Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.     

Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells.

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.     

Endotoxicosis modulates cytosolic free calcium and basal and ACTH-stimulated lipolysis in rat adipocytes

Lipolytic rates and intracellular Ca2+ concentration ([Ca2+]i) were determined under basal conditions and upon stimulation with adrenocorticotropic hormone (ACTH), norepinephrine (NE) and insulin (I), in adipocytes isolated from control and acutely endotoxin (ET)-treated rats (1 mg/100 g body weight, LD50 at 6 h). [Ca2+]i measurements were done using the fluorescent Ca2(+)-indicator Fura-2. NE and ACTH, but not I, produced a marked increase of [Ca2+]i in cells of both control and ET-treated rats. ET treatment elicited a significant increase in [Ca2+]i of resting cells, and enhanced the ACTH effect on this parameter. The changes in lipolytic activity correlated well with changes of [Ca2+]i induced by ACTH. The results indicate that ET-induced alterations in intracellular calcium homeostasis of adipocytes may contribute to the mediation of effects on fat mobilization during endotoxemia.     

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.     

Endothelin-induced mobilization of Ca2+ and the possible involvement of platelet activating factor and thromboxane A2

Endothelin (ET) caused transient and sustained elevations of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat and rabbit vascular smooth muscle cells (VSMC). Specific platelet activating factor (PAF) antagonists (CV-6209 and WEB-2086) and arachidonic acid (AA) cascade blockers (chlorpromazine, indomethacin, CV-4151 and AA-2414) potently inhibited the ET-induced increase in [Ca2+]i. Additionally, these compounds inhibited the PAF-induced increase in [Ca2+]i. These results suggest that PAF and thromboxane A2 (TXA2) may be involved in the mechanism of ET-induced mobilization of Ca2+ in cultured rat and rabbit VSMC.     

Interaction of synthetic sarafotoxin with rat vascular endothelin receptors

The effects of synthetic analogs of sarafotoxin (STX) S6b, a snake venom peptide with a high sequence homology to the endothelium-derived vasoconstrictor endothelin (ET), on ET receptor binding activity and cytosolic free Ca2+ concentration [( Ca2+]i) were studied in cultured rat vascular smooth muscle cells. Binding studies revealed that [Cys1-15, Cys3-11] STX competed with 125I-ET for the binding to its vascular receptors with lower affinity than that of ET, but was far more effective than [Cys1-11, Cys3-15]STX in inhibiting the binding. [Cys1-15, Cys3-11]STX had a less potent effect on increasing [Ca2+]i than ET, whereas [Cys1-11, Cys3-15]STX was inactive. These data suggest that there may exist heterogenous subpopulations of the vascular ET/STX receptors, and that the proper double cyclic structure of STX is essential for interacting with its putative receptors to induce the [Ca2+]i response.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary cultures

We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca2+ concentrations [Ca2+]i) in rat aortic smooth muscle cells in primary culture. In Ca2+-containing medium, ET induced a rapid and sustained elevation of [Ca2+]i. In the latter component, in particular, the elevation of [Ca2+]i was inhibited by diltiazem. In Ca2+-free medium, ET induced a rapid and transient [Ca2+]i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+ store was practically depleted by repeated treatment with caffeine in Ca2+-free media, ET did not elevate [Ca2+]i. Thus, it was suggested that ET induces [Ca2+]i elevation not only by extracellular Ca2+-dependent, mechanisms but also by releasing Ca2+ from the intracellular store, and that the ET-sensitive Ca2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.     

Specific receptor for endothelin in cultured rat cardiocytes

Specific binding sites for the endothelium-derived vasoconstrictor endothelin (ET) and its effect on cytosolic free Ca2+ concentrations [( Ca2+]i) were studied in a primary culture of cardiocytes from neonatal rats. Binding studies using 125I-labeled-porcine ET as a radioligand revealed the presence of a single class of high-affinity binding sites for ET in cardiocytes with an apparent Kd of 6-9 x 10(-10) M and a Bmax of 50,000-80,000 sites/cell. Neither various vasoconstrictors nor Ca2+-channel blockers affected the binding. Pretreatment with ET substantially reduced the total number of ET receptors without changing their affinity. ET dose-dependently increased [Ca2+]i in fura-2-loaded cardiocytes. These data indicate that cardiocytes have specific ET receptors that are controlled by a down-regulation mechanism, and that ET induces a receptor-mediated increase in [Ca2+]i in cardiocytes.     

Differential trafficking and desensitization of human ET(A) and ET(B) receptors expressed in HEK 293 cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET(A) and ET(B) receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET(A) and ET(B) receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET(A) receptors, 83 +/- 2% of these receptors colocalized with pan cadherin. In ET(B) receptor-transfected cells, 54 +/- 2% of the receptor colocalized with pan cadherin. When ET(A) and ET(B) receptors were cotransfected, 97 +/- 1% of ET(B) receptors colocalized with ET(A) receptors and 84 +/- 2% of ET(B) receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET(B) receptor agonist) increased [Ca2+]i in cells transfected with ET(A) or ET(B) receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET(B) receptors desensitized faster (t(1/2) = 21 +/- 1 sec) than ET(A) receptors (t(1/2) = 48 +/- 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ET(B) receptors only (t(1/2) = 17 +/- 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET(A) receptors localize to the cell membrane, whereas ET(B) receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET(B) receptors desensitize faster than ET(A) receptors, but receptor coexpression eliminates desensitization. Finally, ET(A) and ET(B) receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.     

T-cell stimulation through the T-cell receptor/CD3 complex regulates CD2 lateral mobility by a calcium/calmodulin-dependent mechanism

T lymphocyte activation through the T cell receptor (TCR)/CD3 complex alters the avidity of the cell surface adhesion receptor CD2 for its ligand CD58. Based on the observations that activation-associated increases in intracellular [Ca2+] ([Ca2+]i) strengthen interactions between T cells and antigen-presenting cells, and that the lateral mobility of cell surface adhesion receptors is an important regulator of cellular adhesion strength, we postulated that [Ca2+]i controls CD2 lateral mobility at the T cell surface. Human Jurkat T leukemia cells were stimulated by antibody-mediated cross-linking of the TCR/CD3 complex. CD2 was labeled with a fluorescently conjugated monoclonal antibody. Quantitative fluorescence microscopy techniques were used to measure [Ca2+]i and CD2 lateral mobility. Cross-linking of the TCR/CD3 complex caused an immediate increase in [Ca2+]i and, 10-20 min later, a decrease in the fractional mobility of CD2 from the control value of 68 +/- 1% to 45 +/- 2% (mean +/- SEM). One to two hours after cell stimulation the fractional mobility spontaneously returned to the control level. Under these and other treatment conditions, the fraction of cells with significantly elevated [Ca2+]i was highly correlated with the fraction of cells manifesting significantly reduced CD2 mobility. Pretreatment of cells with a calmodulin inhibitor or a calmodulin-dependent kinase inhibitor prevented Ca2+-mediated CD2 immobilization, and pretreatment of cells with a calcineurin phosphatase inhibitor prevented the spontaneous reversal of CD2 immobilization. These data suggest that T cell activation through the TCR/CD3 complex controls CD2 lateral mobility by a Ca2+/calmodulin-dependent mechanism, and that this mechanism may involve regulated phosphorylation and dephosphorylation of CD2 or a closely associated protein.     

Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells

The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ ([Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in [Ca2+]i dependent on extracellular Ca2+, and release of [3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell [Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained [Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of [Ca+]i increases. The temporal pattern of the sustained [Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained [Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying [Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]i). The relative roles of the two PKGI isozymes, PKGIalpha and PKGIbeta, in cyclic GMP-mediated inhibition of [Ca2+]i in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+]i in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGIalpha or PKGIbeta were created, and the ability of PKGI isoforms to inhibit [Ca2+]i in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGIalpha or PKGIbeta, 8-Br-cGMP activation suppressed [Ca2+]i by thrombin receptor activation peptide (TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively (p <0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha is the predominant VSMC PKGI isoform. [Ca2+]i following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]i in Co403, causing a 4-fold increase in the EC50 for [Ca2+]i. In the absence of 8-Br-cGMP, suppression of PKGIalpha levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca2+]i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.     

Endothelin-1 stimulates the release of preloaded [3H]D-aspartate from cultured cerebellar granule cells

We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded [3H]D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca2+, but was unaffected by 1 mM Co2+ or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.     

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

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

The role of extracellular Ca2+ in the response of the hepatocyte to Ca2+-dependent hormones

The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.