protein kinase A-dependent phosphorylation of PDEs, specifically PDE1A, accounts for down-regulation of their activities and elevation in cGMP levels. In conclusion, here we show that GHRH, CRF, and TRH receptors in pituitary cells stimulate sGC activity and that this stimulation also occurs when Ca2+ signaling is abolished. The experiments argue against the action of these receptors on PDE activity and support the hypothesis that de novo cGMP production accounts for a Ca2+-independent increase in cGMP levels. Our results further indicate that the coupling of GHRH and CRF receptors and cross-coupling of the TRH receptor to the Gs signaling pathway represents the main mechanism for stimulation of sGC, whereas the coupling of receptors to the Gi/o signaling pathway leads to inhibition of cGMP production. The results also indicate that Gs-mediated activation of AC and subsequently protein kinase A is required for stimulation of sGC activity. Thus, in addition to [Ca2+]i, up- and down-regulation of AC activity by GPCRs provides an effective mechanism for control of cGMP production." @default.
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• W2079085209 title "Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells" @default.
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• W2079085209 cites W1558298279 @default.
• W2079085209 cites W1561753405 @default.
• W2079085209 cites W1585195212 @default.
• W2079085209 cites W1602807836 @default.
• W2079085209 cites W1607892649 @default.
• W2079085209 cites W1617916547 @default.
• W2079085209 cites W1678800844 @default.
• W2079085209 cites W1925492557 @default.
• W2079085209 cites W1966710180 @default.
• W2079085209 cites W1974790027 @default.
• W2079085209 cites W1980166477 @default.
• W2079085209 cites W1987658927 @default.
• W2079085209 cites W1990435889 @default.
• W2079085209 cites W1991726045 @default.
• W2079085209 cites W1993132699 @default.
• W2079085209 cites W1994898724 @default.
• W2079085209 cites W1995298150 @default.
• W2079085209 cites W2004111202 @default.
• W2079085209 cites W2021549266 @default.
• W2079085209 cites W2022177968 @default.
• W2079085209 cites W2023678585 @default.
• W2079085209 cites W2031295871 @default.
• W2079085209 cites W2041816586 @default.
• W2079085209 cites W2042306678 @default.
• W2079085209 cites W2045519311 @default.
• W2079085209 cites W2045911091 @default.
• W2079085209 cites W2052146020 @default.
• W2079085209 cites W2056679265 @default.
• W2079085209 cites W2063180158 @default.
• W2079085209 cites W2065335664 @default.
• W2079085209 cites W2066058717 @default.
• W2079085209 cites W2067207760 @default.
• W2079085209 cites W2071467078 @default.
• W2079085209 cites W2073139237 @default.
• W2079085209 cites W2074943467 @default.
• W2079085209 cites W2075010505 @default.
• W2079085209 cites W2077322289 @default.
• W2079085209 cites W2084596944 @default.
• W2079085209 cites W2113103296 @default.
• W2079085209 cites W2166389885 @default.
• W2079085209 cites W2167689408 @default.
• W2079085209 cites W2173833672 @default.
• W2079085209 cites W2203609464 @default.
• W2079085209 cites W2437834979 @default.
• W2079085209 cites W89912228 @default.
• W2079085209 cites W2102984773 @default.
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