Regulatory effect of 1,25-dihydroxycholecalciferol on calcium fluxes in thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) on the regulation of calcium fluxes in rat thyroid FRTL-5 cells. The ATP-induced uptake of 45Ca2+ was decreased in cells pretreated with 1,25(OH)2D3 for 48 h. No effect was seen on basal uptake of 45Ca2+. At least a 24 h incubation period was required for the effect of 1,25(OH)2D3 to be expressed. Pretreatment with 1,25(OH)2D3 for 48 h did not change resting intracellular Ca2+ ([Ca2+]i) in fura-2-loaded FRTL-5 cells. However, the ATP-induced increase in [Ca2+]i was significantly enhanced in cells preincubated with 1,25(OH)2D3. The effect of 1,25(OH)2D3 was abolished in Ca(2+)-free buffer. No difference in the ionomycin-induced increase in [Ca2+]i was observed between control cells and cells pretreated with 1,25(OH)2D3. However, in Ca(2+)-free buffer the ionomycin response was decreased in cells incubated with 1,25(OH)2D3. The ATP-induced change in [Ca2+]i was decreased when ATP was added after ionomycin to cells treated with 1,25(OH)2D3. The results suggest that 1,25(OH)2D3 has a regulatory effect on Ca2+ fluxes in FRTL-5 cells, possibly by acting on Ca2+ sequestration.     

Pregnancy-specific modulatory role of mitochondria on adenosine 5'-triphosphate-induced cytosolic [Ca2+] signaling in uterine artery endothelial cells

Vascular endothelial cells respond to extracellular ATP by inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum followed by Ca2+ influx and subsequent synthesis of vasodilators. In this study, the contribution of mitochondria in shaping the ATP-induced Ca2+ increase was examined in ovine uterine artery endothelial cells from nonpregnant and pregnant (late gestation) ewes (NP- and P-UAEC, passage 4). The mitochondrial protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced a rapid mitochondrial depolarization. CCCP also slowly increased cytosolic [Ca2+] ([Ca2+]c), which then gradually declined to 10-20 nM above resting level. Pretreatment with CCCP for 30 min significantly inhibited both ATP and thapsigargin-induced [Ca2+]c, with inhibition in NP-UAEC more effective than in P-UAEC. Pretreatment of mitochondrial permeability transition pore inhibitor cyclosporine A did not affect CCCP-induced mitochondrial depolarization, but delayed CCCP-induced [Ca2+]c for about 12-15 min (we termed this the "window of time"). During the cyclosporine A-delayed window of time of CCCP-induced [Ca2+]c, ATP induced a normal Ca2+ response, but after this window of time, ATP-induced [Ca2+]c was significantly inhibited. Pretreatment of oligomycin B to prevent intracellular ATP depletion by F0F1-ATPase did not reduce the inhibition of ATP-induced [Ca2+]c by CCCP. Ruthenium red, a mitochondrial Ca2+ uptake blocker, did not mimic the inhibition of Ca2+ signaling by CCCP. In conclusion, our data show that mitochondrial Ca2+ depletion after dissipation of mitochondrial membrane potential with CCCP inhibits ATP-induced [Ca2+]c, mediated at the level of Ca2+ release from the endoplasmic reticulum. Moreover, our data revealed that P-UAEC is more resistant to the inhibitory effect of CCCP on [Ca2+]c than NP-UAEC.     

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 (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)     

Effects of ATP and UTP on [Ca2+]i, membrane potential and force in isolated rat small arteries.

We have investigated excitation-contraction coupling mechanisms associated with the activation of purinoceptors and putative pyrimidinoceptors by assessing the effects of ATP and UTP on cytoplasmic Ca2+ activity ([Ca2+]i), membrane potential (Em) and force in rat mesenteric small arteries. UTP induced a sustained concentration-dependent contractions, closely associated with concentration-dependent increases in [Ca2+]i. Superfusion with 0.1 mM UTP caused a sustained depolarisation of 12 +/- 1 mV (SE, n = 8). In Ca(2+)-free medium, the increase in [Ca2+]i and the contraction obtained with UTP (1 mM) were both transient and were inhibited by prior exposure to noradrenaline (NA). In vessels depolarised with KCl, UTP caused no change in Em, but a sustained increase in force and a transient increase in [Ca2+]i were induced, leading to an increased force/[Ca2+]i ratio. Similar effects on [Ca2+]i, Em and force were observed with ATP; but the effect of ATP on force was transient, whereas the effect on [Ca2+]i and Em declined only slowly. There was no crosstachyphylaxis between the responses to ATP and UTP: in the presence of 1 mM of either, the other drug induced contractions in low concentrations, as if they acted through distinct receptors. The results suggest that both UTP and probably ATP release intracellular Ca2+, possibly from the stores emptied by NA. The sustained response to UTP appears to be due to an influx of extracellular Ca2+. UTP but not ATP was found to enhance the force-generating effect of [Ca2+]i.     

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate, a calcium store blocker, increases calcium influx, inhibits alpha-1 adrenergic receptor calcium mobilization, and alters iodide transport in FRTL-5 rat thyroid cells.

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8) is known to inhibit mobilization of calcium from intracellular stores but, more recently, other cellular effects have been described. In the present study, the effects of TMB-8 on cytosolic free calcium [Ca2+]i levels in FRTL-5 rat thyroid cells were determined using the fluorescent dye, Indo-1. TMB-8 increased [Ca2+]i in a dose-dependent manner, with a maximum rise from 120 +/- 7 nM to 229 +/- 16 nM (90 +/- 5% increase) at 5 x 10(-4) M. This effect was considerably reduced in Ca2+ free buffer, demonstrating a dependency upon extracellular calcium influx but not upon membrane potential and which did not involve the Na+/Ca2+ exchanger. In Ca2+ free buffer TMB-8, at a dose which did not affect [Ca2+]i, completely prevented norepinephrine (10(-5) M) from mobilizing intracellular Ca2+. To determine whether TMB-8 affected differentiated functions, iodide uptake and efflux studies were performed with 125I. TMB-8 (10(-4) M) inhibited iodide uptake by approximately 40% without affecting efflux. At 10(-3) M TMB-8, efflux was also enhanced. These studies demonstrate that TMB-8 has at least two effects on [Ca2+]i, promoting calcium influx and preventing alpha-1 adrenergic mobilization from intracellular stores. TMB-8 also has multiple effects on 125I transport, both inhibiting influx and increasing efflux. The results emphasize the importance of characterizing the behavior of this compound in any cell system before using it as a biological probe.     

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.     

12-O-tetradecanoyl-phorbol-13-acetate decreases influx of extracellular Ca2+ induced by depolarization in GH4C1 cells: effects of pretreatment with 1,25-dihydroxycholecalciferol.

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol (1,25(OH)2D3) causes amplification of both the TRH-induced spike phase in cytosolic free calcium [( Ca2+]i) and the increase in [Ca2+]i induced by depolarization with K+. In the present study we investigated the actions of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Ca2(+)-homeostasis in GH4C1 cells pretreated with 1,25(OH)2D3 for 24 h. In control and 1,25(OH)2D3-pretreated cells, incubation with TPA (0.1-300 nM) for 15 min in the presence of 45Ca2+ did not affect the basal uptake of 45Ca2+. However, if the cells were treated with 50 mM K+, TPA induced a time- and concentration-dependent decrease in depolarization-induced net 45Ca2+ uptake. A maximal decrease of 30-50% was observed with 100-300 nM TPA, 1,25(OH)2D3 pretreated cells being more responsive to the action of TPA than control cells. sn-1-Oleoyl-2-acetyl-glycerol, which mimics the action of TPA on protein kinase C (PKC), did not alter depolarization-induced uptake of 45Ca2+. Two agents which inhibit PKC activity, polymyxin B and K252A, did not prevent the effect of TPA on depolarization-induced uptake of 45Ca2+, whereas staurosporin totally inhibited the action of TPA. In Fura-2 loaded cells pretreated with 1,25(OH)2D3, incubation with 200 nM TPA for 9 min decreased the depolarization-induced spike and plateau phases of change in [Ca2+]i; only the spike phase was decreased in control cells. TPA did not affect basal [Ca2+]i in either group. Treatment with TPA for only 3 min decreased the TRH-induced spike in [Ca2+]i only in 1,25(OH)2D3 pretreated cells; however, after a 5-min treatment with TPA, the TRH-induced spike in [Ca2+]i was decreased in both control and 1,25(OH)2D3 pretreated cells. TPA did not affect the spike in [Ca2+] induced by 50 nM ionomycin. Na+/Ca2+ exchange was not altered by TPA, nor did TPA enhance efflux of 45Ca2+ from cells preloaded with 45Ca2+ for 2.5 h. We conclude that, in GH4C1 cells, TPA modulates plasma membrane calcium flux, probably via an inhibitory action on voltage-operated Ca2+ channels. This inhibitory action may be independent of activation of PKC, and 1,25(OH)2D3 pretreated cells are more responsive to the actions of TPA than are control cells. These results are consistent with our previous findings that 1,25(OH)2D3 enhances voltage-dependent Ca2+ channel activity in GH4C1 cells.     

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.     

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.     

Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes

OBJECTIVE: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function. METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+]i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester. RESULTS: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+]i. Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5+/-1.2 s), a transient fall in [Ca(2+]i followed by an abrupt hypercontracture (37.1+/-1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+]i < 250 nM) recovered in only 23.3+/-5.1% of cells and contractions recovered in 15.3+/-2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with [Ca2+]i-free Tyrode or with an inhibitor of Na(+)/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 microM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2+/-3.6% and contractile function to 52+/-4.3%. CONCLUSIONS: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.     

Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium.

When human polymorphonuclear leukocytes (PMN) are placed on various surfaces, they attach and spread rapidly, increasing their diameter severalfold. The spreading is associated with extensive changes in the cytoskeleton. Since many cytoskeletal events are regulated by Ca2+, we measured the cytosolic free calcium concentration ([Ca2+]i) in individual human PMN as they spread. [Ca2+]i was measured in single cells by microspectrofluorometry using the fluorescent Ca2+-sensitive dye fura-2. Immediately before spreading, PMN exhibit a rapid increase in [Ca2+]i, from 69 +/- 51 nM to 547 +/- 190 nM (mean +/- SD, n = 12). [Ca2+]i returns to near resting levels during the next minute, as the cells spread. Neither the spreading nor the [Ca2+]i spike is blocked by removal of extracellular calcium, by verapamil, by calmodulin antagonists, or by mitochondrial or microtubule poisons. Spreading, but not the [Ca2+]i increase, is blocked by the microfilament inhibitor cytochalasin B. Both spreading and the [Ca2+]i spike are blocked by ATP depletion and reversibly blocked by placing the cells in medium containing hypertonic sucrose or sodium chloride. These data strongly suggest that an increase in [Ca2+]i, derived from nonmitochondrial intracellular pools, plays an important role in the microfilament-mediated process of PMN spreading.     

Free intracellular Ca2+ concentration ([Ca2+]i) and growth hormone release from purified rat somatotrophs. I. GH-releasing factor-induced Ca2+ influx raises [Ca2+]i.

This study was carried out to investigate the role of free intracellular Ca2+ ([Ca2+]i) in the action of GH-releasing factor (GRF) by determining whether GRF causes and increase in [Ca2+]i and whether this increase results from changes in Ca2+ influx/efflux and/or mobilization of intracellular Ca2+ stores. We used a purified preparation of normal rat somatotrophs and examined the changes in 45Ca uptake, [Ca2+]i measured with indo-1, intracellular cAMP, and GH release induced by GRF. GRF stimulated a concentration-related biphasic increase in [Ca2+]i. Both the GRF-dependent increase in [Ca2+]i and GH release were blocked by incubation in low Ca2+ medium and by the organic Ca2+ antagonists nifedipine and diltiazem. The measurement of 45Ca uptake, in both steady state and nonsteady state conditions, demonstrated directly that GRF stimulates Ca2+ influx into somatotrophs. These data demonstrate that the GRF-stimulated increase in [Ca2+]i is dependent on Ca2+ influx. Redistribution of intracellularly stored Ca2+ could not be detected, even though intracellular Ca2+ stores were present. Therefore, the increase is due to Ca2+ influx, and the biphasic nature of the increase in [Ca2+]i induced by GRF is due to a difference in the rate of activation of Ca2+ influx and Ca2+ removal from the cytosol.     

Free intracellular Ca2+ concentration and growth hormone (GH) release from purified rat somatotrophs. III. Mechanism of action of GH-releasing factor and somatostatin.

GH-releasing factor (GRF)-stimulated GH release is dependent on a biphasic increase in free intracellular Ca2+ concentration [( Ca2+]i), resulting from an influx of Ca2+ into somatotrophs, while the inhibitory action of somatostatin (SRIF) on basal and GRF-induced GH release results from its ability to lower [Ca2+]i by inhibiting Ca2+ influx. This study was carried out to investigate the mechanism by which GRF and SRIF regulate [Ca2+]i to control GH release. The roles of ion channels, cAMP-dependent processes, and protein kinase-C (PKC) were investigated by measuring changes in [Ca2+]i, 45Ca influx, and GH release when purified rat somatotrophs were exposed to high K+, cAMP analogs, prostaglandin E2, as well as the PKC activators 1,2-dioctanoyl-glycerol and phorbol 12-myristate 13-acetate. High K+ depolarization produced a rapid and transient increase in [Ca2+]i, while cAMP and prostaglandin E2 led to a sustained elevated [Ca2+]i. PKC activators produced a transient increase in [Ca2+]i, followed by a decrease to below baseline. All secretagogues tested raised [Ca2+]i by stimulating Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCC), since the increases in [Ca2+]i were blocked by incubation in Ca2(+)-free medium and by the dihydropyridine Ca2+ antagonist nifedipine. SRIF lowered [Ca2+]i by blocking the Ca2+ influx stimulated by all of these GH secretagogues except high K+. These results are consistent with the model in which GRF initiates its action by increasing Na+ conductance to depolarize the somatotroph via cAMP. This depolarization would stimulate Ca2+ influx through VSCC, which would result in the first phase of the GRF-dependent increase in [Ca2+]i. This increase in [Ca2+]i would stimulate Ca2+ removal from the cytosol by activating Ca-ATPase via Ca-calmodulin and/or PKC. This would result in the lowering of [Ca2+]i to the plateau level of the second phase of the GRF response. SRIF prevents the GRF-induced increase in [Ca2+]i by increasing K+ conductance and, thus, hyperpolarizing the cell. Hyperpolarization would close VSCC, leading to a decrease in Ca2+ influx, with a subsequent drop in [Ca2+]i.     

Potential mechanism of cyclosporine A-induced vascular smooth muscle contraction

Arterial hypertension is a common side effect of cyclosporine A therapy; however, the cellular mechanism of cyclosporine A-induced hypertension is still unknown. The present study, therefore, examined the effect of cyclosporine A on Ca2+ kinetics and contraction in primary cultures of vascular smooth muscle cells. Cyclosporine A (10 micrograms/ml) did not affect resting intracellular free Ca2+ ([Ca2+]i) levels (151 +/- 10 vs. 146 +/- 5 nM, NS), but augmented the 10(-8) M arginine vasopressin-induced increase of [Ca2+]i (delta 76 +/- 4 vs. delta 172 +/- 6 nM, p less than 0.001). This effect of cyclosporine A was also observed in Ca2+-free medium. Arginine vasopressin-stimulated [Ca2+]i efflux within 30 seconds compared with baseline efflux rates (1,644 +/- 146 vs. 2,591 +/- 373 cpm/mg prot/30 sec, p less than 0.005), but this transient effect was significantly greater (p less than 0.001) with arginine vasopressin plus cyclosporine A (1,702 +/- 133 vs. 5,605 +/- 1235 cpm/mg prot/30 sec, p less than 0.01). Basal 45Ca2+ efflux rates were not affected by cyclosporine A, and prior incubation of the cells with cyclosporine A was required to elicit the augmentory effect. 45Ca2+ uptake was measured to examine the mechanism by which cyclosporine A may affect [Ca2+]i stores. Cyclosporine A increased Ca2+ uptake when compared with control (6.38 +/- 0.69 vs. 10.99 +/- 0.59 x 10(3) cpm/mg prot/5 min, p less than 0.001). This effect was not blocked by the Ca2+ antagonist verapamil. Arginine vasopressin (10(-8) M) induced contraction of smooth muscle cells with 25.5% of the cells responding.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

Effect of ouabain on cellular free calcium and cellular cyclic AMP production in response to arginine vasopressin in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration [( Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0.1 mumol/l increased [Ca2+]i to 177.1 +/- 26.2 nmol/l from 92.2 +/- 8.0 nmol/l (P less than 0.01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.     

Bradykinin modulates arginine vasopressin-induced calcium influx in vascular myocytes.

We investigated direct, endothelium-independent effects of bradykinin on arginine vasopressin-induced calcium influx in vascular smooth muscle cells. We studied cultured rat vascular smooth muscle cells by using the whole-cell voltage-clamp and calcium fluorescence imaging methods. Exposing cultured vascular smooth muscle cells (A7r5 cell line) to arginine vasopressin (100 nM) produced a transient increase in [Ca2+]i, followed by a sustained increase in [Ca2+]i. This was readily reversible (n=28). At a holding potential of -40 to -60 mV, arginine vasopressin induced a sustained inward current correlated with a sustained increase in [Ca2+]i. Bradykinin (30 nM to 30 microM) had no effect on arginine vasopressin-induced [Ca2+]i transients. However, during the sustained phase of increased [Ca2+]i, bradykinin reversibly attenuated relative fluorescence and inward current in the presence of arginine vasopressin (n=14). This was concentration dependent and inhibited by [D-Phe7]-bradykinin (30 microM), a kinin receptor antagonist. Also, sustained arginine vasopressin-mediated increases in [Ca2+]i and inward current were attenuated by Ca2+-free or La3+-supplemented perfusate but not by nifedipine (n=5). Conclusions: (1) Bradykinin can attenuate arginine vasopressin-induced and sustained Ca2+ influx and sustained inward current through a novel endothelium-independent process. (2) The direct effect of bradykinin on arginine vasopressin-induced increases in [Ca2+]i sustained Ca2+ influx in vascular smooth muscle cells is concentration dependent and kinin-receptor mediated. (3) Arginine vasopressin-induced sustained [Ca2+]i elevation correlates with the activation of a dihydropyridine-insensitive, Ca2+-conducting inward current.     

Alpha 1-adrenergic stimulation of in vitro growth hormone release and cytosolic free Ca2+ in rat somatotrophs

The effects of alpha 1-adrenergic agents on GH release and intracellular free Ca2+ concentration ([Ca2+]i) were investigated in purified rat somatotroph preparations. Phenylephrine (PHE) stimulated in vitro GH release; the maximal effect (2.5-fold stimulation) occurred at 1 microM PHE. The effect was completely blocked by the alpha-adrenergic antagonist phentolamine and partially counteracted by the beta-antagonist propranolol. Experiments with the fluorescent Ca2+ probe fura 2 show that PHE causes [Ca2+]i to rise from 178 +/- 31 nM (mean +/- SE; n = 25) to 370 +/- 55 nM (n = 9). This effect was complete within 20 sec and was maintained for at least 5-10 min. The rise was rapidly interrupted by administration of 1 microM phentolamine. The beta-receptor agonist isoproterenol caused a small [Ca2+]i rise due to action on alpha 1-adrenoreceptors. The PHE-induced [Ca2+]i rise showed two components: an initial peak due to Ca2+ mobilization from intracellular stores and a subsequent rise due to Ca2+ influx from the extracellular space. Somatostatin (SRIF) lowered both resting [Ca2+]i and Ca2+ influx stimulated by PHE. Pertussis toxin pretreatment did not modify PHE-induced [Ca2+]i changes, while it completely prevented the effect of SRIF on both resting and triggered [Ca2+]i, thus suggesting that a GTP-binding protein sensitive to the toxin is involved in the transduction of SRIF action. The increase in cAMP induced by cholera toxin pretreatment modified neither PHE nor SRIF action on [Ca2+]i. In conclusion, in rat somatotrophs Ca2+ mobilization and influx are stimulated by alpha 1-adrenergic agents, and this triggered [Ca2+]i rise results in a stimulation of GH release. In these cells SRIF is able to reduce both resting [Ca2+]i levels and [Ca2+]i increases induced by alpha 1-adrenergic activation.