pH homeostasis in pituitary GH4C1 cells: basal intracellular pH is regulated by cytosolic free Ca2+ concentration.

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.     

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.     

Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.     

Effect of secretagogues on cytosolic free Ca2+ and insulin release at different extracellular Ca2+ concentrations in the hamster clonal beta-cell line HIT-T15

We have examined the relationship between extracellular Ca2+, cytosolic free Ca2+ and insulin release in the clonal beta-cell line HIT-T15. Glucose-stimulated insulin release was dependent on the extracellular Ca2+ concentration in a dose-related manner; the threshold medium Ca2+ concentration for glucose-stimulated insulin release was 0.5 mM. Both forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased insulin release in the presence of glucose at all extracellular Ca2+ concentration tested (0.1-2.5 mM) but not in the absence of Ca2+. Thus, the threshold medium Ca2+ concentration for glucose-stimulated insulin release was reduced to 0.1 mM by forskolin or TPA. Step-wise increases in the medium Ca2+ concentration in the presence of an initiator of insulin release resulted in a dose-related increase in cytosolic free Ca2+. In the presence of 10 mM glucose, cytosolic free Ca2+ in HIT cells was increased from 60 +/- 5 nM in Ca2+-free medium to 290 +/- 46 nM in medium containing 2.5 mM Ca2+. The effects of increasing extracellular Ca2+ in the presence of 40 mM K+ were similar but considerably more pronounced. Inclusion of either TPA or forskolin in the incubation medium had no significant effect on the steady-state cytosolic free Ca2+ levels in the absence of glucose but in the presence of 10 mM glucose forskolin caused modest (11-18%) increases in steady-state cytosolic free Ca2+ levels at extracellular Ca2+ concentrations of 0.25 mM or above. In contrast, in the presence of glucose TPA significantly reduced the steady-state levels of cytosolic free Ca2+ by 17-21% at extracellular Ca2+ concentrations of 0.25 mM or above. These data provide further evidence that insulin release mediated by activation of beta-cell protein kinases involves primarily an increase in sensitivity of the secretory system to intracellular Ca2+.     

Intracellular Ca2+ mobilization by thyrotropin, carbachol, and adenosine triphosphate in dog thyroid cells

The effect of TSH, carbachol (CC), and ATP on intracellular calcium concentration ([Ca2+]i) in primary cultures of dog thyroid cells was examined using the fluorescent Ca2+ indicator fura-2. TSH caused an increase in [Ca2+]i at 37 C, but not 22 C, while it increased cAMP formation in these cells at both 22 and 37 C. CC and ATP increased [Ca2+]i at both 22 and 37 C. The CC-induced increase in [Ca2+]i was under muscarinic receptor control, and it was biphasic, with an initial spike followed by a sustained increase at a lower level. TSH and ATP were weaker agonists compared to CC, since maximal doses of TSH (100-500 mU/ml) and ATP (100-500 microM) increased [Ca2+]i by 40-70% over basal levels, compared to a 2- to 4-fold increase in [Ca2+] induced by maximal doses of CC (10-50 microM). The TSH-induced increase in [Ca2+]i was transient, returning to basal levels within 1-2 min after application of the agonist. All three agents were able to transiently increase [Ca2+]i to be internal stores. In the presence of the inorganic Ca2+ channel blockers La3+, Ni2+, and Co2+, the peak [Ca2+]i change was little affected, while the persistent response to CC and ATP was blocked, indicating dependence of this phase on influx of Ca2+. Paradoxically, these channel blockers abolished the effect of TSH on [Ca2+]i. TSH stimulation of cAMP formation was also inhibited 80-90% by these blockers, but not in Ca2+-free/EGTA buffer. These results suggest that the Ca2+ channel blockers may have actions in addition to inhibition of Ca2+ entry in these cells. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl] specifically blocked both the initial and sustained increase induced by CC, while having no effect on ATP or TSH-induced [Ca2+]i, suggesting that TMB-8 may not be a general antagonist of Ca2+ mobilization. Activators of protein kinase-C, such as phorbol esters or an analog of diacylglycerol, inhibited the [Ca2+]i rise induced by all the three agonists used, indicating a regulatory role of protein kinase-C activation on [Ca2+]i in these cells. In FRTL-5 cells, [Ca2+]i was also increased by TSH and ATP, but not by CC. ATP, however, was a more effective agonist than in dog thyroid cells, while TSH increased [Ca2+]i by a similar magnitude in both cell types. The results of the present study demonstrate that TSH, albeit of lesser potency than CC, increases [Ca2+]i by causing intracellular Ca2+ mobilization in cultured dog thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.

Treatment of 45Ca2+-loaded GH3 pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two 45Ca2+ pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.     

Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal beta-cell line HIT-T15

We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal beta-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mM increased Cai by 36%, from 89 +/- 4 to 121 +/- 6 nM (mean +/- S.E.M., n = 23). Further raising the medium glucose concentration to 10 mM increased Cai to 139 +/- 6 nM. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mM K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 +/- 48 nM. The increases in Cai in response to glucose of K+ were blocked by the simultaneous presence of verapamil (50 microM). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.     

Platelet cytosolic free Ca2+ concentration and serotonin (5-HT) content in essential hypertension

Cytosolic free [Ca2+] and serotonin (5-HT) content were measured in platelets from 22 untreated hypertensive patients and 16 normotensive subjects. In hypertensive patients, cytosolic free [Ca2+] was significantly higher (239 +/- 13 nmol/l versus 186 +/- 7 nmol/l, n = 22 and 16, P less than 0.01) and 5-HT content was significantly lower than those measured in cells from control subjects (3.22 +/- 0.26 versus 4.99 +/- 0.38 X 10(-7) mol/10(11) cells, n = 22 and 16, P less than 0.001). These two parameters were closely correlated (r = -0.565, n = 38, P less than 0.001). These two concomitant changes in platelet characteristics might result from a common cause, such as cell membrane alterations. As ritanserine, a specific 5-HT2 receptor antagonist, did not modify the cytosolic free [Ca2+], a higher stimulation of 5-HT2 receptors is not likely to be responsible for the enhanced free Ca2+ levels.     

Hypothalamic peptides modulate cytosolic free Ca2+ levels and adenylyl cyclase activity in human nonfunctioning pituitary adenomas.

The effects of hypothalamic peptides (TRH, GnRH, arginine vasopressin, vasoactive intestinal peptide, GHRH, CRH, and SRIH) on cytosolic free calcium concentrations ([Ca2+]i) and adenylyl cyclase (AC) activity were evaluated in 12 nonfunctioning pituitary adenomas. TRH, GnRH, and arginine vasopressin induced a marked [Ca2+]i rise in 10/12, 4/12, and 2/5 tumors, respectively. The transients induced by these peptides were due to both Ca2+ mobilization from the intracellular stores and Ca2+ influx from the extracellular medium. AC activity was evaluated in 10 adenomas; 1 microM vasoactive intestinal peptide induced a 2- to 6-fold stimulation of the enzyme activity in all tumors, while neither GHRH nor CRH were effective. Moreover, in 5/10 tumors 1 microM SRIH reduced both AC activity and [Ca2+]i, while in 2/10 the peptide caused a significant rise in [Ca2+]i despite the AC inhibition and in 3/10 SRIH did not modify either AC activity or [Ca2+]i. This study indicates that in nonfunctioning pituitary adenomas a wide spectrum of hypothalamic peptides modulate [Ca2+]i and AC activity. Moreover, the presence of biologically active receptors may offer a possible target for therapeutic intervention.     

Association of 45Ca2+ mobilization with stimulation of growth hormone (GH) release by GH-releasing factor in dispersed normal male rat pituitary cells

Dispersed normal male rat anterior pituitary cells were prelabeled with 45Ca2+ and perifused to study the influence of GH-releasing factor (GRF) on fractional calcium efflux and GH release. The cells were exposed for 2 min to 0, 0.03, 0.1, 0.3, 1.0, or 10.0 nM GRF in separate perifusion columns, and the response to each concentration was determined by integration of the area under the curve. Concentrations of 0.1 nM GRF and higher produced a simultaneous and significant stimulation of calcium efflux and GH release. The increase in calcium efflux was proportional to GRF concentration and was maximally responsive at 1 nM GRF. The value for the entire integrated response of GH release increased continuously with GRF concentration, but GH released rapidly (0-4 min) in response to GRF achieved a maximal response at 1 nM GRF and was significantly correlated with calcium efflux. Somatostatin (100 nM) abolished the stimulation of GH release and calcium efflux due to 10 nM GRF. We conclude that GRF receptor activation is intimately associated with calcium mobilization, although the relative dependence upon intracellular or extracellular calcium sources has yet to be defined. This interaction occurs at a GRF concentration about 10 times lower than that observed to cause a measurable increase in intracellular biochemical messengers such as cAMP, phosphatidylinositol, or arachidonate. We postulate that GRF-stimulated calcium mobilization is a rapid and very sensitive event contributing to GRF-stimulated GH release.     

3H]-Thyroliberin (TRH) binding to nuclei isolated from a pituitary clonal cell line (GH3).

[3H]Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and nucleus. We studied the [3H]-TRH binding capacity of a highly purified nuclear fraction isolated by an original procedure from GH3 cells. The nuclei still presented their double nuclear envelope. They are able to bind [H]-TRH to the same extent as nuclei isolated from GH3 cells previously exposed to [3H]-TRH. The equilibrium of binding was reached after 2--5 min incubation at 25 degrees or 35 degrees C. The binding is stable at 4 degrees C and partially (50%) dissociated within 15 min at 25 degrees C. 50% of the binding was inhibited by large excess of unlabelled TRH. Nuclei obtained from a variant GH3 cell which has lost its responsiveness to TRH presented only the noncompetitive binding compartment. The binding was found dose dependent and not saturable. Two apparent dissociation constants were evaluated: 1.5--2.5 x 10--8M and 2.10--6M, respectively, for high and low doses of [3H]-TRH. The first one was identical to that previously found for intact GH3 cells. The present data show the existence of specific nuclear binding sites for TRH, establish their characteristics and suggest a possible nuclear site of action for that peptide hormone.     

Importance of transients in cytosolic free calcium concentrations on activation of Na+/H+ exchange in GH4C1 pituitary cells.

In GH4C1 cells, TRH and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been shown to activate Na+/H+ exchange, probably via stimulation of protein kinase C. In the present study, the dependence of changes in intracellular pH (pHi) on transients in the cytosolic free calcium concentration [( Ca2+]i) was investigated using BCECF and fura-2, respectively. In buffer containing 0.4 mM extracellular Ca2+, both TRH and ionomycin induced rapid cytosolic alkalinization in GH4C1 cells acid loaded with nigericin. The action of ionomycin on pHi was abolished by preincubating the cells with 100 microM amiloride or by replacing extracellular Na+ with choline+, indicating that the change in pHi was probably due to activation of Na+/H+ exchange. The actions of both TRH and ionomycin on pHi were blunted in Ca2(+)-free buffer. When acid-loaded cells were stimulated first with ionomycin, to deplete intracellular Ca2+ stores, and then incubated with TRH, the TRH-induced alkalinization was blunted; thus, an increase in [Ca2+]i is needed for full activation of Na+/H+ exchange. To study further the importance of agonist-induced changes in [Ca2+]i on the activation of Na+/H+ exchange, acid-loaded cells were incubated first with TPA, and then with either TRH or ionomycin. TPA induced a rise in pHi, which was further enhanced by TRH, but not ionomycin. The actions of both TRH and ionomycin on Na+/H+ exchange were attenuated, but not abolished, in cells pretreated with TPA for 36 h. Acidification of the cytosol with nigericin increased the resting [Ca2+]i level from 125 +/- 29 to 200 +/- 25 nM (P less than 0.01). The increase in [Ca2+]i was greatly attenuated when extracellular Ca2+ was chelated with EGTA before the addition of nigericin. Both the TRH- and ionomycin-induced increases in [Ca2+]i were blunted in acid-loaded cells. We conclude that in GH4C1 cells, a transient increase in [Ca2+]i can enhance Na+/H+ exchange and cause a rise in pHi, but that to obtain full activation of exchange, protein kinase C activity must also be stimulated. Furthermore, pHi is important in maintaining an adequate store of sequestered intracellular Ca2+ and in the release of Ca2+ from that store in response to TRH and ionomycin.     

Effects of verapamil and nifedipine on gliclazide-induced increase in cytosolic free Ca2+ in pancreatic islet cells

The changes in cytosolic free calcium [Ca2+]i induced by the sulfonylurea gliclazide and potassium in normal rat pancreatic islet cells were measured using the fluorescent Ca2+ indicator fura-2. Both in the absence or presence of 5.6 mM glucose, gliclazide caused a rapid and sustained increase in [Ca2+]i. The phenylalkylamine verapamil reduced these increases, but the Ca2+ channel blocker was more potent in the presence than in the absence of glucose. In contrast, nifedipine, a Ca2+ channel blocker of another chemical type, reduced to a similar extent the increase in [Ca2+]i evoked by gliclazide in the absence and presence of glucose. In the absence of glucose, a rise in extracellular K+ concentration from 5 to 20 or 30 mM also induced a rapid and sustained rise in [Ca2+]i. Verapamil more markedly reduced the rise in [Ca2+]i induced by 30 mM than by 20 mM K+. It is concluded that gliclazide increases Ca2+ inflow into normal islet cells primarily, if not exclusively, by opening voltage-sensitive Ca2+ channels. The differential sensitivity toward verapamil of gliclazide-induced rise in [Ca2+]i can be explained by the use-dependent block exerted by Ca2+ channel blockers of the phenylalkylamine type.     

Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels.

Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. However, direct evidence for activation of plasma-membrane Ca2+ channels by physiological signals in higher plants has not yet been obtained. In this context, several lines of evidence suggest that Ca2+ flux into the cytosol of guard cells is a major factor in the induction of stomatal closing by abscisic acid (ABA). ABA closes stomatal pores, thereby reducing transpirational loss of water by plants under drought conditions. To directly investigate initial events in ABA-induced signal transduction in guard cells, we devised an experimental approach that allows simultaneous photometric measurements of cytosolic Ca2+ and patch-clamp recordings of ion currents across the plasma membrane of single Vicia faba guard cells. Using this approach, we found that the resting cytosolic Ca2+ concentration was 0.19 +/- 0.09 microM (n = 19). In responsive guard cells, external exposure to ABA produced transient repetitive increases in the cytosolic free Ca2+ concentration. These Ca2+ transients were accompanied by concomitantly occurring increases in an inward-directed ion current. Depolarization of the membrane terminated both repetitive elevations in cytosolic Ca2+ and inward-directed ion currents, suggesting that ABA-mediated Ca2+ transients were produced by passive influx of Ca2+ from the extracellular space through Ca2(+)-permeable channels. Detailed voltage-clamp measurements revealed that ABA-activated ion currents could be reversed by depolarizations more positive than -10 mV. Interestingly, reversal potentials of ABA-induced currents show that these currents are not highly Ca2(+)-selective, thereby permitting permeation of both Ca2+ and K+. These results provide direct evidence for ABA activation of Ca2(+)-permeable ion channels in the plasma membrane of guard cells. ABA-activated ion channels allow repetitive elevations in the cytosolic Ca2+ concentration, which, in turn, can modulate cellular responses promoting stomatal closure.     

1,25-Dihydroxycholecalciferol enhances both the bombesin-induced transient in intracellular free Ca2+ and the bombesin-induced secretion of prolactin in GH4C1 pituitary cells

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances both the synthesis of PRL and the TRH-induced transient increase in cytosolic free calcium ( [Ca2+]i). In the present report we investigated whether 1,25-(OH)2D3 could enhance the effect of the tetradecapeptide bombesin (BBS) in GH4C1 cells. Pretreatment of the cells with 1 nM 1,25-(OH)2D3 for 24 h enhanced the BBS-induced transient increase in [Ca2+]i compared to that in control cells, while having no significant effect on the plateau phase of [Ca2+]i. Addition of the Ca2+ channel blocker nimodipine or chelating extracellular Ca2+ with EGTA did not abolish the enhancement of the BBS response in 1,25-(OH)2D3-pretreated cells. Furthermore, the BBS-induced efflux of 45Ca2+ from cells preequilibrated with 45Ca2+ was larger in cells treated with 1,25-(OH)2D3. Incubating GH4C1 cells with 1,25-(OH)2D3 alone or in combination with BBS for up to 72 h did not stimulate synthesis of PRL. However, the BBS-induced secretion of PRL was enhanced in cells pretreated with 1,25-(OH)2D3 for 24 h compared with that in vehicle-treated control cells. The effect of 1,25-(OH)2D3 on BBS-induced secretion was dose dependent, with 10(-11) M 1,25-(OH)2D3 enhancing the stimulated secretion of PRL. We conclude that in GH4C1 cells, pretreatment with 1,25-(OH)2D3 enhances the BBS-induced transient increase in [Ca2+]i. This effect may be due to a modulation of the availability of sequestered intracellular Ca2+ and/or membrane Ca2+ conductance. Furthermore, pretreatment with 1,25-(OH)2D3 enhanced secretion of PRL stimulated by BBS. The enhanced transient increase in [Ca2+]i may be the factor inducing the enhanced BBS-induced secretion of PRL.     

Thyrotropin-releasing hormone (TRH) stimulates biphasic elevation of cytoplasmic free calcium in GH3 cells. Further evidence that TRH mobilizes cellular and extracellular Ca2+

TRH stimulation appears to be coupled to PRL secretion, at least in part, by elevation of the concentration of Ca2+ free in the cytoplasm [( Ca2+]i). We employed an intracellularly trapped fluorescent probe of Ca2+, Quin 2, to measure [Ca2+]i in GH3 cells, cloned rat pituitary tumor cells. Basal [Ca2+]i in GH3 cells incubated in medium containing 1.5 mM Ca2+ was 148 +/- 8.6 nM (mean +/- SE). TRH caused a biphasic elevation of [Ca2+]i to 517 +/- 29 nM at less than 10 sec after TRH addition, followed by a decline towards the resting level over 1.5 min (first phase) and then a sustained elevation to 261 +/- 14 nM (second phase). We attempted to determine whether mobilization of cellular calcium or enhanced influx of extracellular Ca2+, or both, were involved in the elevation of [Ca2+]i during each of the two phases. In all experiments, the elevation of [Ca2+]i stimulated by TRH was compared with that induced by depolarization of the plasma membrane with high extracellular K+, which enhances Ca2+ influx. In medium with 1.5 mM Ca2+, K+-depolarization caused an elevation of [Ca2+]i to 780 +/- 12 nM. When the concentration of Ca2+ in the medium was lowered to 0.1 mM and 0.01 mM, basal [Ca2+]i was lowered to 114 +/- 3.4 and 110 +/- 11 nM, respectively. In medium with 0.1 and 0.01 mM Ca2+, peak K+ depolarization-induced elevation of [Ca2+]i was lowered to 30 +/- 3.9% and 7.3 +/- 2.0% of control, respectively. The peak second phase increase caused by TRH was reduced to 33 +/- 2.8% and 16 +/- 5.6% of control, respectively, whereas the peak first phase elevation of [Ca2+]i was lowered only to 79 +/- 5.5% and 52 +/- 10% of control in medium with 0.1 mM and 0.01 mM Ca2+, respectively. When cells were incubated in medium with 1.5 mM Ca2+ containing the Ca2+-channel blocking agents, nifedipine and verapamil, basal [Ca2+]i was not affected. Nifedipine plus verapamil, each at a maximally effective dose, lowered K+ depolarization-induced elevation of [Ca2+]i to 6.5 +/- 1.0% of control, the peak second phase increase caused by TRH to 28 +/- 4.3% of control, but the peak first phase elevation only to 64 +/- 3.7% of control. The decrease in the first phase response to TRH caused by the channel blockers appeared to be secondary to partial depletion of an intracellular, nonmitochondrial calcium pool.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.     

Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.

The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximately equal to 20 nM), with both C5a and ionomycin, cytosolic Ca2+ increases to less than 200 nM, and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O-2 when exposed to ionomycin but not to C5a. These results and other considerations suggest that (i) activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM; (ii) C5a receptor-mediated activation of Ca2+ influx may require cooperation between the neutrophil surface and some cytoplasmic organelle and/or redistribution of the C5a-receptor complexes on the cell surface; and (iii) the phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.     

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.     

Changes in cytosolic free Ca++ by histamine stimulation in isolated guinea-pig parietal cells (the second report)

It was demonstrated in our previous report that cytosolic free Ca++ concentration showed increase following histamine stimulation in the dose dependent manner, by double wave length excitation method using fura-2 as an intracellular Ca++ probe. In this paper, the investigation about the source of cytosolic free Ca++ coming out following histamine stimulation was performed. Both Ca++ channel blocker (Verapamil and Diltiazem) and an intracellular Ca++ antagonist (TMB-8) suppressed the increase of cytosolic Ca++ concentration following histamine stimulation. 10(-4)M verapamil, 10(-4)M diltiazem and 10(-4)M TMB-8 suppressed cytosolic Ca++ concentration from 355.9 +/- 5.7 nM to 213.8 +/- 4.9 nM, from 330.1 +/- 7.3 nM to 149.8 +/- 1.7 nM and from 324.9 +/- 6.1 nM to 151.3 +/- 3.8 nM respectively in the values at the point of 20 min after 10(-4)M histamine stimulation. The time course study revealed that the course of increase consisted of two components: the "initial rise" which was observed in the first 1 min. followed by the "continuous rise". The initial rise may reflect the Ca++ coming out from the intracellular Ca++ pool and the continuous rise may reflect the Ca++ flowed into cell from outside. Verapamil and diltiazem did not suppress the initial rise, while TMB-8 suppressed it. It is concluded that increase of cytosolic Ca++ concentration following histamine stimulation may be concerned with both extra- and intracellular Ca++ and that at first, intracellular Ca++ may be mobilized.