Evidence for receptor-mediated bivalent-cation entry in A10 vascular smooth-muscle cells.
ABSTRACT: In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.
Project description:Undifferentiated monolayers of the hamster vas deferens smooth-muscle cell line, DDT1MF-2, were grown on glass coverslips and loaded with the Ca(2+)-sensitive fluorescent dye fura-2. Stimulation with histamine produced a rapid and maintained increase in intracellular free Ca2+ ([Ca2+]i), with an EC50 of 7.0 +/- 0.7 microM. The initial rise in [Ca2+]i can be attributed to Ca2+ release from intracellular stores, whereas the maintained or plateau phase is due to influx of extracellular Ca2+. The Ca2+ influx associated with the plateau phase required the continued presence of histamine on the receptor, since the H1-antagonist mepyramine (10 microM) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re-applied after the cells had been stimulated with histamine, in experiments performed in nominally Ca(2+)-free buffer. Pretreatment with the inorganic Ca(2+)-channel blockers Ni2+ (1 mM) and Co2+ (1 mM) inhibited the influx component, whereas the organic voltage-operated Ca(2+)-channel antagonists nifedipine (10 microM) and PN-200-110 (10 microM) had no effect. These data suggest that histamine stimulates Ca2+ influx through an H1-receptor-activated Ca2+ channel. Experiments with Mn2+ indicated that the receptor-mediated Ca(2+)-influx pathway(s) is impermeable to Mn2+. Furthermore, the refilling of Ca2+ stores can occur independently of H1-receptor-mediated influx, since store refilling can be demonstrated even when the receptor-mediated Ca2+ entry is blocked by mepyramine. In conclusion, H1-receptor activation in the smooth-muscle cell line DDT1MF-2 stimulates both release of Ca2+ from intracellular stores [inositol 1,4,5-triphosphate (InsP3)-mediated] and Ca2+ influx through a receptor-activated Ca2+ channel. The subsequent refilling of the InsP3-sensitive intracellular Ca2+ store is independent of histamine H1-receptor stimulation (mepyramine-insensitive) and occurs without an observable rise in cytosolic free Ca2+.
Project description:The relationship between histamine-induced Ca2+ mobilization and Ca2+ entry in bovine adrenal chromaffin cells has been investigated. Stopped-flow fluorimetry of fura-2-loaded chromaffin cell populations revealed that 10 microM histamine promoted entry of Ca2+ or Mn2+ without measurable delay (< or = 20 ms), through a pathway that was insensitive to the dihydropyridine antagonist nifedipine. In the absence of extracellular Ca2+, or in the presence of 100 microM La3+, a blocker of receptor-mediated Ca2+ entry, 10 microM histamine triggered an elevation in intracellular calcium concentration ([Ca2+]i), but only after a delay of approx. 200 ms, which presumably represented the time required to mobilize intracellular Ca2+. These data suggested that histamine-induced bivalent-cation entry precedes extensive Ca2+ mobilization in chromaffin cells. In order to confirm that histamine can promote Ca2+ entry largely independently of mobilizing intracellular Ca2+, the ability of histamine to promote Ca2+ entry into cells whose intracellular Ca2+ store had been largely depleted was assessed. Fura-2-loaded chromaffin cells were treated with 10 microM ryanodine together with 40 mM caffeine, to deplete the hormone-sensitive Ca2+ store. This resulted in an approx. 95% inhibition of histamine-induced Ca2+ release. Under these conditions, histamine was still able to promote an entry of Ca2+ that was essentially indistinguishable from that promoted in control cells. In single cells, introduction of heparin (100 mg/ml), but not de-N-sulphated heparin (100 mg/ml), abolished the histamine-induced rise in [Ca2+]i. All these data suggest that histamine can induce G-protein- or inositol phosphate-dependent rapid (< or = 20 ms) Ca2+ entry without an extensive intracellular mobilization response in chromaffin cells, which points to activation of an entry mechanism distinct from the Ca(2+)-release-activated Ca2+ channel found in non-excitable cells.
Project description:In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.
Project description:Populations of hepatocytes in primary culture were loaded with fura 2 and the effects of extracellular heavy-metal ions were examined under conditions that allowed changes in fura 2 fluorescence (R340/360, the ratio of fluorescence recorded at 340 and 360 nm) to be directly attributed to changes in cytosolic free [Ca2+] ([Ca2+]i). In Ca2+-free media, Ni2+ [EC50 (concentration causing 50% stimulation) approximately 24+/-9 microM] caused reversible increases in [Ca2+]i that resulted from mobilization of the same intracellular Ca2+ stores as were released by [Arg8]vasopressin. The effects of Ni2+ were not mimicked by increasing the extracellular [Mg2+], by addition of MnCl2, CoCl2 or CdCl2 or by decreasing the extracellular pH from 7.3 to 6.0; nor were they observed in cultures of smooth muscle, endothelial cells or pituitary cells. CuCl2 (80 microM), ZnCl2 (80 microM) and LaCl3 (5 mM) mimicked the ability of Ni2+ to evoke Ca2+ mobilization. The response to La3+ was sustained even in the absence of extracellular Ca2+, probably because La3+ also inhibited Ca2+ extrusion. Although Ni2+ entered hepatocytes, from the extent to which it quenched fura 2 fluorescence the free cytosolic [Ni2+] ([Ni2+]i) was estimated to be <5 nM at the peak of the maximal Ni2+-evoked Ca2+ signals and there was no correlation between [Ni2+]i and the amplitude of the evoked increases in [Ca2+]i. We conclude that extracellular Ni2+, Zn2+, Cu2+ and La3+, but not all heavy-metal ions, evoke an increase in [Ca2+]i in hepatocytes by stimulating release of the hormone-sensitive intracellular Ca2+ stores and that they may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing hepatocytes to contribute to regulation of plasma heavy-metal ions and may mediate responses to Zn2+ released into the portal circulation with insulin.
Project description:Rat hepatocytes respond to glycogenolytic stimuli acting via phosphoinositide breakdown (e.g. alpha 1-adrenergic agonists, vasopressin) by oscillations of the free intracellular Ca2+ concentration ([Ca2+]i). We have investigated the action of metformin and phenformin, two anti-diabetic drugs of the biguanide type, on phenylephrine-induced [Ca2+]i oscillations. Metformin and phenformin lowered the frequency of the [Ca2+]i oscillations in a concentration-dependent manner with an IC50 of 0.1 mM and 1 microM, respectively. Simultaneous addition of the biguanides and insulin resulted in a further reduction of the frequency. By contrast, agents which increase the cellular cyclic AMP (cAMP) concentration (glucagon, forskolin, N,2'-O-dibutyryl-cAMP) reversed this inhibition. Furthermore, we investigated whether biguanides influenced the agonist-induced Ca2+ influx across the plasma membrane. When hepatocytes were loaded with the acetoxymethyl ester of fura-2 (fura-2/AM), addition of Mn2+ led to a quench of cellular fura-2, measured at the isosbestic excitation wavelength of 360 nm, until a new steady state was reached. Surprisingly, however, this addition of Mn2+ caused a marked increase of the fluorescence ratio simultaneously measured at 340 and 380 nm during the approach of the 360 nm signal to a new steady state. This observation can be understood on the basis of a compartmentalization of fura-2/AM into intracellular stores sensing the [Ca2+] therein. Subsequent application of phenylephrine resulted in a further decline of the fura-2 signal at 360 nm and a concomitant decrease of the fluorescence ratio. This second phase of the Mn2+ quench and the decrease of the fluorescence ratio could be diminished by addition of either 3 mM metformin or 30 microM phenformin. By contrast, when hepatocytes were loaded with fura-2/pentapotassium salt via a patch pipette, only the initial Mn(2+)-induced quench, measured at 360 nm, but no change of the fluorescence ratio, could be observed. The subsequent addition of phenylephrine and biguanides during the on-going quench caused no further changes, except for a fading oscillatory response. After loading hepatocytes with fluo-3 acetoxymethyl ester, the cells were permeabilized with 5 microM digitonin. Addition of inositol-1,4,5-trisphosphate (IP3) caused a rapid decrease of the remaining cellular fluorescence which could be effectively inhibited by 20 micrograms/ml heparin, indicating a release of Ca2+ from intracellular compartments mediated by IP3. This IP3-induced release of Ca2+ from intracellular stores could be diminished by prior addition of metformin and phenformin.(ABSTRACT TRUNCATED AT 400 WORDS)
Project description:Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction. In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the ?1-adrenergic agonist phenylephrine (5 ?M) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 ?M) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 ?M), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.
Project description:Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.
Project description:Receptor-mediated Ca2+ entry was studied in fura-2-loaded isolated hepatocytes. Emptying of internal Ca2+ stores by treatment with either the Ca(2+)-mobilizing hormone vasopressin or the inhibitors of the microsomal Ca2+ pump, 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) or thapsigargin, stimulated Ca2+ entry, as indicated by a rise in the cytosolic free Ca2+ concentration after Ca2+ was added to cells suspended in nominally Ca(2+)-free medium. The enhancement of Ca2+ entry was proportional to the degree of depletion of the intracellular Ca2+ pool and occurred also after removal of vasopressin from its receptor. In contrast, the stimulation of Mn2+ entry by vasopressin required the continuous presence of the agonist, since it was prevented by the addition of vasopressin receptor antagonist. This effect was observed under conditions where refilling of the agonist-sensitive pool was prevented by using nominally Ca(2+)-free medium. Unlike vasopressin, tBuBHQ or thapsigargin did not stimulate Mn2+ entry. These results suggest the existence of two pathways for receptor-mediated Ca2+ entry in hepatocytes, a 'capacitative' pathway that is sensitive to the Ca2+ content in the Ins(1,4,5)P3-sensitive Ca2+ pool and does not allow Mn2+ entry, and a second pathway that depends on receptor occupation, seems to require a second messenger for activation, and permits influx of Mn2+.
Project description:The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.
Project description:Recent studies by Moneer and Taylor [(2002) Biochem. J. 362, 13-21] have proposed a reciprocal regulation of two Ca2+-entry pathways by AVP ([Arg8]-vasopressin) in A7r5 vascular smooth-amuscle cells. Their model proposes that AVP inhibits CCE (capacitative Ca2+ entry) and predicts a rebound of CCE after the removal of AVP. In the present study, we used whole-cell perforated patch-clamp techniques to measure ISOC (store-operated current) corresponding to CCE in A7r5 cells. When 100 nM AVP is present, it activates ISOC with no apparent rebound on removal of AVP. ISOC activated by thapsigargin or cyclopiazonic acid was not inhibited by 100 nM AVP. We also used fura 2 fluorescence techniques to re-examine the model of Moneer and Taylor, specifically focusing on the proposed inhibition of CCE by AVP. We find that 100 nM AVP activates capacitative Mn2+ entry and does not inhibit thapsigargin- or cyclopiazonic acid-activated Mn2+ entry. Moreover, Ca2+ entry after depletion of intracellular Ca2+ stores is enhanced by AVP and we detect no rebound of Ca2+ or Mn2+ entry after AVP removal. On the basis of these findings, we conclude that AVP does not inhibit CCE in A7r5 cells.