Uncoupling of Calcium Mobilization and Entry Pathways in Endothelin-stimulated Pituitary Lactotrophs

Abstract

In cells expressing Ca2+-mobilizing receptors, InsP3-induced Ca2+ release from intracellular stores is commonly associated with extracellular Ca2+ influx. Operation of these two Ca2+ signaling pathways mediates thyrotropin-releasing hormone (TRH) and angiotensin II (AII)-induced prolactin secretion from rat pituitary lactotrophs. After an initial hyperpolarization induced by Ca2+ mobilization from the endoplasmic reticulum (ER), these agonists generated an increase in the steady-state firing of action potentials, further facilitating extracellular Ca2+ influx and prolactin release. Like TRH and AII, endothelin-1 (ET-1) also induced a rapid release of Ca2+ from the ER and a concomitant spike prolactin secretion during the first 3–5 min of stimulation. However, unlike TRH and AII actions, Ca2+ mobilization was not coupled to Ca2+ influx during sustained ET-1 stimulation, as ET-1 induced a long-lasting abolition of action potential firing. This lead to a depletion of the ER Ca2+ pool, a prolonged decrease in [Ca2+] i, and sustained inhibition of prolactin release. ET-1-induced inhibition and TRH/AII-induced stimulation of Ca2+ influx and hormone secretion were reduced in the presence of the L-type Ca2+ channel blocker, nifedipine. Basal [Ca2+] i and prolactin release were also reduced in the presence of nifedipine. Furthermore, TRH-induced Ca2+ influx and secretion were abolished by ET-1, as TRH was unable to reactivate Ca2+ influx and prolactin release in ET-1-stimulated cells. Depolarization of the cells during sustained inhibitory action of ET-1, however, increased [Ca2+] i and prolactin release. These results indicate that L-type Ca2+ channel represents a common Ca2+ influx pathway that controls basal [Ca2+] i and secretion and is regulated by TRH/AII and ET-1 in an opposite manner. Thus, the receptor-mediated uncoupling of Ca2+ entry from Ca2+ mobilization provides an effective control mechanism in terminating the stimulatory action of ET-1. Moreover, it makes electrically active lactotrophs quiescent and unresponsive to other calcium-mobilizing agonists. In cells expressing Ca2+-mobilizing receptors, InsP3-induced Ca2+ release from intracellular stores is commonly associated with extracellular Ca2+ influx. Operation of these two Ca2+ signaling pathways mediates thyrotropin-releasing hormone (TRH) and angiotensin II (AII)-induced prolactin secretion from rat pituitary lactotrophs. After an initial hyperpolarization induced by Ca2+ mobilization from the endoplasmic reticulum (ER), these agonists generated an increase in the steady-state firing of action potentials, further facilitating extracellular Ca2+ influx and prolactin release. Like TRH and AII, endothelin-1 (ET-1) also induced a rapid release of Ca2+ from the ER and a concomitant spike prolactin secretion during the first 3–5 min of stimulation. However, unlike TRH and AII actions, Ca2+ mobilization was not coupled to Ca2+ influx during sustained ET-1 stimulation, as ET-1 induced a long-lasting abolition of action potential firing. This lead to a depletion of the ER Ca2+ pool, a prolonged decrease in [Ca2+] i, and sustained inhibition of prolactin release. ET-1-induced inhibition and TRH/AII-induced stimulation of Ca2+ influx and hormone secretion were reduced in the presence of the L-type Ca2+ channel blocker, nifedipine. Basal [Ca2+] i and prolactin release were also reduced in the presence of nifedipine. Furthermore, TRH-induced Ca2+ influx and secretion were abolished by ET-1, as TRH was unable to reactivate Ca2+ influx and prolactin release in ET-1-stimulated cells. Depolarization of the cells during sustained inhibitory action of ET-1, however, increased [Ca2+] i and prolactin release. These results indicate that L-type Ca2+ channel represents a common Ca2+ influx pathway that controls basal [Ca2+] i and secretion and is regulated by TRH/AII and ET-1 in an opposite manner. Thus, the receptor-mediated uncoupling of Ca2+ entry from Ca2+ mobilization provides an effective control mechanism in terminating the stimulatory action of ET-1. Moreover, it makes electrically active lactotrophs quiescent and unresponsive to other calcium-mobilizing agonists. Typically, in anterior pituitary cells operated by calcium-mobilizing agonists, release of Ca2+ from intracellular Ca2+ stores is associated with extracellular Ca2+ influx. The coordinate actions of these two pathways provide long-lasting Ca2+ signals and secretion during sustained agonist stimulation (1Stojilkovic S.S. Catt K.J. Endocr. Rev. 1992; 13: 256-280Crossref PubMed Scopus (154) Google Scholar). Endothelins (ETs) 1The abbreviations used are: ET, endothelin; TRH, thyrotropin-releasing hormone; AII, angiotensin II; ER, endoplasmic reticulum; SOCC, store-operated calcium channel(s); VGCC, voltage-gated calcium channel(s); AP, action potential. are common calcium-mobilizing agonists for secretory pituitary cells and operate through ETA receptors (2Kanyicska B. Freeman M.E. Am. J. Physiol. 1993; 265: E601-E608PubMed Google Scholar, 3Samson W.K. Biochem. Biophys. Res. Commun. 1992; 187: 590-595Crossref PubMed Scopus (34) Google Scholar) coupled to phospholipase C- (4Stojilkovic S.S. Balla T. Fukuda S. Cesnjaj M. Merelli F. Krsmanovic L.Z. Catt K.J. Endocrinology. 1992; 130: 465-474Crossref PubMed Google Scholar) but not phospholipase D-dependent signaling pathways (5Cesnjaj M. Zheng L. Catt K.J. Stojilkovic S.S. Mol. Biol. Cell. 1995; 9: 1037-1047Crossref Scopus (24) Google Scholar). In lactotrophs, the ET-1-induced release of Ca2+ from intracellular pools is associated with a rapid and transient increase in prolactin secretion (6Stojilkovic S.S. Iida T. Merelli F. Catt K.J. Mol. Pharmacol. 1991; 39: 762-770PubMed Google Scholar, 7Samson W.K. Skala K.D. Endocrinology. 1992; 130: 2964-2970Crossref PubMed Scopus (21) Google Scholar) followed by a prolonged inhibition to below basal levels (8Kanyicska B. Livingstone J.D. Freeman M.E. Endocrinology. 1995; 136: 990-994Crossref PubMed Scopus (24) Google Scholar). This bidirectional effect of a calcium-mobilizing agonist on secretion is uncommon among cells expressing phospholipase C-coupled receptors and is unique for cells expressing ETA receptors. The mechanism underlying the paradoxical action of ETs on hormone secretion is still unknown. It is unlikely that a rapid desensitization of ETA receptors could explain the inhibition of secretion observed in lactotrophs. Therefore, as the sustained phase in hormone secretion is affected in ET-1-stimulated lactotrophs, we addressed the alternate hypothesis that Ca2+ influx is uncoupled from Ca2+ mobilization in ET-1-stimulated lactotrophs, leading to depletion of the ER calcium pool and inhibition of hormone secretion. To test this hypothesis, rat pituitary lactotrophs were employed. These cells exhibit spontaneous, extracellular Ca2+-dependent action potential (AP) activity (9Ozawa S. Sand O. Physiol. Rev. 1986; 66: 887-952Crossref PubMed Scopus (157) Google Scholar), which is tightly coupled to basal prolactin secretion (10Stojilkovic S.S. Izumi S. Catt K.J. J. Biol. Chem. 1988; 263: 13054-13061Abstract Full Text PDF PubMed Google Scholar). In addition to ETA receptors, lactotrophs express thyrotropin-releasing hormone (TRH) receptors (11Gershengorn M.C. Osman R. Physiol. Rev. 1996; 76: 175-191Crossref PubMed Scopus (143) Google Scholar), the calcium-mobilizing actions of which have been well characterized (12Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Angiotensin II (AII) also stimulates prolactin release, presumably through activation of AT1B receptors (13Kakar S.S. Sellers J.C. Devor D.C. Musgrove L.C. Neill J.D. Biochem. Biophys. Res. Commun. 1992; 183: 1090-1096Crossref PubMed Scopus (311) Google Scholar). Two calcium entry pathways are proposed to operate in lactotrophs: voltage-gated (VGCC) and store-operated (SOCC) calcium channels (12Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Our results indicate that Ca2+ influx through VGCC rather than through SOCC is essential for the sustained secretagogue actions of TRH and AII. In contrast to these agonists, ET-1 inhibits voltage-gated Ca2+ influx, leading to a depletion of the ER calcium pool, a decrease in [Ca2+] i, and prolonged inhibition of prolactin release. GnRH, TRH, AII, and ET-1 were obtained from Peninsula Laboratories (Belmont, CA). Fura 2-AM was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma. Anterior pituitary glands from adult female Sprague-Dawley rats obtained from Charles River Inc. (Wilmington, MA) were dispersed into single cells by a trypsin/DNase treatment procedure (14Tomic M. Cesnjaj M. Catt K.J. Stojilkovic S.S. Endocrinology. 1994; 135: 1762-1771Crossref PubMed Scopus (44) Google Scholar). All experiments were performed in either mixed pituitary cell populations or purified lactotroph populations. Cell purification of dispersed pituitary cells was done by sedimentation on a ficol gradient as described previously (15Stojilkovic S.S Kukuljan M. Iida T. Rojas E. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4081-4085Crossref PubMed Scopus (72) Google Scholar). For cell column perifusion, 2 × 107 cells were incubated with preswollen cytodex-1 beads in 60-mm culture dishes for 2 days. The cells were then loaded into temperature-controlled chambers and perifused at 37 °C with Hanks' M199 containing 20 mmHEPES and 0.1% bovine serum albumin for 60 min at a flow rate of 0.6 or 1.0 ml/min, after which a stable basal secretion rate was established. During the test period, 3-s or 1-min fractions were collected, and the perifusate was subsequently stored at −20 °C. Prolactin content in each fraction was determined by radioimmunoassay using the reagents and standards provided by the National Pituitary Agency (Baltimore). Cells were plated on coverslips coated with poly-l-lysine and cultured in medium 199 containing Earle's salts, sodium bicarbonate, horse serum, and antibiotics for 24–48 h. For cytosolic Ca2+ measurements, the cells were incubated for 60 min at 37 °C with 2 μm fura 2-AM in phenol red-free medium 199 containing Hanks' salts, 20 mmsodium bicarbonate, and 20 mm HEPES. Coverslips with cells were washed with phenol red-free physiological buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to an 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 505 nm was measured. [Ca2+] i is expressed as the ratio of 340 nm/380 nm (F(340)/F(380)). Membrane potential (V m ) was measured using the perforated patch-clamp recording technique (16Kukuljan M. Stojilkovic S.S. Rojas E. Catt K.J. FEBS Lett. 1992; 301: 19-22Crossref PubMed Scopus (66) Google Scholar). Before recording, the culture medium was replaced with a solution containing (in mm) 140 NaCl, 4 KCl, 2.6 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose (pH adjusted to 7.36 using NaOH). Patch-clamp pipettes were fabricated from borosilicate glass (type 7740; World Precision Instruments, Sarasota, FL) using a horizontal micropipette puller (model P-87; Sutter Instrument Co., Novato, CA) and heat polished to a final tip resistance of 2–4 MΩ. The composition of the pipette solutions was (in mm) 70 KCl, 70 K+-aspartate, 1 MgCl2, 10 HEPES (pH adjusted to 7.2 with KOH). Amphotericin B (Sigma) was added from a stock solution to obtain a final concentration of 240 μg/ml. The signals were recorded under the current-clamp mode using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). m was the solution and the were at 2 using a and using the 1.0 in with a (Axon All were for a potential of 10 using the J. 1994; PubMed Scopus Google Scholar). For calcium and were either to the or perifused at a rate of 2 ml/min, during which solution the cells In cell column stimulation with nm ET-1 induced a rapid and transient increase in prolactin release 1 the of which was determined by agonist concentration not The spike phase was followed by a sustained inhibitory during which prolactin secretion was below that observed in controls 1 of TRH and AII receptors also induced a rapid of prolactin however, unlike the ET-1 the spike was followed by a sustained in which prolactin secretion basal levels 1 To the of [Ca2+] i signaling in to ET-1, TRH, and AII that of we in [Ca2+] i in single rat lactotrophs using the Ca2+ fura induced a rapid in [Ca2+] i, followed by a decrease to below basal levels 1 In in and lactotrophs, the spike phase of the [Ca2+] i was followed by sustained phase 1 During of dispersed pituitary cells at a flow rate of 0.6 with the of 1-min the stimulatory action of ET-1 for 3–5 min 1 This is in contrast to the [Ca2+] i, which was basal levels for To the hormone release to ET-1 with that of in [Ca2+] i, the of ET-1 were further examined using a rapid in which the flow rate was adjusted to 1.0 ml/min, and 3-s fractions were The secretory was then with an [Ca2+] i from lactotrophs with nm ET-1. these the hormone release that of the in [Ca2+] i This further the that the of ET-1-induced in [Ca2+] i that of prolactin release. in ET-1-stimulated cells was to the of after the of The of TRH and AII was followed by a in the [Ca2+] i and secretory not to basal levels In at min was for the of basal [Ca2+] i and prolactin secretion ET-1 These indicate that TRH and AII operate in a for Ca2+-mobilizing agonists, which of an spike and sustained the stimulatory actions are In ET-1 on [Ca2+] i and secretion. This an stimulation followed by a sustained the for a prolonged of the bidirectional of ET-1 on [Ca2+] i and secretion were observed in single lactotrophs in medium In cells in ET-1 induced a increase in prolactin secretion and [Ca2+] i and hormone also induced a increase in [Ca2+] i 4 of extracellular Ca2+ in but not ET-1-stimulated cells lead to an increase in [Ca2+] of extracellular Ca2+ reduced [Ca2+] i and prolactin that Ca2+ entry is coupled to basal [Ca2+] i and prolactin secretion in lactotrophs Thus, it is that a common Ca2+ influx pathway controls basal [Ca2+] i and secretion and is by TRH/AII and by ET-1. it been that VGCC and SOCC in TRH-induced Ca2+ mobilization in lactotrophs in P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). To test for the of VGCC in the inhibitory action of ET-1 on the the Ca2+ channel and and ET-1 was The inhibitory action of ET-1 on prolactin release was but not in the presence of and not the of ET-1-induced inhibition of prolactin release was reduced in the presence of it that these VGCC and ET-1 at the to secretion. also reduced basal prolactin release in a with that observed in cells of extracellular Ca2+ 4 and The of were concentration a concentration of ET-1 was still to prolactin release, but the of inhibition was of that observed in control cells In the phase of [Ca2+] i was also reduced by in the presence of nm nifedipine. These are in with the hypothesis that L-type voltage-gated channels are common Ca2+ influx channels in the control of [Ca2+] i and secretion in and lactotrophs. Thus, the Ca2+ influx through these channels is by it further by ET-1. To further test this hypothesis, potential was measured in single lactotrophs. the in this lactotrophs which was by addition of activity was abolished in ET-1 as well as in cells and However, the of this inhibition the two agonists. hormone induced a transient hyperpolarization and of firing followed by an increase in the In ET-1 induced a long-lasting hyperpolarization with the of which min The of hyperpolarization of lactotrophs by ET-1 was which is to the potential for under This that inhibition of activity is to activation of a which the cells. Depolarization of cells by or the addition of an L-type calcium channel agonist not increased [Ca2+] i and prolactin further that inhibition of voltage-gated is activation of voltage-gated Ca2+ influx in ET-1-stimulated lactotrophs. increase in [Ca2+] i in a lactotroph to nm ET-1. of on prolactin secretion in perifused pituitary During the ET-1-induced inhibition of TRH was unable to further hyperpolarization or action potential In addition of ET-1 to cells with TRH induced an inhibition of sustained extracellular Ca2+-dependent [Ca2+] i and prolactin release and This that activation of receptors is for the inhibitory action on Ca2+ influx. Furthermore, the of the spike [Ca2+] i and prolactin to ET-1 in cells and as well as to not that the ER calcium pool is The of TRH-induced [Ca2+] i also in cells to nm ET-1 in as well as medium not The of and Ca2+ were reduced in cells with ET-1, that the intracellular Ca2+ pool is min of stimulation not of TRH 20 min after stimulation with ET-1 not increase prolactin secretion The of Ca2+ influx to a pathway is commonly observed in and cells operated by Ca2+-mobilizing receptors. This is to the of intracellular and during sustained agonist stimulation E.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, S.S. Iida T. Merelli F. A. Krsmanovic L.Z. Catt K.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). In Ca2+ influx through SOCC in to and depletion of the ER calcium pool J. Endocr. Rev. 1993; PubMed Scopus Google Scholar, Biochem. J. 1995; PubMed Scopus Google Scholar). In Ca2+ influx in these cells is from the mechanism of depletion of the ER calcium pool 1991; PubMed Scopus Google Scholar, O. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In VGCC are in sustained Ca2+ In these the of Ca2+ release from intracellular stores the of firing and associated voltage-gated Ca2+ entry (1Stojilkovic S.S. Catt K.J. Endocr. Rev. 1992; 13: 256-280Crossref PubMed Scopus (154) Google Scholar). of channels a in the of Ca2+ mobilization with the Ca2+ entry during agonist stimulation (9Ozawa S. Sand O. Physiol. Rev. 1986; 66: 887-952Crossref PubMed Scopus (157) Google Scholar). The of these however, not the of in lactotrophs, calcium entry also been J. J. Physiol. 1995; PubMed Scopus Google Scholar). Pituitary lactotrophs and cell express Ca2+-mobilizing receptors that operate in a for this of receptors, is coupled to Ca2+ TRH receptors are the are coupled to a phospholipase pathway through a (11Gershengorn M.C. Osman R. Physiol. Rev. 1996; 76: 175-191Crossref PubMed Scopus (143) Google Scholar). of this pathway to a increase in [Ca2+] i and hormone the phase on and the sustained phase on Ca2+ entry J. Biol. Chem. Full Text PDF PubMed Google Scholar, E.J. P.M. Endocrinology. 1994; 135: PubMed Scopus Google Scholar, Biochem. J. 1988; PubMed Scopus Google Scholar) and 4 voltage-gated and Ca2+ entry have been proposed to in TRH-induced Ca2+ influx in these cells. The of VGCC and the of TRH-induced activity is well M.C. P.M. Mol. Endocrinol. 1992; Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), the of SOCC (12Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). The other agonist for these AII, signals through AT1B receptors that are also coupled to a phospholipase pathway (13Kakar S.S. Sellers J.C. Devor D.C. Musgrove L.C. Neill J.D. Biochem. Biophys. Res. Commun. 1992; 183: 1090-1096Crossref PubMed Scopus (311) Google Scholar). Our results indicate that this in a with that observed during TRH Furthermore, lactotrophs express and receptors, the activation of which are also associated with a of Ca2+ signaling and prolactin secretion W.K. Biol. 1995; Google Scholar, T. M. S. S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The is an of calcium-mobilizing receptors expressed in lactotrophs as well as in other pituitary cell S.S. Catt K.J. Trends Pharmacol. Sci. 1992; 13: Full Text PDF PubMed Scopus (44) Google Scholar). The of this to a phospholipase pathway in pituitary cells (4Stojilkovic S.S. Balla T. Fukuda S. Cesnjaj M. Merelli F. Krsmanovic L.Z. Catt K.J. Endocrinology. 1992; 130: 465-474Crossref PubMed Google Scholar) provides a transient increase in [Ca2+] i and hormone release (6Stojilkovic S.S. Iida T. Merelli F. Catt K.J. Mol. Pharmacol. 1991; 39: 762-770PubMed Google Scholar). In contrast to TRH and AII however, the stimulatory action of ET-1 is with a prolonged inhibition of prolactin release. This was observed by W.K. Skala K.D. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) and subsequently by other (6Stojilkovic S.S. Iida T. Merelli F. Catt K.J. Mol. Pharmacol. 1991; 39: 762-770PubMed Google Scholar, B. Freeman M.E. Biochem. Biophys. Res. Commun. 1991; PubMed Scopus Google Scholar, J. M. 1992; PubMed Scopus Google Scholar). In with the (4Stojilkovic S.S. Balla T. Fukuda S. Cesnjaj M. Merelli F. Krsmanovic L.Z. Catt K.J. Endocrinology. 1992; 130: 465-474Crossref PubMed Google Scholar), it was that inhibition was by ETA receptors (2Kanyicska B. Freeman M.E. Am. J. Physiol. 1993; 265: E601-E608PubMed Google Scholar, 3Samson W.K. Biochem. Biophys. Res. Commun. 1992; 187: 590-595Crossref PubMed Scopus (34) Google Scholar, S.S. Balla T. Fukuda S. Cesnjaj M. Merelli F. Krsmanovic L.Z. Catt K.J. Endocrinology. 1992; 130: 465-474Crossref PubMed Google Scholar). In addition to the pituitary inhibition of prolactin release by ET-1 was also observed in cells S. Endocrinology. 1993; PubMed Scopus Google Scholar), which express ETA receptors as well J. A. L. J. Pharmacol. 1993; PubMed Scopus Google Scholar). Our that uncoupling of Ca2+ influx from Ca2+ mobilization the bidirectional action of ETs on prolactin release. Thus, ET-1 two opposite on Ca2+ stimulation and inhibition of Ca2+ influx The of of TRH and AII to Ca2+ influx in ET-1-stimulated cells and the of ET-1 to Ca2+ entry in cells that these receptors control the Ca2+ influx pathway but the opposite mode of Furthermore, the channels control basal [Ca2+] i and prolactin release. of and indicate that Ca2+ influx through calcium channels rather than SOCC is by these calcium-mobilizing receptors. Basal prolactin release is by and by depletion of extracellular activity in lactotrophs and cells is also by and extracellular F. B. PubMed Scopus Google Scholar). TRH-induced Ca2+ influx, and prolactin secretion in these cells are J. Biol. Chem. Full Text PDF PubMed Google Biochem. J. 1988; PubMed Scopus Google Scholar, M.C. P.M. Mol. Endocrinol. 1992; Google Scholar). ET-1 a prolonged inhibition of activity and Ca2+ influx through The of the L-type Ca2+ channels blocker, and to the inhibitory action of ET-1 on secretion also that ET-1 on VGCC to Ca2+ Ca2+ entry by depletion of the ER calcium pool in cells expressing is a depletion of the ER calcium pool in and but this action is with a action on Ca2+ influx by these two receptors, inhibition by ET-1 and stimulation by The results also indicate that inhibition of Ca2+ entry through L-type Ca2+ channels is to activation of leading to a hyperpolarization and abolition of the of channels in lactotrophs been B. Freeman M.E. Endocrinology. PubMed Scopus Google Scholar). These channels are also by TRH and are for a rapid inhibition of activity in lactotrophs (9Ozawa S. Sand O. Physiol. Rev. 1986; 66: 887-952Crossref PubMed Scopus (157) Google Scholar). these channels are by [Ca2+] i, however, the inhibition of firing is transient S. Proc. Natl. Acad. Sci. U. S. A. 76: PubMed Scopus Google Scholar) and explain the phase in hyperpolarization of these cells. Thus, the sustained inhibition of activity by experiments of this as well as the mechanism of activation by as with or through intracellular pathways B. Trends 1994; Full Text PDF PubMed Scopus Google Scholar, Physiol. Rev. 1995; PubMed Scopus Google Scholar). In the presence of ET-1 is still to a inhibition of prolactin release. of basal extracellular Ca2+-dependent hormone secretion is by Ca2+ influx through calcium This influx could by other VGCC of which the is the characterized in these cells J. Physiol. PubMed Scopus Google Scholar, Endocrinology. 1988; PubMed Scopus Google Scholar). It is also that the calcium is to the other of as a sodium channel in lactotrophs and cells S. Am. J. Physiol. 1996; PubMed Google Scholar). VGCC and the sodium channels are of the activity and are to the control of firing of This provide the for the of a Ca2+ influx in cells and inhibition and by ET-1 and TRH, Our results also that and TRH-induced Ca2+ mobilization to depletion of the ER Ca2+ pool but that the rate of depletion not the [Ca2+] i In cells in of TRH or during the phase of [Ca2+] i in an release of Ca2+ influx is coupled to Ca2+ mobilization in this pathway not the cells from a decrease in the ER calcium pool, as by the of ET-1 and but is to the steady-state phase in [Ca2+] i This is with a in which the [Ca2+] i in and ET-1-stimulated lactotrophs represents the and Ca2+ influx. In ET-1-stimulated Ca2+ mobilization is and Ca2+ influx is leading to the bidirectional and sustained in [Ca2+] i and a depletion in the intracellular Ca2+ In pathways are leading to the and in [Ca2+] i but by a decrease in the intracellular Ca2+ pool to a steady-state However, experiments are to the Ca2+ in lactotrophs. In results indicate that the signaling through ETA receptors is essential not for stimulatory but also for inhibitory actions of ETs on [Ca2+] i and prolactin secretion. The of these receptors is by the uncoupling of Ca2+ mobilization and voltage-gated Ca2+ entry uncoupling to a unique physiological for a Ca2+-mobilizing unresponsive cells. The ER calcium pool is to Ca2+-mobilizing action of the a other calcium-mobilizing agonists are unable to Ca2+ hyperpolarization of lactotrophs also these agonists to voltage-gated Ca2+ influx in these cells. basal activity and hormone secretion are also This makes lactotrophs leading to a sustained In this a transient stimulatory action of ET-1 represents a first in the of a quiescent of these cells.