Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.     

Mentoring: a professional commitment

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca2+ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca2+]i, with half-maximal effect (EC50) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ETA receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca2+ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca2+]i to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i response to these peptides. ETs exhibited homologous desensitization of the Ca2+ response, but partial heterologous desensitization of the Ca2+ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca2+ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by these peptides is due to the activation of ETA receptors and subsequently the release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)     

ETA and ETB receptors on vascular smooth muscle cells from mesenteric vessels of spontaneously hypertensive rats

1. To investigate the contribution of ETA and ETB receptors, calcium responses to the ETB agonist, IRL-1620, to endothelin-1 (ET-1) and to the ETA antagonist, BQ-123, were examined in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric vessels of 3, 9 and 17 week old spontaneously hypertensive rats (SHR), Wistar and Wistar-Kyoto (WKY) rats using Fura-2 methodology. 2. IRL-1620 (10(-7) mol/L) and ET-1 (10(-9) mol/L) increased [Ca2+]i in all strains and ages. Responses to ET-1 and IRL-1620 were blunted in 17 week SHR. BQ-123 significantly reduced ET-1-stimulated [Ca2+]i. In endothelium-denuded mesenteric vessels, ET-1 and IRL-1620 induced significant [Ca2+]i responses. 3. Binding of ET-1 was significantly lower in mesenteric artery membranes from 17 week SHR compared to controls. 4. Thus, ETA and ETB receptors are present in rat mesenteric VSMC. In adult SHR, a reduced density of ET receptors results in decreased responses to IRL-1620 and to ET-1.     

Endothelin-induced calcium signaling and secretion in chief cells and fibroblasts from pathological human parathyroid glands

Endothelins (ETs) are 21 amino acid peptides with vasoactive and mitogenic properties. The three isopeptides (ET-1, -2, and -3) and their receptors (E1A and ETB subtypes) display expression in numerous tissues and possibly mediate autocrine/paracrine actions. The present investigation shows that ET-1 triggers biphasic increases of the concentration of cytoplasmic Ca2+ ([Ca2+]i) in pathological human parathyroid cells. Both the peak and sustained [Ca2+]i increase, as well as the proportion of responding cells, are dose-dependent in the 10(-10)-10(-7) mol/L range of ET-1. In absence of external Ca2+, the ET-1-induced [Ca2+]i peak is attenuated. ET-3 has no effect on [Ca2+]i indicating functional dominance of the ETA receptor subtype. ET-1 (10 nmol/L) lowers parathyroid hormone secretion in 0.5 mmol/L but not in higher external Ca2+ concentrations, and parathyroid cell ET release is inhibited by increases of external Ca2+. Fibroblasts overgrowing the parathyroid chief cells during monolayer culture respond to ET-1 with biphasic [Ca2+]i increases or repetitive [Ca2+]i spikes, but show no response to elevation of external Ca2+. These findings imply that ET secretion and ET receptor expression may constitute an autocrine/paracrine mechanism in the regulation of human PTH secretion.     

Histamine-induced calcium released from cultured human mucosal microvascular endothelial cells from nasal inferior turbinate

Changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human mucosal microvascular endothelial cells (HMMECs) from nasal inferior turbinate were measured using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy. Histamine caused a transient increase in intracellular free Ca2+ in cell populations and in individual cells, followed by a decrease to a sustained elevation. Histamine (100 microM) elevated [Ca2+]i in HMMECs up to 563 +/- 20 nM from a resting level of 60 +/- 45 nM (means +/- SD, n = 31). Promethazine (a histamine H1 receptor antagonist) inhibited [Ca2+]i increase during histamine stimulation, whereas cimetidine (a H2 receptor antagonist) and thioperamide (a H3 receptor antagonist) showed no inhibition. These results suggest that the histamine increase [Ca2+]i in HMMECs induces both a Ca2+ release from stores and a Ca2+ influx through activation of the H1 receptor.     

Intracellular free Ca2+ elevations in cultured astroglia induced by neuroligands playing a role in cerebral ischemia

For better understanding of glial participation in cerebral ischemia, spectrofluorimetric analysis using the calcium indicator Fura-2AM was applied to examine the role of intracellular free Ca2+ ([Ca2+])i elevation induced by different neuroactive substances in cultured rat brain astrocytes. The activation by the general receptor agonist glutamate resulted in a biphasic cell response in [Ca2+]i. We couldn't observe N-methyl-D-aspartate-evoked [Ca2+]i response at all. Quisqualate triggered a complex [Ca2+]i response in astrocytes consisting of mobilization of Ca2+ from the intracellular stores and also Ca2+ influx from the extracellular space. Kainate elicited a markedly different Ca2+ signal an external Ca(2+)-dependent sustained [Ca2+]i rise resulting from the activation of the ionotropic glutamate receptor. According to our results two types of glutamate receptors, the quisqualate-specific metabotropic and kainate-specific ionotropic receptor, are involved in [Ca2+]i elevation in these cultures. We could monitor agonist-specific cell response to noradrenaline, serotonin, vasopressin and ATP as well in these cultured rat astrocytes.     

Ecomycins, unique antimycotics from Pseudomonas viridiflava

While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+]i) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ET(A) receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+]i levels but led to a significant increase in peak [Ca2+]i response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 (an ET(B) selective agonist), ANG II, and vasopressin remained unaffected. ET-1-evoked peak [Ca2+]i responses were significantly attenuated by the inclusion of the ET(A) antagonist, BQ123, in both groups. The ET(B) antagonist, BQ788, abolished [Ca2+]i responses to IRL-1620 but failed to affect the exaggerated [Ca2+]i responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by 125I-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by 125I-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ET(A) mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide. Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [3H]BQ-123 revealed a twofold higher number of ET(A) specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+]i responses via increased vascular ET(A) receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

Activation of calcium sparks by angiotensin II in vascular myocytes

Contraction in smooth muscle is triggered by an increase in cytoplasmic free calcium ([Ca2+]i) which depends on both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from the sarcoplasmic reticulum (SR). Two mechanisms have been shown to be involved in SR Ca2+ release, one is stimulated by Ca2+ and involved ryanodine-sensitive Ca2+-release channels; the other is stimulated by an increase in inositol 1,4,5-trisphosphate (InsP3) generation induced by various mediators and involved InsP3-sensitive Ca2+ release channels. Here, we examined the effects of angiotensin II on [Ca2+]i in single rat portal vein myocytes using both the whole cell patch-clamp method and a laser scanning confocal microscope. Elementary Ca2+ release events (Ca2+ sparks) were obtained spontaneously or in response to L-type Ca2+ channel current activation, and resulted from activation of ryanodine-sensitive Ca2+-release channels in the SR. We show that angiotensin AT1 receptors stimulate Ca2+ sparks through activation of L-type Ca2+ channels without involving InsP3-induced Ca2+ release. This novel transduction pathway may be a common mechanism for vasoconstrictors which do not stimulate generation of chemical second messengers.     

Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries: comparison with noradrenaline and potassium

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

Fluorescent monitoring of Jurkatt cell intracellular magnesium during metabolic poisoning

Divalent cation movement characterizes the final common pathway of cellular death from ischemic or metabolic injury. The influx of calcium is an essential step in cellular death. We hypothesized that intracellular magnesium levels may change during the progression to cellular death. Verapamil-sensitive changes in free ionized intracellular Mg2+ ([Mg2+[i) and Ca2+ ([Ca2+]i) levels were estimated in transformed T-lymphocytes exposed to metabolic inhibitors. Separate experiments used a Mg(2+)-sensitive fluoroprobe, fura-2 (Ex 1,344, Ex 2,376, Em 500), and a Ca(2+)-sensitive fluoroprobe, fura-2 (Ex 1,340, Ex 2,380, Em 510). Chemical anoxia (sodium cyanide 1 mM, iodoacetic acid 10 mM) caused a gradual increase in [Ca2+]i (control 126 +/- 13 nM) to > 1 mM by 10 min. This increase in [Ca2+]i was not affected by verapamil treatment. In separate experiments, [Mg2+]i levels were monitored during chemical anoxia. The specificity of mag-fura for Mg2+ over Ca2+ was reflected in the absence of a response to the lymphocyte Ca2+ mobilizer OKT-3. Uncorrected control [Mg2+]i levels (.4 +/- .1 mM) were not affected by the combined cyanide-iodoacetate treatment. A small increase in mag-fura-2 fluorescence was noted, probably due to binding of Ca2+ to the fluoroprobe when [Ca2]i exceeded 1 mM. Elimination of Ca2+ from the extracellular buffer increased the resting estimate of intracellular [Mg2+] to 1.6 + .1 mM. These results indicate that 1) extracellular Ca2+ can interfere with the fluorescent determination of intracellular magnesium concentration, and 2) intracellular free Mg2+ concentrations do not change in this cell line during chemical anoxia.     

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.     

Regulation of Ca2+ release by InsP3 in single guinea pig hepatocytes and rat Purkinje neurons

The repetitive spiking of free cytosolic [Ca2+] ([Ca2+]i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP3-evoked Ca2+ release. The kinetics of both processes were studied with flash photolytic release of InsP3 and time resolved measurements of [Ca2+]i in single cells. InsP3 evoked Ca2+ flux into the cytosol was measured as d[Ca2+]i/dt, and the kinetics of Ca2+ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP3 concentrations greater than 0.1-0.2 microM. A comparison with photolytic release of metabolically stable 5-thio-InsP3 suggests that metabolism of InsP3 is important in determining the minimal concentration needed to produce Ca2+ release. A distinct latency or delay of several hundred milliseconds after release of low InsP3 concentrations decreased to a minimum of 20-30 ms at high concentrations and is reduced to zero by prior increase of [Ca2+]i, suggesting a cooperative action of Ca2+ in InsP3 receptor activation. InsP3-evoked flux and peak [Ca2+]i increased with InsP3 concentration up to 5-10 microM, with large variation from cell to cell at each InsP3 concentration. The duration of InsP3-evoked flux, measured as 10-90% risetime, showed a good reciprocal correlation with d[Ca2+]i/dt and much less cell to cell variation than the dependence of flux on InsP3 concentration, suggesting that the rate of termination of the Ca2+ flux depends on the free Ca2+ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca2+ flux, up to 50 microM. s-1 and in Purkinje neurons at high flux up to 1,400 microM. s-1. Experiments in which [Ca2+]i was controlled at resting or elevated levels support a mechanism in which InsP3-evoked Ca2+ flux is inhibited by Ca2+ inactivation of closed receptor/channels due to Ca2+ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP3-evoked Ca2+ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP3 receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP3 receptor density promotes fast rising, rapidly inactivating InsP3-evoked [Ca2+]i transients.     

Role of Ca2+ in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells

The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.