Hypoxia induces an increase in intracellular magnesium via transient receptor potential melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.     

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.     

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration ([Ca(2+)](i), fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 microM) stimulated a rapid increase in [Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in [Ca(2+)](i) and a slow increase in intracellular NO levels. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.     

Mechanisms of Mg(2+) transport in cultured ruminal epithelial cells

Net Mg(2+) absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg(2+) transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)), the intracellular Na(+) concentration ([Na(+)](i)), and the intracellular pH (pH(i)) of REC under basal conditions, after stimulation with butyrate and HCO(-)(3), and after changing the transmembrane chemical gradients for Mg(2+), H(+), and Na(+). REC had a mean resting pH(i) of 6.83 +/- 0.1, [Mg(2+)](i) was 0.56 +/- 0. 14 mM, and [Na(+)](i) was 18.95 +/- 3.9 mM. Exposure to both HCO(-)(3) and HCO(-)(3)/butyrate led to a stimulation of Mg(2+) influx that amounted to 27.7 +/- 5 and 29 +/- 10.6 microM/min, respectively, compared with 15 +/- 1 microM/min in control solution. The increase of [Mg(2+)](i) was dependent on extracellular Mg(2+) concentration ([Mg(2+)](e)). Regulation of pH(i) has been demonstrated to be Na(+) dependent and is performed, for the most part, by a Na(+)/H(+) exchanger. The recovery of pH(i) was fully blocked in nominally Na(+)-free media, even if [Mg(2+)](e) was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg(2+)](i) was observed after reversing the transmembrane Na(+) gradient. This rise in [Mg(2+)](i) was pH independent, K(+) insensitive, dependent on [Mg(2+)](e), imipramine and quinidine sensitive, and accompanied by a decrease of [Na(+)](i). The results are consistent with the existence of a Na(+)/Mg(2+) exchanger in the cell membrane of REC. The coupling between butyrate, CO(2)/HCO(-)(3), and Mg(2+) transport may be mediated by another mechanism, perhaps by cotransport of Mg(2+) and HCO(-)(3).     

Polyvalent cation-sensing mechanism increased Na(+)-independent Mg(2+) transport in renal epithelial cells

Extracellular Ca(2+)/polyvalent cation-sensing receptor (CaSR) is capable of monitoring changes in extracellular polyvalent cation concentrations. In the present study, we investigated whether CaSR agonists reinforce the decrease of intracellular free Mg(2+) concentration ([Mg(2+)](i)) induced by extracellular Mg(2+) plus Na(+) removal. Interestingly, exposure of NRK-52E renal epithelial cells to increasing extracellular Mg(2+) concentrations from 0.8 to 15 mM for 1-2 days resulted in a twofold increase in the levels of CaSR mRNA and protein. By fluorophotometer (with mag-fura 2 fluorescent dye) and atomic absorption spectrophotometer, we confirmed that activation of CaSR by neomycin (0.5 mM) or gadolinium (1 mM) reinforced the decrease of [Mg(2+)](i) induced by Mg(2+) removal in the cells cultured in 10 mM Mg(2+)-containing medium. The neomycin-induced [Mg(2+)](i) decrease was inhibited by nicardipine (50 microM), but not by verapamil (50 microM) or amiloride (0.1 mM). These results indicate that CaSR monitors extracellular Mg(2+) concentration, and probably cause activation of Na(+)-independent Mg(2+)-transport system.     

AMPA receptor-induced intracellular calcium response in the paraventricular nucleus is modulated by nitric oxide: Calcium imaging in a hypothalamic organotypic cell culture model

An organotypic cell culture (OCC) model of the rat hypothalamic paraventricular nucleus (PVN) was established to monitor intracellular calcium levels ([Ca(2+)](i)) of magnocellular neurons in response to glutamate and nitric oxide (NO). The histoarchitectural organization of these cultures was characterized either by immunohistochemical labeling of vasopressin, neuronal nitric oxide synthase (nNOS) and the neuronal marker NeuN or by the enzyme histochemical NADPH-diaphorase staining. A distinct NeuN positive cell population in 14-days old OCC's was confirmed as being the PVN by its vasopressin- and nNOS-immunostained neurons as well as by its NADPH-diaphorase labeling. Life cell imaging was performed using the [Ca(2+)](i) sensor Fluo-4 to measure [Ca(2+)](i) transients in response to bath applications of glutamate, high potassium (60 mM), and ATP. The glutamate-induced [Ca(2+)](i) response was mimicked by AMPA but not NMDA in the PVN. NMDA, however, elicited a [Ca(2+)](i) transient in a different area of the OCC that corresponds to the suprachiasmatic nucleus indicating the potential effectiveness of the stimulus. The AMPA-receptor blocker NBQX abolished the glutamate-induced response in the PVN. An inhibition of endogenous NO production by the NOS inhibitor L-NAME decreased the amplitude of AMPA- and glutamate-induced [Ca(2+)](i) rises. Taken together, these data suggest that AMPA mediates the glutamate-induced [Ca(2+)](i) rises within the PVN, where endogenous NO is able to modulate such glutamate signaling in OCC.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Low [Mg(2+)](o) induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases

The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg(2+)](o)) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg(2+)](o) medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg(2+)](o), the stronger the contractions. The low-[Mg(2+)](o) medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC(50) levels obtained for these five antagonists are consistent with reported inhibitor constant (K(i)) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg(2+)](o) medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca(2+)](i)) peaks followed by a slow, sustained, and elevated plateau of [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries. Low-[Mg(2+)](o) medium induces rapid and stable increases in [Ca(2+)](i); these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg(2+)](o)-induced contractions or the elevation of [Ca(2+)](i). The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg(2+)](o)-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO

In vitro extracellular Mg(2+) concentration ([Mg(2+)](0)) produces endothelium-dependent and endothelium-independent relaxations in rat aorta in a concentration-dependent manner. These relaxant effects of Mg(2+) on intact rat aortic rings, but not denuded rat aortic rings, were suppressed by either N(G)-monomethyl-L-arginine (L-NMMA), N(omega)-nitro-L-arginine methyl ester (L-NAME), or methylene blue. The inhibitory effects of L-NMMA and L-NAME could be reversed partly by L-arginine. [Mg(2+)](0)-induced dilatation in vivo in rat mesenteric arterioles and venules was almost completely inhibited by N(G)-nitro-L-arginine and L-NMMA. Removal of extracellular Ca(2+) concentration ([Ca(2+)](0)) or buffering intracellular Ca(2+) concentration in endothelial cells, with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, markedly attenuated the relaxant effects of Mg(2+). Mg(2+) produced nitric oxide (NO) release from the intact aortic rings in a concentration-dependent manner. Removal of [Ca(2+)](0) diminished the increased NO release induced by elevated levels of [Mg(2+)](0). In vivo infusion of increasing doses (1-30 microM/min) of MgSO(4), directly into the femoral veins of anesthetized rats, elicited significant concentration-dependent sustained increases in serum total Mg and concomitant decreases in arterial blood pressure. Before and after employment of various doses of MgSO(4), intravenous administration of either L-NMMA (10 mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the MgSO(4)-lowered blood pressure markedly, and intravenous injection of L-arginine restored partially the increased blood pressure effects of both L-NMMA and L-NAME. Our results suggest that 1) small blood vessels are very dependent on NO release for Mg(2+) dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg(2+) is mediated by release of endothelium-derived relaxing factor-NO from the endothelium, and requires Ca(2+) and formation of guanosine 3',5'-cyclic monophosphate.     

Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.     

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.     

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.     

Mechanisms of endothelial P2Y(1)- and P2Y(2)-mediated vasodilatation involve differential [Ca2+]i responses

The present study was designed to evaluate the role of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in the difference between P2Y(1)- and P2Y(2)-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca(2+) indicator, for simultaneous measurement of endothelial [Ca(2+)](i) and diameter. Luminal administration of 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), an endothelial P2Y(1) agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca(2+)](i) increases up to approximately 300 nM (resting [Ca(2+)](i) = 145 nM). UTP, an endothelial P2Y(2) agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca(2+)](i) increases to >400 nM. In the presence of N(G)-nitro-L-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca(2+)](i) threshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y(1) and P2Y(2) purinoceptors result in part from differential [Ca(2+)](i) responses. Consistent with this finding, these studies also demonstrate a higher [Ca(2+)](i) threshold for EDHF-dependent responses compared with NO.     

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.