Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells)

Changes in the cytosolic free Ca2+ concentration, [Ca2+]i, have been proposed to mediate the regulation of the secretion of pituitary hormones by hypothalamic peptides. Using an intracellularly trapped fluorescent Ca2+ probe, quin2, [Ca2+]i was monitored in GH3 cells. Somatostatin lowers [Ca2+]i in a dose dependent manner from a prestimulatory level of 120 +/- 4 nM (SEM, n = 13) to 78 +/- 9 nM (n = 5) at 10(-7)M; the effect is half maximal at 2 X 10(-9) M somatostatin. The decrease in [Ca2+]i occurs rapidly after somatostatin addition and a lowered steady state [Ca2+]i is maintained for several minutes. Somatostatin does not inhibit the rapid rise in [Ca2+]i elicited by thyrotropin releasing hormone (TRH) and can still cause a decrease in [Ca2+]i in the presence of TRH (10(-7)M). Concomitantly with its action on [Ca2+]i somatostatin causes hyperpolarization of GH3 cells assessed with the fluorescent probe bis-oxonol. The lowering of [Ca2+]i by somatostatin is however not only due to reduced Ca2+ influx through voltage dependent Ca2+ channels, since it persists in the presence of the channel blocker verapamil. These results suggest that somatostatin may exert its inhibitory action on pituitary hormone secretion by decreasing [Ca2+]i.     

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

Platelet-activating factor affects cytosolic free calcium concentration and prolactin secretion in GH4C1 rat pituitary cells.

Platelet-activating factor (PAF) is a naturally occurring pleiotropic mediator which acts via specific membrane receptors. In certain target cells, PAF causes elevations in cytosolic free Ca2+ concentration ([Ca2+]i); however, little is known of the effects of PAF on endocrine cells. Therefore, we have investigated the actions of PAF on [Ca2+]i in prolactin-secreting GH4C1 cells and have compared the effects with the well documented actions on these cells of thyrotropin-releasing hormone (TRH). GH4C1 cells were loaded with quin2/AM and fluorescence was measured in suspended populations. PAF induced a dose-dependent (10-100 microM) rise in [Ca2+]i which was slower in onset than that caused by TRH, peaking (200 to 400% above basal [Ca2+]i) at about 12 sec, and decaying over about 3 min to basal [Ca2+]i. Unlike TRH, PAF did not cause a secondary plateau phase of rise in [Ca2+]i. The terpene PAF receptor antagonist BN52021 inhibited the action of PAF on [Ca2+]i. Voltage-dependent Ca2+ channel blocker, verapamil (200 microM), antagonized the action of PAF on [Ca2+]i as did chelation of extracellular Ca2+. PAF also stimulated the secretion of prolactin in a dose-dependent manner (10 to 50 microM). The concentrations of PAF required to evoke responses in GH4C1 cells were considerably higher than those required in several other known PAF target cell types. The high concentration requirement in GH4C1 cells may be due to rapid degradation of PAF or the presence of low affinity receptors. We conclude that PAF can act, via cell surface receptors, on pituitary GH4C1 cells to alter [Ca2+]i by a pathway that enhances influx of extracellular Ca2+ through voltage-gated channels and then to enhance the secretion of prolactin.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. II. An increase in potassium conductance initiates somatostatin-induced inhibition of prolactin secretion

The neuropeptide somatostatin inhibits prolactin release from GH4C1 pituitary cells via two mechanisms, inhibition of stimulated adenylate cyclase activity and an undefined cAMP-independent process. Somatostatin also hyperpolarizes GH4C1 cells and reduces their intracellular free Ca2+ concentration ([Ca2+]i) in a cAMP-independent manner. To determine whether these ionic changes were involved in the cAMP-independent mechanism by which somatostatin inhibited secretion, changes in cAMP levels were prevented from having any biological consequences by performing experiments in the presence of a maximal concentration of a cAMP analog. Under these conditions, inhibition of prolactin release by somatostatin required a transmembrane concentration gradient for K+ but not one for either Na+ or Cl-. However, elimination of the outward K+ gradient did not prevent somatostatin inhibition of vasoactive intestinal peptide-stimulated hormone release. Therefore, somatostatin's cAMP-mediated mechanism does not require a K+ gradient, whereas its cAMP-independent inhibition of secretion appears to result from a change in K+ conductance. Consistent with this conclusion, membrane hyperpolarization with gramicidin (1 microgram/ml) mimicked somatostatin inhibition of prolactin release. In addition, the K+ channel blocker tetrabutylammonium prevented the effects of somatostatin on the membrane potential, the [Ca2+]i and hormone secretion. Nonetheless, a K+ gradient was not sufficient for somatostatin action. Even in the presence of a normal K+ gradient, somatostatin was only able to inhibit prolactin release when the extracellular Ca2+ concentration was at least twice the [Ca2+]i. Furthermore, the calcium channel blocker, nifedipine (10 microM), which prevents the action of somatostatin to reduce the [Ca2+]i, specifically blocked inhibition of prolactin release via somatostatin's cAMP-independent mechanisms. Therefore, a decrease in Ca2+ influx through voltage-dependent Ca2+ channels produces both the fall in [Ca2+]i and inhibition of hormone secretion in response to somatostatin.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

Synergism in cytosolic Ca2+ mobilization between bradykinin and agonists for pertussis toxin-sensitive G-protein-coupled receptors in NG 108-15 cells.

Bradykinin (BK) induced a transient and pertussis toxin (PT)-insensitive increase in cytosolic Ca2+ ([Ca2+]i) in NG 108-15 neuroblastoma x glioma hybrid cells, whereas leucine-enkephalin (EK), somatostatin, norepinephrine or carbachol showed a weak but PT-sensitive action. When any one of the latter agonists was applied to the cells treated with low doses of BK, however, the level of [Ca2+]i rise caused by the agonist was remarkably increased in a PT-sensitive manner. The decreasing of extracellular Ca2+ only slightly influenced the actions of these agonists. Thus, synergism between a BK receptor and PT-sensitive G-protein-coupled receptors results in marked intracellular Ca2+ mobilization by the latter agonists.     

Adenosine A1 receptors inhibit both adenylate cyclase activity and TRH-activated Ca2+ channels by a pertussis toxin-sensitive mechanism in GH3 cells

The present study has examined the effects of adenosine A1 receptors on second messenger processes in GH3 cells. A1 receptors are present which are shown to inhibit adenylate cyclase in a GTP-requiring manner. Hormone (VIP) stimulation is also absolutely required for the observation of inhibition. Adenosine A1 receptor analogues also inhibit TRH-stimulated [Ca2+]i-mobilization in GH3 cells. Both effects of the adenosine receptor agonists are apparently mediated by pertussis toxin substrates, of which there are two--41,000 and 40,000 daltons respectively--in these cells. Somatostatin exerts analogous effects to the adenosine agonists in GH3 cells. Thus it may turn out that a general property of 'cyclase inhibitory receptors' is also to inhibit [Ca2+]i-mobilization in the same cells, when such mechanisms are present.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.     

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.     

An acute release of Ca2+ from sequestered intracellular pools is not the primary transduction mechanism causing the initial burst of PRL and TSH secretion induced by TRH in normal rat pituitary cells.

With 1.5 mM [Ca2+]e, 10 nM TRH induced a prompt high-amplitude burst of hormone secretion and an initial high-amplitude [Ca2+]i burst (first phase) followed by a sustained low-amplitude [Ca2+]i increment (second phase) in both tumor-derived GH4C1 and normal adenohypophyseal (AP) cells. With less than 2 microM [Ca2+]e, in both cell types the TRH-induced first phase rise in [Ca2+]i was suppressed 30% while the second phase rise was completely abolished; however, hormone secretion was inhibited only 20-30% in GH4C1 but greater than 80% in AP cells. Thapsigargin induced a first-phase rise in [Ca2+]i in AP cells equal to that induced by 10 nM TRH but only 20% as much first-phase hormone secretion. Blocking Ca2+ channels with nifedipine inhibited TRH-induced secretion in AP cells significantly more than in GH4C1 cells. Our data indicate that the TRH-induced first-phase spike in [Ca2+]i from intracellular Ca2+ stores may play a major transduction role in hormone secretion in GH4C1 cells but not in normal AP cells. Transduction mechanisms coupled to Ca2+ influx through Ca2+ channels in the plasmalemma are apparently a much more important component of TRH-induced secretion in normal than in tumor-derived pituitary cells.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.     

Pretreatment with muscarinic receptor agonist activates a calcium-inhibitable adenylate cyclase in GH3 pituitary cells

We have demonstrated previously that pretreatment of GH3 pituitary cells with muscarinic agonists may induce a higher cAMP formation in response to vasoactive intestinal peptide (VIP) or forskolin. In the present study, we further examined the adenylate cyclase (AC) that may be involved. We found that carbachol-pretreatment enhanced both VIP- and forskolin-activated AC activities. The addition of calcium ions to the incubation buffer diminished this enhancing effect. Carbachol was found to induce a decrease in intracellular calcium concentration [Ca2+]i by inhibiting calcium influx through L-type Ca2+ channels. However, the incubation of cells in Ca(2+)-free buffer or in the presence of L-type Ca2+ channel blockers had no influence on forskolin-stimulated cAMP formation, although both treatments induced decreases in [Ca2+]i as carbachol did. On the other hand, incubation in the presence of LaCl3 at a low concentration not being able to enter cells, forskolin-stimulated cAMP formation as well as the enhancing effect of carbachol-pretreatment on this response, were both suppressed. Similar phenomena were observed when membrane-bound AC activities were measured in the presence of LaCl3. Taken together, these results seem to suggest that pretreatment of GH3 cells with muscarinic receptor agonist may activate a Ca(2+)-inhibitable AC for a higher stimulated response. Low intracellular calcium concentrations are essential but not sufficient for this effect.     

Adenosine triphosphate, bradykinin, and thyrotropin-releasing hormone regulate the intracellular Ca2+ concentration and the 45Ca2+ efflux of human thyrocytes in primary culture

The hormonal stimulation of phospholipase C and the consequent activation of the Ca2+-phosphatidylinositol cascade in eukaryotic cells is associated with modifications of the [Ca2+]i (intracellular Ca2+ concentration) which modulates cellular functions. In this study, these modifications were investigated in primary cultures of human thyroid cells. The mean apparent basal [Ca2+]i of human thyrocytes measured using the intracellularly trapped fluorescent indicator Quin-2 was found to be 89 +/- 16 nM (n = 49). ATP and, to a lesser extent, ADP, but not AMP or adenosine, elicited a concentration-dependent biphasic rise in human thyrocytes [Ca2+]i and increased their 45Ca2+ efflux. The first transient phase of the [Ca2+]i rise induced by ATP was resistant to extracellular Ca2+ depletion, whereas the second sustained phase was abolished in these conditions. This suggests that although the first phase of this response involves a release of Ca2+ from intracellular stores, the second phase requires extracellular Ca2+ influx. The response of human thyrocytes to analogs of ATP is compatible with a P2-purinergic effect of ATP on these cells. Bradykinin and TRH affected the human thyrocyte [Ca2+]i and 45Ca2+ efflux similarly to ATP. The human thyrocyte [Ca2+]i and the 45Ca2+ efflux were not modified by carbachol, a nonhydrolyzable analog of acetylcholine. The present results suggest the presence of P2-purinergic receptors to ATP and of receptors to TRH and bradykinin on human follicular thyroid cells. They also confirm that the Ca2+-phosphatidylinositol cascade is present in these cells and suggest that this cascade is modulated by ATP, TRH, and bradykinin. As this cascade is involved in the regulation of protein iodination, and therefore of thyroid hormones synthesis, these agents might have an important role in the regulation of the thyroid function.     

Dual actions of phorbol esters on cytosolic free Ca2+ concentrations and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses

We have used phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), to study the actions of protein kinase C (a TPA receptor) on cytosolic free Ca2+ concentrations [( Ca2+]i) and hormone secretion in rat pituitary cells (GH cells), and to elucidate the role of diacylglycerol (a protein kinase C activator) in thyrotropin-releasing hormone (TRH) action. TPA had a dual action on [Ca2+]i, inducing a stimulatory phase from 300 (basal) to 420 nM, which was interrupted in 30-60 s by an inhibitory phase which transiently lowered [Ca2+]i to 240 nM and rose in 3-10 min to yield the stimulatory phase. TPA-mediated changes in [Ca2+]i were induced by other phorbol esters and mezerein but not by phorbol or activators of kinases different from protein kinase C. Both phases of TPA action on [Ca2+]i were abolished by 5-min pretreatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.33 mM) or Ca2+ channel antagonists (verapamil or nifedipine). TPA also enhanced the rate of sustained hormone secretion without inducing a burst of hormone release (unlike TRH). Also, stimulation of secretion by TPA was not inhibited by Ca2+ channel antagonists and was resistant (10%) to EGTA. Simultaneous addition of TPA with the ionophore ionomycin (100 nM) reconstituted a TRH-like spike, nadir and plateau of [Ca2+]i. Ionomycin generated the spike in [Ca2+]i by releasing TRH-sensitive Ca2+ stores, while TPA induced the nadir (inhibitory phase), and a nifedipine/verapamil-sensitive plateau of [Ca2+]i (stimulatory phase). Concurrent (but not separate) addition of ionomycin and TPA also reconstituted a TRH-like burst of hormone secretion. These and previous results indicate that activation of protein kinase C by TPA or diacylglycerol (which is elevated by TRH) and a simultaneous spike in [Ca2+]i are required for burst secretion. Diacylglycerol may also mediate the TRH-induced nadir and plateau of [Ca2+]i; the latter process contributes to Ca2+-dependent stimulation of steady secretion by TRH.     

Control of the intracellular Ca(2+)-concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: evidence for different kinetics of Ca(2+)-phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production

Carbachol, through a muscarinic receptor, thyrotropin-releasing hormone (TRH), prostaglandin F2 alpha (PGF2 alpha), bradykinin, and adenosine triphosphate (ATP) increased the apparent [Ca2+]i (intracellular free Ca2(+)-concentration) of dog thyrocytes in primary culture. The [Ca2+]i measured by the Quin-2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+]i declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2 alpha and in the case of bradykinin, TRH, and ATP, the [Ca2+]i returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+]i followed by a decline of the [Ca2+]i often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF2 alpha, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2 alpha, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2 alpha, bradykinin, and ATP through their action on the Ca2(+)-phosphatidylinositol cascade.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells. Interaction with Ca2+

Prolactin (PRL) release in permeable GH3 pituitary cells was stimulated by the protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). Both agents stimulated secretion at 10 nM Ca2+, but higher [Ca2+] (greater than 0.1 microM) potentiated TPA and OAG action. Maximal potentiation occurred at 1 microM calculated free Ca2+, and a similar value was obtained when the cytoplasmic [Ca2+] was measured with the Ca2+-sensitive dye Quin 2. Release of a secretory sulfated proteoglycan was also stimulated by TPA and OAG in permeable GH3 cells, with characteristics similar to those for PRL release. Trifluoroperazine, polymyxin B, neomycin, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate all inhibited both TPA- and Ca2+-stimulated PRL release, but in each case the half-maximal inhibitory concentrations were approximately 2-fold higher for TPA-stimulated release compared to Ca2+-stimulated release. Thyrotropin-releasing hormone (TRH) and guanosine 5'-Q-thiotriphosphate, which stimulate polyphosphoinositide breakdown in permeable cells, were found to be only weak stimulators of PRL release, compared to TPA and exogenous diacylglycerol. However, a much stronger effect of TRH was seen if cells were briefly treated with TRH prior to permeabilization. PRL release from TRH-pretreated permeable cells resembled TPA- and OAG-stimulated secretion, with [Ca2+] greater than 0.1 microM potentiating the effect of TRH pretreatment. These studies support the hypothesis that PRL release in GH3 cells can be stimulated directly by a diacylglycerol-activated secretory mechanism whose activity is modulated by [Ca2+].