Thyrotropin-releasing hormone stimulation of prolactin secretion is coordinately but not synergistically regulated by an elevation of cytoplasmic calcium and 1,2-diacylglycerol

The precise roles of the calcium and lipid pathways in TRH-stimulated PRL secretion from rat pituitary (GH3) cells are controversial. In particular, it is debated whether elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i) is sufficient to cause burst secretion (0-2 min) or whether an increase in 1,2-diacylglycerol must accompany the Ca2+ elevation. In this study, the effects of TRH, which elevates 1,2-diacylglycerol, on [Ca2+]i and stimulation of burst secretion were compared with those of depolarization by high extracellular K+, which does not increase 1,2-diacylglycerol. A maximal concentration of TRH (1 microM) and depolarization by 17.5 mM K+ caused elevation of [Ca2+]i from the resting level of 140 +/- 20 nM to 470 +/- 70 nM and 514 +/- 60 nM, respectively, and stimulated burst secretion from 0.6 +/- 0.2 ng/10(6) cells/min to 3.3 +/- 0.8 and 3.1 +/- 0.4 ng/10(6) cells/min, respectively, when a small component of TRH-stimulated secretion that is independent of elevation of [Ca2+]i was subtracted. A detailed comparison of multiple levels to which [Ca2+]i was elevated (up to 600 nM) and the degree of stimulation of burst phase secretion demonstrated the same positive linear correlation (correlation coefficient = 0.96) for TRH and K+ depolarization. Hence, elevation of [Ca2+]i is sufficient to cause burst secretion irrespective of elevation of 1,2-diacylglycerol. Optimal stimulation by TRH of sustained secretion of PRL did not depend on elevation of [Ca2+]i; sustained PRL secretion stimulated by 10 nM TRH was 2.6 +/- 0.4 and 2.7 +/- 0.2 ng/10(6) cells/min in control cells and arachidonic acid-pretreated cells in which [Ca2+]i was not elevated, respectively. The data from this and previous studies demonstrate that elevation of [Ca2+]i and 1,2-diacylglycerol may act coordinately, but not synergistically, to mediate TRH stimulation of PRL secretion from GH3 cells.     

Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells

TRH stimulation of prolactin secretion is thought to be mediated by an elevation of free cytoplasmic Ca2+. However, whether TRH-induced influx of extracellular Ca2+ is required to elevate cytoplasmic Ca2+ remains controversial. We measured cytoplasmic free Ca2+ concentration in GH3 cells with an intracellularly trapped fluorescent indicator, Quin 2. In unstimulated cells incubated in medium containing 1.5 mM Ca2+, cytoplasmic free Ca2+ concentration was 118 +/- 18 nM (mean +/- SD). TRH (1 microM) caused a rapid transient elevation of free cytoplasmic Ca2+ to a level estimated to be at least 500 nM. High extracellular K+, which induces extracellular Ca2+ influx, caused an elevation of free cytoplasmic Ca2+ which was greater and longer in duration that that caused by TRH. When cells were incubated in medium containing 3 mM EGTA, the K+ depolarization-induced increase in free cytoplasmic Ca2+ was abolished. By contrast, the TRH-induced increase was not affected by incubating cells in medium with 3 mM EGTA, or high K+, or both; incubation of cells in medium with EGTA and high K+ abolishes the electrochemical driving force for Ca2+ influx. These data demonstrate that Ca2+ influx is not required for TRH-induced elevation of free cytoplasmic Ca2+ in GH3 cells. We conclude that in GH3 cells TRH induces an elevation of free cytoplasmic Ca2+ leading to stimulated prolactin secretion by mobilizing cellular Ca2+.     

Fura-2 imaging of thyrotropin-releasing hormone and dopamine effects on calcium homeostasis of bovine lactotrophs

Dual wavelength digital imaging microscopy to detect fura-2 has been employed to characterize in normal bovine PRL-secreting cells the effects of TRH and dopamine on the intracellular ionized calcium concentration [( Ca2+]i). Concentrations of TRH greater than 10 nM caused a rapid but transient increase in [Ca2+]i, arising mainly from intracellular calcium stores, since it was unaffected by lowering extracellular calcium with EGTA or blocking calcium channels with Co2+. The threshold for TRH action was close to 0.1 nM. TRH action was dose dependent, with lower concentrations (less than 1-10 nM) slowing the time to peak [Ca2+]i response. The TRH-induced [Ca2+]i rise had a Q10 of about 2. TRH caused multiple transient increases in [Ca2+]i, but a recovery time of 10-15 min was required for full restoration of the TRH-induced response. In some cells the [Ca2+]i response to TRH was polarized to one region of the cell, suggesting the following possibilities, none of them exclusive: 1) Ca2+ release sites may be localized within the cell; or 2) an efficient local mechanism exists for lowering Ca2+ once it is liberated inside the cells; or 3) barriers may exist to diffusion of Ca2+ released within the cell. Extracellular application of Co2+, Mn2+, and EGTA under basal conditions resulted in lowering of [Ca2+]i within seconds, consistent with tonic Ca2+ influx under resting conditions which could contribute to the basal release of hormone. Dopamine, a PRL release-inhibiting factor, also lowered [Ca2+]i under basal conditions. However, the [Ca2+]i response of lactotrophs to TRH was unaffected by dopamine. This suggests that dopamine and TRH act via separate intracellular pathways to modulate hormone secretion. Applications of forskolin preceding the TRH-induced transient rise in [Ca2+]i resulted in a prolonged plateau rise in [Ca2+]i. This was mainly due to increased influx of Ca2+ since addition of Co2+ or EGTA-containing or Ca(2+)-free medium during this phase of response lowered the plateau concentration of [Ca2+]i.     

TRH raises cytosolic Ca2+ in human adenomatous lactotrophs

The effect of TRH on cytosolic free calcium concentrations, [Ca2+]i, was evaluated on cell suspensions obtained from 6 human PRL secreting pituitary adenomas. In these cells resting [Ca2+]i levels were variable (mean +/- SE; 103.8 +/- 6.5, n = 25); the addition of 100 nM TRH caused a marked [Ca2+]i rise within 20 sec., the peak values ranging from 200 to 437 nM (285 +/- 10.8 nM, n = 10). The transients induced by TRH were composed by a rapid increase, due to mobilization of calcium from intracellular stores, followed within a few seconds by a lower plateau which was due to stimulated influx from the extracellular space. In fact, when EGTA and verapamil were applied after TRH they caused the Ca2+ plateau to dissipate rapidly. The addition of 1 microM dopamine (DA) caused a substantial decrease of resting [Ca2+]i (about 10-30%) as well as an inhibition of the plateau phase induced by TRH. The effect of DA completely depended on extracellular Ca2+. The TRH-induced transients observed in adenomatous cells were quite similar in size and time course to those recorded in normal rat lactotrophs. As previously observed in rat lactotrophs, in adenomatous cells treatment with pertussis toxin (PTx, 1 microgram/ml for 4 h) was unable to affect the [Ca2+]i transients induced by TRH while completely abolished the effect of DA. The effects of TRH on in vivo and in vitro PRL secretion were also evaluated. Before surgery, no patient showed a positive response to the iv administration of 200 micrograms TRH (serum PRL levels: 95 +/- 62 ng/ml in basal conditions vs 124 +/- 92 after TRH, P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of thyrotropin-releasing hormone on phosphoinositides and cytoplasmic free calcium in thyrotropic pituitary cells

We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion.     

Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2(+)-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10(-6) M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 +/- 11 nM in PMA-treated cells compared to 703 +/- 44 nM in control cells. High extracellular Ca2(+)-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10(-6) M) increased [Ca2+]i to an initial peak of 738 +/- 49 nM followed by a sustained increase to 501 +/- 30 nM in control cells (n = 15). After exposure to PMA (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 +/- 5 and peaked at 323 +/- 15 nM with ionomycin (10(-6) M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 +/- 19 nM observed with ionomycin (10(-6) M) in control cells (P less than 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2(+)-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potassium depolarization elevates cytosolic free calcium concentration in rat anterior pituitary cells through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels

Changes in membrane potential may influence Ca2+-dependent functions through changes in cytosolic free calcium concentration [( Ca2+]i). This study characterized pharmacologically those voltage-dependent Ca2+ channels in normal rat anterior pituitary cells that are involved in the elevation of [Ca2+]i upon high potassium-induced membrane depolarization. The [Ca2+]i was monitored directly by means of the intracellularly trapped fluorescent indicator fura-2. The addition of K+ (6-100 mM) increased [Ca2+]i in a concentration-dependent manner. The fluorescent signal reached a peak within seconds and then decayed to form a new elevated plateau. K+ at the highest concentration used (100 mM) raised [Ca2+]i by about 450 nM. The K+-induced increase in [Ca2+]i was absent in a Ca2+-free medium. BAY K 8644, a 1,4-dihydropyridine Ca2+ channel agonist, also caused an increase in [Ca2+]i. The maximum response in [Ca2+]i upon stimulation with BAY K 8644 (100 nM) was about 40 nM. The half-maximally effective concentration of BAY K 8644 (100 nM) was about 20 nM. The response in [Ca2+]i upon BAY K 8644-stimulation was abolished in a Ca2+-free medium. Predepolarization with various K+ concentrations enhanced the effect of BAY K 8644 (1 microM) on [Ca2+]i. Pretreatment with BAY K 8644 (1 microM) enhanced the response in [Ca2+]i induced by K+ (25 mM). The addition of Mg2+ (30 mM) and nifedipine (1 microM) lowered the resting [Ca2+]i by about 40 and 20 nM, respectively. Mg2+, nifedipine, nimodipine, G? 5438, verapamil, and diltiazem inhibited the K+ (25 mM)-induced increase in [Ca2+]i; the order of potency (and half-maximally inhibitory concentrations) were nimodipine = G? 5438 = nifedipine (approximately 100 nM) greater than verapamil (900 nM) greater than diltiazem (greater than 10 microM) greater than Mg2+ (6 mM). Omega-Conotoxin (100 nM) did not inhibit the K+ (25 mM)-induced increase in [Ca2+]i. These data demonstrate that, over a wide range, membrane depolarization induced by high potassium concentration is indeed associated with increases in [Ca2+]i in normal rat anterior pituitary cells. This elevation of [Ca2+]i is mainly due to an influx of Ca2+ through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels (L-type).     

U-73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing hormone effects in GH3 rat pituitary cells.

TRH increases cytosolic-free calcium ([Ca2+]i) by activating phospholipase C(PL-C), which induces phosphoinositol hydrolysis, leading to Ca2+ mobilization from inositol trisphosphate (IP3) sensitive stores, and by increasing Ca2+ influx. Increases in [Ca2+]i stimulate PRL secretion. We investigated the effects of U-73122, an aminosteroid inhibitor of PL-C dependent processes, on TRH-stimulated second messenger pathways and on PRL secretion in GH3 rat pituitary cells. [Ca2+]i was monitored by Indo-1 fluorescence, and IP3 and metabolites separated on ion exchange columns. In Ca(2+)-free buffer, [Ca2+]i was 96 +/- 6 nM and increased to 323 +/- 23 nM (P less than 0.001) after TRH (100 nM). U-73122 dose dependently inhibited the TRH effect (IC50 = 967 nM; complete inhibition at 3-5 microM). Subsequent addition of monensin (100 microM) increased [Ca2+]i from 107 +/- 4 to 142 +/- 4 nM (P < 0.001), confirming our previous findings of a non-TRH regulated Ca2+ pool in GH3 cells. Pretreatment (15 sec) with U-73122 partly inhibited the TRH effect on [Ca2+]i; complete suppression occurred with 70 sec of pretreatment. An inactive analog (U-73343) had no inhibitory effect at 5 microM. U-73122 acted noncompetitively, as the mean maximum velocity (expressed as percent increase in [Ca2+]i after TRH) was reduced from 225 to 91 while the Michaelis-Menten constant for TRH was unchanged (15.4 vs. 13.8 nM, n = 3). Of note, U-73122, at 3-5 microM, increased basal [Ca2+]i from 109 +/- 5 to 120 +/- 5 nM (P less than 0.001). In 1.3 mM Ca2+ buffer containing nifedipine (1 microM) and verapamil (50 microM), similar effects of U-73122 (5 microM) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 +/- 12%, 148 +/- 23%, and 167 +/- 39% of control, 30 sec after TRH (100 nM); these responses were prevented by 1 microM U-73122. At 5 microM, U-73122 also significantly increased IP3 levels. TRH (100 nM) increased 4-h PRL secretion from 16.3 +/- 1.4 to 27.6 +/- 3.2 ng/well (P less than 0.05). U-73122 (5 microM) increased basal PRL secretion to 35.9 +/- 3.2 ng/well (P less than 0.05), but abolished the TRH effect. In contrast, U-73343 (with Ca2+ channel blockers) did not inhibit the TRH effect on PRL (control: 24.3 +/- 2.1; TRH: 51.0 +/- 6.3 ng/well).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of phosphatidic acid on parathyroid hormone release, intracellular free Ca2+, and inositol phosphates in dispersed bovine parathyroid cells

The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionophore bromo-A23187 reveals cellular calcium stores in single pituitary somatotropes

Free cytosolic calcium concentration, [Ca2+]i, in single rat pituitary cells can be measured with the fluorescent, calcium-sensitive probe fura-2 and digital image analysis. A reverse hemolytic plaque assay (RHPA) identifies somatotropes in the mixed population of pituitary cells. Previous studies showed that growth hormone releasing factor (GRF) stimulates growth hormone (GH) release from pituitary somatotropes by increasing the influx of calcium into the cell. Somatostatin reduced [Ca2+]i and inhibits hormone release presumably by closing calcium channels in the membrane. The calcium-ionophore bromo-A23187 rapidly increased [Ca2+]i from a baseline of 226 +/- 38 nM to a peak of 842 +/- 169 nM (mean +/- SEM) which was reached 30 s after exposure to the drug. This spike was followed by a sustained phase of elevated [Ca2+]i approximately 370 nM. When somatostatin (SRIF) (10 nM) was combined with ionophore treatment, the initial rise was preserved. However, the second phase was abolished and SRIF lowered [Ca2+]i to 57 +/- 7 nM. Depolarizing the cellular membrane with high extracellular potassium (60 mM) increased cytosolic calcium as well (797 +/- 178 nM); however, this was not affected by the addition of SRIF (988 +/- 71 nM). KCl depolarization in calcium-free medium (+1.5 mM EGTA) provoked no rise in cytosolic calcium. In contrast, after ionophore, the initial spike was preserved while the sustained phase of elevated [Ca2+]i was abolished. We conclude from these data that (1) membrane depolarization and ionophore treatment lead to an influx of calcium into the cytosol of normal pituitary somatotropes. (2) SRIF inhibits calcium influx induced by ionophore but not influx after depolarization with high potassium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose induces insulin release and a rise in cytosolic calcium concentration in a transplantable rat insulinoma

An important role for calcium in the cellular events leading to insulin secretion is supported by many studies. However, simultaneous measurements of changes in intracellular free Ca2+ concentrations [( Ca2+]i) and insulin release in response to secretagogues have not been performed. Using cells isolated from a glucose-responsive insulinoma, changes in [Ca2+]i were measured with the fluorescent calcium probe quin2. With the nutrient secretagogues glucose (30 mM) and D,L-glyceraldehyde (GA; 20 mM), [Ca2+]i increased slowly, reaching a peak approximately 15 min after addition of the stimulus, while KCl (25 mM) and carbachol (2 mM) led to a rapid but transient increase in [Ca2+]i. Glucose increased [Ca2+]i from 104 +/- 6 (mean +/- SEM) to 248 +/- 31 mM (n = 13), and GA caused a rise in [Ca2+]i from 96 +/- 6 to 280 +/- 39 nM (n = 4). KCl and carbachol caused a rise from 107 +/- 6 to 184 +/- 5 nM and from 98 +/- 5 to 157 +/- 5 nM, respectively (n = 5 each). When insulin release was measured simultaneously with changes in [Ca2+]i and compared to unstimulated cells, the following results were obtained. During the first 5 min of stimulation, high glucose caused a 90 +/- 12% increase in insulin release and a 72 +/- 11% rise in [Ca2+]i (n = 5). GA evoked a 122 +/- 30% increase in insulin secretion, with a 82 +/- 17% rise in [Ca2+]i (n = 3). Both KCl and carbachol caused a 58 +/- 9% increase in insulin release, with 7 +/- 4% and 50 +/- 2% rises in [Ca2+]i, respectively (n = 4 each). Insulin release was also measured in a perifusion system. It was shown that glucose (30 mM), GA (20 mM), and alpha-ketoisocaproate (30 mM) caused a biphasic release of insulin, while KCl (25 mM) and carbachol (2 mM) caused a monophasic release. The results show that [Ca2+]i increases during the stimulation of insulin secretion when measured simultaneously on the same beta-cells. However, while these changes coincide, a simple direct quantitative relationship between insulin release and the rise in [Ca2+]i could not be demonstrated.     

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.     

Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone

The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.6]GnRH. GnRH elevated [Ca2+]i only in gonadotroph-enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca2+]i in mammotroph- and somatotroph-enriched cells fractions, respectively. A rapid increase (first phase) in [Ca2+]i induced by GnRH was observed in Ca2+-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca2+ to the medium induced a second slower rise in [Ca2+]i (plateau phase). Addition of K+ caused a rapid rise in [Ca2+]i, which was dependent on extracellular Ca2+, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca2+]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca2+]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca2+]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca2+ pools and partly from influx of Ca2+ across the cell membrane.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for Ca2+ entry after stimulation with ATP

The relationship between ATP-induced uptake of 45Ca2+ and the ATP-induced changes in [Ca2+]i was investigated in rat FRTL-5 thyroid cells. Addition of 1 microCi 45Ca2+/ml together with ATP induced a time- and dose-dependent increase in uptake of 45Ca2+, the uptake being still significantly above control after 30 min. Resting intracellular free Ca2+ levels ([Ca2+]i), measured using Fura-2, was determined to be 60 +/- 14.3 nM (mean +/- SE). ATP induced a rapid, transient increase in [Ca2+]i (785 +/- 56.2 nM) followed by a plateau phase (127 +/- 34.3 nM). In a Ca(2+)-free buffer, the ATP-induced transient was significantly decreased (357 +/- 57.4 nM, p less than 0.05), and the plateau phase was abolished. The results suggested that stimulating FRTL-5 cells with ATP induced an influx of Ca2+, possibly by a mechanism dependent on a transient increase in [Ca2+]i. To further test this possibility, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was tested. In cells loaded with BAPTA, the ATP-induced uptake of 45Ca2+ was greatly enhanced, while the ATP-induced transient increase in [Ca2+]i was almost totally abolished. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after termination of the ATP response induced a rapid increase in [Ca2+]i. Furthermore, addition of Mn2+ to cells stimulated with ATP induced a more rapid quenching of Fura-2, compared to that seen in control cells. The results indicate that stimulating FRTL-5 cells with ATP induces a rapid release of Ca2+ from intracellular stores, followed immediately by an increase in plasma membrane conductance and influx of extracellular Ca2+. The ATP-induced change in [Ca2+]i may function as a signal enhancing influx of extracellular Ca2+, although some other unknown mechanism(s) is also needed.     

Lidocaine and procaine inhibit the increase in cytosol Ca2+ induced by thyrotropin-releasing hormone or K+ depolarization in GH4C1 cells

Lidocaine at greater than or equal to 1 mM and procaine at greater than or equal to 2.5 mM exerted dose-dependent inhibition of the increment in [Ca2+]i induced by 100 nM thyrotropin-releasing hormone (TRH) or 30 mM K+ in GH4C1 cells. The rise in [Ca2+]i induced by K+ was more sensitive to this inhibition than that induced by TRH. Lidocaine was more potent than procaine in inhibiting the [Ca2+]i increment induced by secretagogues. Maximal lidocaine inhibition of the TRH-induced [Ca2+]i increment occurred within 15-20 min and a normal response to secretagogues returned within 20 min after removal of lidocaine from the incubation medium. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced intracellular Ca2+ mobilization, depolarization of the cell membrane, and the opening of voltage-dependent Ca2+ channels. This may explain the depression of secretagogue-stimulated hormone secretion induced by these agents.     

Mechanisms involved in the effects of TRH on GHRH-stimulated growth hormone release from ovine and bovine pituitary cells

Incubation of cultured ovine pituitary cells with growth hormone-releasing hormone (GHRH) (10(-12)-10(-7) M) stimulated growth hormone secretion up to 3-fold. At a maximal stimulatory concentration of GHRH (10(-10) M), thyrotropin-releasing hormone (TRH) (10(-7) M) caused an inhibition of growth hormone release to approx. 50% of the response obtained with GHRH alone (during a 15 min incubation period). TRH also caused a small inhibition of the GHRH-stimulated cellular cyclic AMP level but this effect was only significant at a relatively high concentration of GHRH (10(-9) M). Incubation of cultured bovine pituitary cells with GHRH (10(-11)-10(-8) M) plus TRH (10(-7) M) caused a significant stimulation of growth hormone release by up to 40%, compared with the response obtained with GHRH alone (at all concentrations of GHRH). TRH (10(-7) M) had no effect on GHRH (10(-8) M)-stimulated cellular cyclic AMP levels in a partially purified bovine pituitary cell preparation. The effects of varying extracellular [Ca2+] (0.1-10 mM) on intracellular [Ca2+] and on the responsiveness to releasing hormones were also determined using ovine pituitary cells. GHRH (10(-10) M)-stimulated growth hormone release was inhibited when cells were incubated at both high (10 mM) and low (0.1 mM) [Ca2+] (compared with 1 mM or 3 mM Ca2+) with or without TRH (10(-7) M). At 1 mM Ca2+, TRH produced a synergistic effect with GHRH to stimulate growth hormone release. However, at 3 mM Ca2+ TRH inhibited GHRH-stimulated growth hormone release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of basal and corticotropin-releasing hormone-stimulated adenylate cyclase activity and cytosolic Ca2+ levels by somatostatin in human corticotropin-secreting pituitary adenomas

The effects of CRH and somatostatin (SRIH) on adenylate cyclase (AC) activity, intracellular free calcium concentrations [( Ca2+]i) and in vitro ACTH release were investigated in six human ACTH-secreting pituitary adenomas. In all tumors, CRH induced a marked stimulation (from 69-210% at 10 nM), whereas SRIH caused a definite inhibition (from 29-50% at 100 nM) of membrane AC. When added together, CRH and SRIH caused a purely additive effect on AC. In adenomatous corticotrophs CRH (10 nM) caused [Ca2+]i to rise from 160 +/- 30 nM (mean +/- SD) to 410 +/- 95 nM. CRH-induced transients were biphasic, with an initial peak predominantly due to redistribution from intracellular Ca2+ stores and a secondary phase due to Ca2+ influx. The effects of CRH on [Ca2+]i were totally independent of the stimulation of AC. In fact, cAMP-elevating agents other than CRH did not modify [Ca2+]i. SRIH (100 nM) decreased resting [Ca2+]i (approximately 20-40%) as well as [Ca2+]i rises induced by CRH, arginine vasopressin, or high K+. The effect of SRIH on [Ca2+]i was maintained in presence of high cAMP levels, while was totally abolished after pertussis toxin pretreatment. CRH (10 nM) stimulated ACTH release (from 22.5 +/- 3.5 to 45.0 +/- 8.5 pmol/L) by an extent similar to that elicited by calcium ionophore and forskolin. By contrast, SRIH (0.1 microM) inhibited both basal and CRH-stimulated ACTH release. In conclusion, in human adenomatous corticotrophs SRIH exerts an inhibitory action by reducing both AC activity and, independently, [Ca2+]i. In this way, SRIH can efficiently counteract the stimulatory action of CRH that in these cells involves activation of both cAMP and Ca2+ pathways.     

Influx of extracellular calcium mediates 1,25-dihydroxyvitamin D3-dependent transcaltachia (the rapid stimulation of duodenal Ca2+ transport)

We investigated the role of extracellular Ca2+ in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] rapid stimulation of intestinal Ca2+ transport (termed transcaltachia) in the perfused duodenal of vitamin D-replete chicks. The carboxylic ionophore ionomycin (2 microM) was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25-(OH)2D3. The increase in duodenal 45Ca2+ transport caused by 1,25-(OH)2D3 was dependent on the presence of medium Ca2+, since it was abolished by prior addition of EGTA and was restored upon the addition of Ca2+. Depolarization of the basal lateral membrane of intestinal epithelial cells with 70 mM K+ caused a rapid increase in 45Ca2+ transport (30% above control values within 2 min and 250% after 20 min of vascular perfusion). The rise was also abolished by prior addition of EGTA. Intracellular calcium concentrations ([Ca2+]i) were measured in isolated duodenal cells from vitamin D-replete chicks using the fluorescent dye fura 2. A 1-min incubation with physiological concentrations of 1,25-(OH)2D3 (130 pM) caused an increase in [Ca2+]i from a basal level of 168 +/- 23 nM to 363 +/- 44 nM. Pretreatment of intestinal epithelial cells with the protein kinase-C activator tetradeconyl-phorbol acetate (100 nM) or the adenylate cyclase activator forskolin (10 microM), both shown to induce acute stimulation of intestinal 45Ca2+ transport in the perfused duodenum, also mimicked the stimulatory effect of 1,25-(OH)2D3 on [Ca2+]i. The increase in [Ca2+]i elicited by the 1,25-(OH)2D3 was due to Ca2+ influx from the extracellular medium, since it was blocked by the Ca2+ chelator EGTA (5 mM) and the Ca2+ channel antagonist nifedipine (1 microM). These results suggest that the acute effects of 1,25-(OH)2D3 on duodenal 45Ca2+ transport are triggered by the influx of Ca2+ through voltage-operated Ca2+ channels and that both protein kinase-C and protein kinase-A play an important role in mediating or modulating 1,25-(OH)2D3 effects on transcaltachia.     

The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.

The effects of the muscarinic cholinergic receptor agonist carbachol on intracellular calcium ion concentration ([Ca2+]i) and progesterone production was determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the calcium-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 13). There was a 4- to 8-fold increase in [Ca2+]i in 85% (n = 80) of the cells within 10 sec after the addition of a maximally stimulating concentration (2 mM) of carbachol. The initial [Ca2+]i spike was followed by a sustained, but lower, calcium elevation, with superimposed oscillations which returned to resting level after several minutes. Both phases of the calcium transient were inhibited by pretreating the cells with atropine (1 microM), pirenzepine (2 microM) or 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). The sustained phase of the response with its superimposed oscillations, but not the initial spike, was inhibited by pretreating the cells with the calcium channel blockers lanthanum (1 mM), cobalt (5 mM), or methoxyverapamil (50 microM), or by incubating the cells in calcium-free medium. Nifedipine (0.5-20 microM) did not affect the carbachol-induced calcium transient. 8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (50 microM) blocked the sustained-oscillatory phase of the carbachol-induced [Ca2+]i transient, but did not affect the initial spike when added before carbachol (2 mM). Despite its ability to stimulate [Ca2+]i surges, carbachol (10(-6)-10(-3) M) did not affect basal or LH-stimulated cAMP or progesterone production in 24-h cultures. These studies demonstrate that activation of chicken granulosa cell muscarinic receptors causes a rapid increase in [Ca2+]i through the release of Ca2+ from intracellular stores, followed by a sustained elevation of Ca2+ with superimposed oscillations caused by the influx of extracellular Ca2+. These results also indicate that an increase in [Ca2+]i in chicken granulosa cells does not alone stimulate steroidogenesis, since the carbachol-induced increases in [Ca2+]i were not accompanied by increased progesterone production.