Endothelin-induced, Long Lasting, and Ca2+ Influx-independent Blockade of Intrinsic Secretion in Pituitary Cells by Gz Subunits
2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês
10.1074/jbc.m502226200
ISSN1083-351X
AutoresSilvana A. Andrić, Dragoslava Živadinović, Arturo E. González-Iglesias, Agnieszka Lachowicz, Melanija Tomić, Stanko S. Stojilković,
Tópico(s)Cellular transport and secretion
ResumoThe G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the Gi/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to Gi/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity. The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the Gi/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to Gi/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity. Calcium is the primary intracellular signaling molecule controlling the fusion of secretory vesicles with the plasma membrane to release transmitters from neurons and hormones from endocrine cells (1Martin T.F. Biochim. Biophys. Acta. 2003; 1641: 157-165Crossref PubMed Scopus (94) Google Scholar). This process is termed regulated exocytosis and is mediated by complex protein machinery that is conserved in organisms ranging from yeast to mammals. These proteins participate in docking, ATP-dependent priming, and fusion of vesicle membranes through interactions that are still not fully characterized (2Rettig J. Neher E. Science. 2002; 298: 781-785Crossref PubMed Scopus (275) Google Scholar, 3An S.J. Almers W. Science. 2004; 306: 1042-1046Crossref PubMed Scopus (114) Google Scholar, 4Di Paolo G. Moskowitz H.S. Gipson K. Wenk M.R. Voronov S. Obayashi M. Flavell R. Fitzsimonds R.M. Ryan T.A. De Camilli P. Nature. 2004; 431: 415-422Crossref PubMed Scopus (303) Google Scholar). In regulated exocytosis, an increase in intracellular calcium concentration ([Ca2+]i) is required for two steps: priming the secretory vesicles, which occurs on the time scale of tens of seconds; and triggering the fusion, which occurs within 1–2 s (1Martin T.F. Biochim. Biophys. Acta. 2003; 1641: 157-165Crossref PubMed Scopus (94) Google Scholar). In excitable cells, both the depolarization-driven Ca2+ entry (5Felmy F. Neher E. Schneggenburger R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15200-15205Crossref PubMed Scopus (43) Google Scholar) and the agonist-induced Ca2+ mobilization from intracellular stores (6Tse A. Tse F.W. Almers W. Hille B. Science. 1993; 260: 82-84Crossref PubMed Scopus (218) Google Scholar) can trigger the fusion of primed secretory vesicles. The intracellular messenger cascades involving G proteins (7Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (514) Google Scholar) have prominent effects on secretion, predominantly by modulating voltage-gated Ca2+ influx (VGCI) 1The abbreviations used are: VGCI, voltage-gated Ca2+ influx; PRL, prolactin; ET, endothelin; PTX, pertussis toxin; PMA, phorbol 12-myristate 13-acetate. and Ca2+ mobilization (8Stojilkovic S.S. Catt K.J. J. Neuroendocrinol. 1995; 7: 739-757Crossref PubMed Scopus (85) Google Scholar, 9Ashworth R. Hinkle P.M. Endocrinology. 1996; 137: 5205-5212Crossref PubMed Scopus (33) Google Scholar, 10Bluet-Pajot M.T. Epelbaum J. Gourdji D. Hammond C. Kordon C. Cell. Mol. Neurobiol. 1998; 18: 101-123Crossref PubMed Scopus (113) Google Scholar, 11Freeman M.E. Kanyicska B. Lerant A. Nagy G. Physiol. Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1858) Google Scholar, 12Ben-Jonathan N. Hnasko R. Endocr. Rev. 2001; 22: 724-763Crossref PubMed Scopus (606) Google Scholar). Other intracellular messengers triggered by G protein-coupled receptors, including cAMP/protein kinase A (13Sakaba T. Neher E. Nature. 2003; 424: 775-778Crossref PubMed Scopus (197) Google Scholar, 14Nagy G. Reim K. Matti U. Brose N. Binz T. Rettig J. Neher E. Sorensen J.B. Neuron. 2004; 41: 417-429Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), diacylglycerol/protein kinase C (15Gillis K.D. Mossner R. Neher E. Neuron. 1996; 16: 1209-1220Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 16Yang Y. Udayasankar S. Dunning J. Chen P. Gillis K.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17060-17065Crossref PubMed Scopus (75) Google Scholar, 17Zhu H. Hille B. Xu T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17055-17059Crossref PubMed Scopus (54) Google Scholar), and phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3-kinase (4Di Paolo G. Moskowitz H.S. Gipson K. Wenk M.R. Voronov S. Obayashi M. Flavell R. Fitzsimonds R.M. Ryan T.A. De Camilli P. Nature. 2004; 431: 415-422Crossref PubMed Scopus (303) Google Scholar, 18Grishanin R.N. Kowalchyk J.A. Klenchin V.A. Ann K. Earles C.A. Chapman E.R. Gerona R.R. Martin T.F. Neuron. 2004; 43: 551-562Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), can influence Ca2+-triggered secretion by regulating the size of the releasable secretory pool and the rate of exocytosis. Furthermore, it has been suggested that G proteins may inhibit synaptic transmission in neuromuscular junction downstream of Ca2+ entry mechanisms (19Silinsky E.M. J. Physiol. 1984; 346: 243-256Crossref PubMed Scopus (155) Google Scholar, 20Silinsky E.M. Solsona C.S. J. Physiol. 1992; 457: 315-328Crossref PubMed Scopus (61) Google Scholar). It appears that the Ca2+-independent inhibition of neurotransmission occurs through Gβγ subunits and their binding partners, syntaxin 1B and SNAP25B (21Blackmer T. Larsen E.C. Takahashi M. Martin T.F. Alford S. Hamm H.E. Science. 2001; 292: 293-297Crossref PubMed Scopus (209) Google Scholar). At the present time, however, the identity of G proteins and role of Gα subunits associated with Gβγ subunits responsible for such inhibition are unknown. The potential relevance of G subunits in inhibiting the fusion of dense core vesicles and the duration of such a blockade during the sustained firing of action potentials have also not been clarified. Here we studied the relevance of G proteins in controlling the VGCI-triggered exocytosis of dense core vesicles in spontaneously firing pituitary lactotrophs. In these cells, the action potential-dependent fluctuations in [Ca2+]i account for high basal prolactin (PRL) release (22Van Goor F. Zivadinovic D. Martinez-Fuentes A.J. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 33840-33846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Such an intrinsic PRL release is controlled in vivo by three types of Gi/o protein-coupled receptors: dopamine, somatostatin, and endothelin (ET) (11Freeman M.E. Kanyicska B. Lerant A. Nagy G. Physiol. Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1858) Google Scholar). Parallelism in the actions of ET-1 in a nanomolar concentration range on Ca2+ signaling and secretion in pituitary lactotrophs (23Lachowicz A. Van Goor F. Katzur A.C. Bonhomme G. Stojilkovic S.S. J. Biol. Chem. 1997; 272: 28308-28314Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and somatotrophs (24Tomic M. Zivadinovic D. Van Goor F. Yuan D. Koshimizu T. Stojilkovic S.S. J. Neurosci. 1999; 19: 7721-7731Crossref PubMed Google Scholar) suggests that the rate of exocytosis in these cells reflects the pattern of Ca2+ signaling. Our present results, however, indicate that ET receptors can inhibit PRL release independently of the status of Ca2+ signaling. We also present evidence that ET-1 desensitizes Ca2+ secretion coupling through Gz signaling pathway. Cell Cultures—Experiments were performed on anterior pituitary cells from normal postpubertal female Sprague-Dawley rats obtained from Taconic Farm (Germantown, NY). Pituitary cells were dispersed and cultured as mixed cells or enriched lactotrophs in medium 199 containing Earle's salts, sodium bicarbonate, 10% heat-inactivated horse serum, penicillin (100 units/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 studies was done by the addition of thyrotropin-releasing hormone (25Koshimizu T.A. Tomic M. Wong A.O. Zivadinovic D. Stojilkovic S.S. Endocrinology. 2000; 141: 4091-4099Crossref PubMed Scopus (42) Google Scholar). PRL and cAMP Measurements—Cells (1 million/well) were plated in 24-well plates in serum-containing M199 and incubated overnight at 37 °C under 5% CO2-air and saturated humidity. Prior to the experiments, cells were washed with serum-free medium and stimulated at 37 °C under 5% CO2-air and saturated humidity for 120 min if not otherwise stated. Hormone secretion was also monitored using cell column perifusion experiments. Briefly, 1.2 × 107 cells were incubated with preswollen Cytodex-1 beads in 60-mm Petri dishes for 24 h. The beads were then transferred to 0.5-ml chambers and perifused with Hanks' M199 containing 25 mm HEPES, 0.1% bovine serum albumin, and penicillin (100 units/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 at 1-min intervals, stored at –20 °C, and later assayed for PRL and cAMP contents using radioimmunoassay. Primary antibody and standard for PRL assay were provided by the National Pituitary Agency and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA); 125I-labeled PRL was purchased from PerkinElmer Life Sciences and secondary antibody from Sigma. Cyclic AMP was determined in both media and cell contents using specific antiserum provided by Albert Baukal (NICHD, National Institutes of Health, Bethesda, MD). Single Cell Calcium Measurements—For [Ca2+]i measurements, cells were incubated in Hanks' M199 supplemented with 2 μm fura-2 AM (Molecular Probes, Eugene OR) at 37 °C for 60 min. Coverslips with cells were then washed 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 at the rate of 1 point/s. The [Ca2+]i was calibrated as described previously (22Van Goor F. Zivadinovic D. Martinez-Fuentes A.J. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 33840-33846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Western Blot Analysis—Crude membranes were prepared as described previously (26Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Crossref PubMed Google Scholar). Protein concentration was estimated by the Bradford method with bovine serum albumin as a standard (Pierce). Equal amounts of proteins were run on one-dimensional SDS-PAGE using a discontinuous buffer system (Novex), and proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA) with a wet transfer and following the manufacturer's recommendation. The immunodetection of G proteins was done with Gz, Gs (Santa Cruz Biotechnology, Santa Cruz, CA), Gq, Gi1–Gi3, Go, G12, and G13 (Calbiochem) α-protein-specific antibodies. β-Actin was detected using monoclonal antibody produced by Oncogene Research Products (San Diego, CA). The secondary antibodies were a goat anti-rabbit IgG or anti-mouse IgG-IgM (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Secondary antibodies were linked to horseradish peroxidase. The reactive bands were always determined with a luminol-based kit (Pierce), and the reaction was detected by an enhanced chemiluminescence system using x-ray film. Gz Antisense Experiments—The oligodeoxynucleotides were from Invitrogen. The sequence of Gzα antisense oligodeoxynucleotide was 5′-CGTGATCTCACCCTTGCTCTCTGCCGGGCT-3′, whereas the sequence of the missense oligodeoxynucleotide was 5′-CCCTTATTTACTTTCGCC-3′ (27Serres F. Li Q. Garcia F. Raap D.K. Battaglia G. Muma N.A. Van de Kar L.D. J. Neurosci. 2000; 20: 3095-3103Crossref PubMed Google Scholar). Both sequences were phosphorothioate-modified only at positions 5′-CC and GC-3′ (28Sanchez-Blazquez P. Garcia-Espana A. Garzon J. J. Pharmacol. Exp. Ther. 1995; 275: 1590-1596PubMed Google Scholar). Oligodeoxynucleotides were dissolved in 1× TE buffer, containing 10 mm Tris, pH 8.0, and 0.1 mm EDTA, according to the recommendation of the producer. A purified lactotroph fraction of cells were plated in 24-well plates (106 cells/ml/well) and cultured for 6 h in culture medium enriched with 10% horse serum. Then the medium was replaced, and 0.25 nmol of antisense or missense oligodeoxynucleotides in 2 μl of TE buffer/well was applied every hour during the following 36-h time period, whereas the cells in control wells received an equal volume of TE buffer alone. After that medium was changed, the procedure was repeated for an additional 36 h. Calculations—The time course of PRL release was fitted to a single exponential function (ae–kt + b) using GraphPad Prism (GraphPad Software, Inc., San Diego, CA) to generate the rates of signaling desensitization (k). Concentration-response relationships were fitted to a four-parameter logistic equation using a nonlinear curve-fitting program, which derives 50% efficient concentrations (EC50) and 50% inhibitory concentrations (IC50) (Kaleidagraph, Synergy Software Technologies, Reading, PA). In the figures, the results shown are means ± S.E. from sextuplicate determination in one of at least three similar experiments, and asterisks indicate a significant difference (p < 0.01) among means, estimated by Student's t test. ET Inhibits Extracellular Ca2+-dependent Basal PRL Release—Lactotrophs exhibit spontaneous firing of action potentials; the associated Ca2+ transients are sufficient to maintain PRL release at high and steady levels in cells in population for many hours (22Van Goor F. Zivadinovic D. Martinez-Fuentes A.J. Stojilkovic S.S. J. Biol. Chem. 2001; 276: 33840-33846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Such secretion is termed intrinsic, spontaneous, or basal (12Ben-Jonathan N. Hnasko R. Endocr. Rev. 2001; 22: 724-763Crossref PubMed Scopus (606) Google Scholar). Fig. 1A, left, illustrates the extracellular Ca2+ dependence of basal PRL release in pituitary cells perifused at a flow rate of 0.8 ml/min. Perifusion chambers containing 12 × 106 cells (with an estimated number of lactotrophs of 2 × 106) released between 30 and 90 ng of PRL/min (mean value from 40 experiments = 55.2 ± 4.4 ng/min), corresponding to ∼15–45 fg/min PRL released per cell. Depletion of extracellular Ca2+ induced a decrease in PRL release to 6.3 ± 0.8 ng/ml (n = 8), and the reconstitution of Ca2+ led to a full recovery of secretion with a transient overshot (Fig. 1A). There was also a progressive accumulation of PRL in the medium during a 16-h incubation of cells in static cultures (Fig. 1B). The estimated rate of release per cell ranged between 20 and 40 fg/min. Basal PRL secretion was not immediately affected by the inhibition of de novo PRL synthesis, as documented in experiments with the application of brefeldin and cycloheximide (Fig. 1C). Spontaneous PRL release may be enhanced by the Gq/11-coupled thyrotropin-releasing hormone and angiotensin II receptors (9Ashworth R. Hinkle P.M. Endocrinology. 1996; 137: 5205-5212Crossref PubMed Scopus (33) Google Scholar, 23Lachowicz A. Van Goor F. Katzur A.C. Bonhomme G. Stojilkovic S.S. J. Biol. Chem. 1997; 272: 28308-28314Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and inhibited by Gi/o-coupled dopamine and somatostatin receptors (11Freeman M.E. Kanyicska B. Lerant A. Nagy G. Physiol. Rev. 2000; 80: 1523-1631Crossref PubMed Scopus (1858) Google Scholar). G protein-coupled ET receptors (29Kedzierski R.M. Yanagisawa M. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 851-876Crossref PubMed Scopus (624) Google Scholar) are also present in pituitary cells and participate in the control of secretion (30Lange M. Pagotto U. Renner U. Arzberger T. Oeckler R. Stalla G.K. Exp. Clin. Endocrinol. Diabetes. 2002; 110: 103-112Crossref PubMed Scopus (13) Google Scholar). Several reports suggest that the ET-A subtype of these receptors is expressed in anterior pituitary cells (24Tomic M. Zivadinovic D. Van Goor F. Yuan D. 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Perifusion experiments revealed the presence of two phases in the action of ET-1 on PRL release: a rapid and transient increase in PRL release and a sustained decrease in secretion below the basal levels (Fig. 1A, center; hereafter the bidirectional response). As in cells perifused with Ca2+-deficient medium, PRL secretion dropped to 5.9 ± 0.7 ng/ml (n = 36) during the sustained ET-1 application, clearly showing that this agonist blocks Ca2+ influx-dependent PRL release. In cells perifused with Ca2+-deficient medium, ET induced only monophasic response (Fig. 1A, right). In cells in static cultures, the application of 100 nm ET-1 induced a long lasting inhibition of PRL release, whereas the stimulatory effect was not visible (Fig. 1B). The ET-induced decrease in PRL release was not related to the status of PRL synthesis or the depletion of the secretory pool (Fig. 1C). Our previous work (23Lachowicz A. Van Goor F. Katzur A.C. Bonhomme G. Stojilkovic S.S. J. Biol. Chem. 1997; 272: 28308-28314Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) shows the parallelism in the actions of 100 nm ET-1 on [Ca2+]i and PRL secretion. Fig. 1D illustrates such an example. The Ca2+ trace shown represents the mean value from 15 single lactotrophs, and the secretory profile was generated from 5 perifusion experiments. Both the extracellular Ca2+ dependence of PRL release (Fig. 1A) and the bidirectional effects of ET-1 on Ca2+ and PRL release (Fig. 1D) are consistent with the hypothesis that the rate of PRL release in these cells reflects changes in [Ca2+]i. ET Inhibits Basal PRL Release Downstream of VGCI—Single cell Ca2+ analysis showed that the pattern of ET-1-induced Ca2+ response varies among cells. In the majority of spontaneously active lactotrophs, [Ca2+]i fluctuated between 0.7 and 1.5 values when expressed as F340/F380 with a mean value of 0.94 ± 0.08 (n = 21), which corresponds to about 350 nm. 100 nm ET-1 induced an initial spike increase in [Ca2+]i and a sustained decrease in [Ca2+]i below the resting level (Fig. 2A, center). The mean value of [Ca2+]i during the sustained ET application was 0.63 ± 0.06 (n = 35), which corresponded to about 100 nm. Depletion of extracellular Ca2+ also decreased [Ca2+]i to comparable levels (F340/F380 = 0.62 ± 0.05; n = 21). In some cells, only the second phase was observed (Fig. 2A, right). In silent cells, the mean F340/F380 value of [Ca2+]i (0.67 ± 0.05, n = 14) was highly comparable with that observed in ET-stimulated cells during the sustained application. In such cells, ET induced a monophasic stimulatory response (Fig. 2A, left). The transient spike phase was observed upon ET-1 stimulation with concentrations of 1 nm or higher but with variable occurrences: 71% (24 of 34) of the cells responded to 100 nm, 28% (9 of 32) of the cells responded to 10 nm, and 9% (3 of 34) of the cells responded to 1 nm. The spike response was not observed in any cells stimulated with 0.1 nm (Fig. 2B) and lower ET-1 concentrations. The inhibition of spontaneous VGCI also occurred in the 1–100 nm ET-1 concentration range. For example, Ca2+ transients were stopped in 56% (19 of 34) of the cells stimulated with 100 nm ET-1, in 25% (8 of 32) of the cells stimulated with 10 nm ET-1, and in none of the cells stimulated with 0.1 nm (Fig. 2B) and lower concentrations. The lack of effect of picomolar concentrations of ET on [Ca2+]i was independent of the duration of agonist application, as no changes in the pattern of signals in spontaneously active cells were observed during a 30-min incubation (mean value of [Ca2+]i, 0.93 ± 0.07 or about 350 nm, n = 17). The roughly estimated EC50 and IC50 values were on the order of 10 nm in magnitude. In contrast to Ca2+, ET-1 inhibited PRL release in static cultures with a calculated IC50 of 9 pm (Fig. 2C), a thousand-fold leftward shift in the potency of ET-1 to stop spontaneous Ca2+ transients. In perifused pituitary cells, ET-1 was also able to inhibit PRL release when added in 0.1 and 0.01 nm concentrations, whereas the spike PRL response was observed only if the concentration of ET-1 exceeded 0.1 nm (Fig. 2D), with a calculated EC50 of 13 nm (Fig. 2E). The detailed kinetic analysis of PRL secretion (Fig. 2F) revealed that the rate of inhibition increased with the elevation of agonist concentration (Fig. 2G, left). On the other hand, the plateau levels of inhibition after 20 min of stimulation were comparable in a 1 pm to 1 μm concentration range of ET-1 (Fig. 2G, right), amounting to 6.2 ± 0.7 ng/ml (n = 12) during the sustained application of 10 nm and, when stimulated with 100 pm, to 6.8 ± 0.9 ng/ml (n = 6). In cells perifused with ET-1 for longer than 30 min, there was a gradual recovery in PRL release, and the rates of this recovery inversely correlated with agonist concentrations (data not shown). These studies indicate that in the picomolar concentration range ET-induced blockade of PRL secretion is independent of the VGCI status. ET Inhibits PRL Release in a Phospholipase C- and Tyrosine Kinase-independent Manner—Pituitary cells express the full repertoire of Gα proteins (Fig. 3A). In contrast to other cell types (26Krsmanovic L.Z. Mores N. Navarro C.E. Saeed S.A. Arora K.K. Catt K.J. Endocrinology. 1998; 139: 4037-4043Crossref PubMed Google Scholar, 40Zou Y. Akazawa H. Qin Y. Sano M. Takano H. Minamino T. Makita N. Iwanaga K. Zhu W. Kudoh S. Toko H. Tamura K. Kihara M. Nagai T. Fukamizu A. Umemura S. Iiri T. Fujita T. Komuro I. Nat. Cell Biol. 2004; 6: 499-506Crossref PubMed Scopus (527) Google Scholar), the application of agonist in pituitary cells did not decrease the plasma membrane localization of any of the α subunits, indicating that this method cannot be used to identify the G proteins activated by ET receptors (Fig. 3A). The agonist-induced blockade of PRL release was not affected in cells in which tyrosine kinases were inhibited by genistein (Fig. 3B). Previous studies showed the coupling of pituitary ET receptors to Gq/11 and activation of phospholipase C, leading to an increase in inositol 1,4,5-triphosphate production and subsequently to Ca2+ mobilization from intracellular stores (23Lachowicz A. Van Goor F. Katzur A.C. Bonhomme G. Stojilkovic S.S. J. Biol. Chem. 1997; 272: 28308-28314Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 41Zheng L. Paik W.Y. Cesnjaj M. Balla T. Tomic M. Catt K.J. Stojilkovic S.S. Endocrinology. 1995; 136: 1079-1088Crossref PubMed Scopus (44) Google Scholar). Consistent with these findings, in cells treated with U73122, a phospholipase C inhibitor, the spike phase of ET-induced PRL release was reduced, but the inhibitory phase was not affected (Fig. 3C). Furthermore, the ET-induced bidirectional pattern of PRL release was not changed in cells with protein kinase C inhibited by bisindolylmaleimide (Fig. 3D). Thus, the coupling of ET receptors to the phospholipase C signaling pathway is not responsible for sustained inhibition of basal PRL release. Coupling of ET Receptors to Gi/o Proteins—Experiments with the pertussis toxin (PTX) indicated that the coupling of ET receptors to Gi/o proteins provides an effective mechanism for the inhibition of spontaneous VGCI by activating inwardly rectifying K+ channels (39Tomic M. Van Goor F. He M.L. Zivadinovic D. Stojilkovic S.S. Mol. Pharmacol. 2002; 61: 1329-1339Crossref PubMed Scopus (17) Google Scholar). As shown in Fig. 4A, the bidirectional response of [Ca2+]i typically observed in control cells during the application of ET-1 (top left) was replaced with a biphasic response in cells treated overnight with PTX, which was composed of an early spike phase and a sustained plateau phase of elevated [Ca2+]i (top right). In contrast to Ca2+ signaling, the bidirectional pattern of ET-induced PRL secretion was not affected by PTX treatment, and the levels of sustained secretion inhibition were comparable between controls and PTX-treated cells (Fig. 4A, bottom). The inhibition of PRL release by ET-1 was also observed in PTX-treated static cultures of pituitary cells (Fig. 4B, bottom). Furthermore, the ET-1 was unable to decrease [Ca2+]i in high K+-depolarized cells but decreased PRL release to the levels observed in control cells (Fig. 4, C and D). Thus, the ET-induced blockade of PRL secretion is independent of the VGCI status, and the G proteins other than Gi/o and Gq/11 play a role in stopping PRL release at elevated [Ca2+]i. Coupling of ET Receptors to Gz Proteins—Interestingly, the coupling of pituitary ET receptors to the Gi/o signaling pathway did not account for the attenuation of adenylyl cyclase activity. In control cells in a static culture, ET-1 decreased cAMP production in a dose-dependent manner with an IC50 of ∼10 pm (Fig. 4B, top left) comparable with that observed for the inhibition of PRL release in the same experiment (bottom left). In cells treated overnight with PTX, the ET-induced attenuation of adenylyl cyclase activity (Fig. 4B, top right), as well as its blockade on PRL release (bottom right), was maintained. The lack of effect of PTX-treatment on ET-induced inhibition of cAMP production is consistent with published findings in other cell types, indicating a role of PTX-insensitive Gzα in control of adenylyl cyclase activity (42Wong Y.H. Conklin B.R. Bourne H.R. Science. 1992; 255: 339-342Crossref PubMed Scopus (232) Google Scholar). To prevent the coupling of ET receptors to Gz-dependent signaling pathways, we used two experimental approaches. In the first series of experiments, cells were stimulated with phorbol esters, which should silence the Gz signaling pathway through the protein kinase C-dependent phosphorylation of the Gzα subunits (43Fields T.A. Casey P.J. J. Biol. Chem. 1995; 270: 23119-23125Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 44Wang J. Frost J.A. Cobb M.H. Ross E.M. J. Biol. Chem. 1999; 274: 31641-31647Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Pituitary cells in static cultures were treated with 100 nm ET-1 in the presence or absence of 100 nm phorbol 12-myristate 13-acetate (PMA). As shown in Fig. 5A, left, cAMP accumulation in control cells was inhibited by ET-1 in a time-dependent manner. In PMA-treated cel
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