Ca(2+) entry into primary cultured pig coronary smooth muscle cells after previous store depletion by repetitive P2Y purinoceptor stimulation

Store-operated Ca(2+) entry, stimulated by depletion of intracellular Ca(2+) pools, has not been fully elucidated in vascular smooth muscle cells of pig coronary arteries. Therefore, [Ca(2+)](i) was measured in cultured cells derived from extramural pig coronary arteries using the Fura-2/AM fluorometry. Divalent cation entry was visualized with the Fura-2 Mn(2+)-quenching technique. Ca(2+) stores were depleted either by repetitive stimulation of P2Y purinoceptors with ATP (10 micromol/L), or by the sarcoendoplasmic Ca(2+)-ATPase inhibitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1 micromol/L) in Ca(2+)-free medium (EGTA 1 mmol/L). Addition of Ca(2+)(1 mmol/L) induced refilling of ATP-sensitive Ca(2+) stores and an increase in [Ca(2+)](i) in the presence of BHQ. Both could be significantly diminished by Ni(2+)(5 and 1mmol/L), La(3+)(10 micromol/L), Gd(3+)(10 micromol/L), and Mg(2+)(5.1 mmol/L). In contrast to the BHQ-mediated rise in [Ca(2+)](i), refilling of ATP-depleted stores was affected by neither flufenamate (0.1 mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1 micromol/L). The data suggest that after store depletion in pig coronary smooth muscle cells ATP and BHQ may converge on a common, Ni(2+)-, La(3+)-, Gd(3+)-, and Mg(2+)- sensitive Ca(2+) entry pathway, i.e. on a store-operated Ca(2+) entry. An additional contribution of the Na(+)/Ca(2+) exchanger cannot be excluded. Flufenamate-sensitive non-selective cation channels and dihydropyridine-sensitive L-type Ca(2+) channels are not involved in refilling of Ca(2+) stores after previous depletion by repetitive P2Y purinoceptor stimulation. The store-operated Ca(2+) entry in-between repetitive purinoceptor stimulation, i.e. in the absence of the agonist, may be responsible for the maintenance of agonist-induced rhythmic Ca(2+) responses.     

Mibefradil improves beta-adrenergic responsiveness and intracellular Ca(2+) handling in hypertrophied rat myocardium

The present study investigated the effects of mibefradil, a novel T-type channel blocker, on ventricular function and intracellular Ca(2+) handling in normal and hypertrophied rat myocardium. Ca(2+) transient was measured with the bioluminescent protein, aequorin. Mibefradil (2 microM) produced nonsignificant changes in isometric contraction and peak systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal rat myocardium. Hypertrophied papillary muscles isolated from aortic-banded rats 10 weeks after operation demonstrated a prolonged duration of isometric contraction, as well as decreased amplitudes of developed tension and peak Ca(2+) transient compared with the sham-operated group. Additionally, diastolic [Ca(2+)](i) increased in hypertrophied rat myocardium. The positive inotropic effect of isoproterenol stimulation was blunted in hypertrophied muscles despite a large increase in Ca(2+) transient amplitude. Afterglimmers and corresponding aftercontractions were provoked with isoproterenol (10(-5) and 10(-4) M) stimulation in 4 out of 16 hypertrophied muscles, but were eliminated in the presence of mibefradil (2 microM). In addition, hypertrophied muscles in the presence of mibefradil had a significant improvement of contractile response to isoproterenol stimulation and a reduced diastolic [Ca(2+)](I), although a mild decrease of peak Ca(2+)-transient was also shown. However, verapamil (2 microM) did not restore the inotropic and Ca(2+) modulating effects of isoproterenol in hypertrophied myocardium. Mibefradil partly restores the positive inotropic response to beta-adrenergic stimulation in hypertrophied myocardium from aortic-banded rats, an effect that might be useful in hypertrophied myocardium with impaired [Ca(2+)](i) homeostasis.     

Effect of hydrogen peroxide on reoxygenation-induced Ca2+ accumulation in rat cardiomyocytes

Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca(2+) homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H(2)O(2)) on Ca(2+) accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca(2+) ([Ca(2+)](i)) and Mg(2+)([Mg(2+)](i)), and mitochondrial membrane potential (Deltapsim). Cell Ca(2+) was determined by (45)Ca(2+) uptake. Free [Mg(2+)](i) and [Ca(2+)](i) and Deltapsim were measured by flow cytometry. Reoxygenation-induced Ca(2+) accumulation was attenuated by 23 and 34% by 10 and 25 microM H(2)O(2), respectively, added at reoxygenation. H(2)O(2) at 100 and 250 microM increased cell Ca(2+) by 50 and 83%, respectively, whereas 500 microM H(2)O(2) decreased cell Ca(2+) by 20%. H(2)O(2) at (25 microM) reduced LDH release and [Mg(2+)](i) and increased Deltapsim, indicating cell protection, whereas 250 microM H(2)O(2) increased LDH release and [Mg(2+)](i) and decreased Deltapsim, indicating cell damage. Clonazepam (100 microM) attenuated the increase in Ca(2+) accumulation, the elevation of [Ca(2+)](i), and the decrease in Deltapsim induced by 100 and 250 microM H(2)O(2) during reoxygenation. We report for the first time that 25 microM H(2)O(2) attenuates Ca(2+) accumulation, LDH release, and dissipation of Deltapsim during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.     

ATP modulation of mitogen activated protein kinases and intracellular Ca2+ in breast cancer (MCF-7) cells

In the breast tumor cell line MCF-7, extracellular nucleotides induce transient elevations in intracellular calcium concentration ([Ca(2+)](i)). In this study we show that stimulation with ATP or UTP sensitizes MCF-7 cells to mechanical stress leading to an additional transient Ca(2+) influx. ATP> or =ATPgamma-S> or =UTP>ADP=ADPbeta-S elevate [Ca(2+)](i), proving the presence of P2Y(2)/P2Y(4) purinergic receptor subtypes. In addition, cell stimulation with ATP, ATPgamma-S or UTP but not ADPbeta-S induced the phosphorylation of ERK1/2, p38 and JNK1/2 mitogen activated protein kinases (MAPKs). The use of Gd(3+), La(3+) or a Ca(2+)-free medium, inhibited ATP-dependent stress activated Ca(2+) (SAC) influx, but had no effect on MAPK phosphorylation. ATP-induced activation of MAPKs was diminished by two PI-PLC inhibitors and an IP(3) receptor antagonist. These results evidence an ATP-sensitive SAC influx in MCF-7 cells and indicate that phosphorylation of MAPKs by ATP is dependent on PI-PLC/IP(3)/Ca(2+)(i) release but independent of SAC influx in these cells, differently to other cell types.     

S-nitrosoglutathione-induced decrease in calcium sensitivity of airway smooth muscle

The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca(2+) sensitivity [i.e., the amount of force for a given intracellular Ca(2+) concentration ([Ca(2+)](i))]. We further investigated whether GSNO decreases Ca(2+) sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca(2+)](i) and the relationship between force and rMLC phosphorylation. GSNO (100 microM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca(2+) sensitivity in comparison to control strips without significantly decreasing [Ca(2+)](i). GSNO reduced the amount of rMLC phosphorylation for a given [Ca(2+)](i) but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca(2+) sensitivity by affecting the level of rMLC phosphorylation for a given [Ca(2+)](i), suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.     

Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.

Acetylcholine-evoked secretion from the parotid gland is substantially potentiated by cAMP-raising agonists. A potential locus for the action of cAMP is the intracellular signaling pathway resulting in elevated cytosolic calcium levels ([Ca(2+)](i)). This hypothesis was tested in mouse parotid acinar cells. Forskolin dramatically potentiated the carbachol-evoked increase in [Ca(2+)](i), converted oscillatory [Ca(2+)](i) changes into a sustained [Ca(2+)](i) increase, and caused subthreshold concentrations of carbachol to increase [Ca(2+)](i) measurably. This potentiation was found to be independent of Ca(2+) entry and inositol 1,4,5-trisphosphate (InsP(3)) production, suggesting that cAMP-mediated effects on Ca(2+) release was the major underlying mechanism. Consistent with this hypothesis, dibutyryl cAMP dramatically potentiated InsP(3)-evoked Ca(2+) release from streptolysin-O-permeabilized cells. Furthermore, type II InsP(3) receptors (InsP(3)R) were shown to be directly phosphorylated by a protein kinase A (PKA)-mediated mechanism after treatment with forskolin. In contrast, no evidence was obtained to support direct PKA-mediated activation of ryanodine receptors (RyRs). However, inhibition of RyRs in intact cells, demonstrated a role for RyRs in propagating Ca(2+) oscillations and amplifying potentiated Ca(2+) release from InsP(3)Rs. These data indicate that potentiation of Ca(2+) release is primarily the result of PKA-mediated phosphorylation of InsP(3)Rs, and may largely explain the synergistic relationship between cAMP-raising agonists and acetylcholine-evoked secretion in the parotid. In addition, this report supports the emerging consensus that phosphorylation at the level of the Ca(2+) release machinery is a broadly important mechanism by which cells can regulate Ca(2+)-mediated processes.     

Low [Mg(2+)](o) induces contraction and [Ca(2+)](i) rises in cerebral arteries: roles of ca(2+), PKC, and PI3

Removal of extracellular Ca(2+) concentration ([Ca(2+)](o)) and pretreatment of canine basilar arterial rings with either an antagonist of voltage-gated Ca(2+) channels (verapamil), a selective antagonist of the sarcoplasmic reticulum Ca(2+) pump [thapsigargin (TSG)], caffeine plus a specific antagonist of ryanodine-sensitive Ca(2+) release (ryanodine), or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]- mediated Ca(2+) release antagonist (heparin) markedly attenuates low extracellular Mg(2+) concentration ([Mg(2+)](o))-induced contractions. Low [Mg(2+)](o)-induced contractions are significantly inhibited by pretreatment of the vessels with G?-6976 [a protein kinase C-alpha (PKC-alpha)- and PKC-betaI-selective antagonist], bisindolylmaleimide I (Bis, a specific antagonist of PKC), and wortmannin or LY-294002 [selective antagonists of phosphatidylinositol-3 kinases (PI3Ks)]. These antagonists were also found to relax arterial contractions induced by low [Mg(2+)](o) in a concentration-dependent manner. The absence of [Ca(2+)](o) and preincubation of the cells with verapamil, TSG, heparin, or caffeine plus ryanodine markedly attenuates the transient and sustained elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by low-[Mg(2+)](o) medium. Low [Mg(2+)](o)-produced increases in [Ca(2+)](i) are also suppressed markedly in the presence of G?-6976, Bis, wortmannin, or LY-294002. The present study suggests that both Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores [both Ins(1,4,5)P(3) sensitive and ryanodine sensitive] play important roles in low-[Mg(2+)](o) medium-induced contractions of isolated canine basilar arteries. Such contractions are clearly associated with activation of PKC isoforms and PI3Ks.     

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Angiotensin II type I receptor modulates intracellular free Mg2+ in renally derived cells via Na+-dependent Ca2+-independent mechanisms

Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) with a concomitant decrease in cytosolic free Mg(2+) concentration ([Mg(2+)](i)). In the present study we demonstrate that this hormone-induced decrease in [Mg(2+)](i) is independent of [Ca(2+)](i) but dependent on extracellular Na(+). [Mg(2+)](i), [Ca(2+)](i), and [Na(+)](i) were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg(2+)](i) and increased [Na(+)](i) in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT(1) receptor blocker) but not by PD123319 (selective AT(2) receptor blocker). Imipramine and quinidine (putative inhibitors of the Na(+)/Mg(2+) exchanger) and removal of extracellular Na(+) abrogated Ang II-mediated [Mg(2+)](i) effects. In cells pretreated with thapsigargin (reticular Ca(2+)-ATPase inhibitor), Ang II-stimulated [Ca(2+)](i) transients were attenuated (p < 0.01), whereas agonist-induced [Mg(2+)](i) responses were unchanged. Clamping the [Ca(2+)](i) near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca(2+)](i) increases but failed to alter Ang II-induced [Mg(2+)](i) responses. Benzamil, a selective blocker of the Na(+)/Ca(2+) exchanger, inhibited [Na(+)](i) but not [Mg(2+)](i) responses. Our data demonstrate that in MDCK cells, AT(1) receptors modulate [Mg(2+)](i) via a Na(+)-dependent Mg(2+) transporter that is not directly related to [Ca(2+)](i). These data support the notion that rapid modulation of [Mg(2+)](i) is not simply a result of Mg(2+) redistribution from intracellular buffering sites by Ca(2+) and provide evidence for the existence of a Na(+)-dependent, hormonally regulated transporter for Mg(2+) in renally derived cells.     

Synaptosomal plasma membrane Ca(2+) pump activity inhibition by repetitive micromolar ONOO(-) pulses.

A sustained increase of intracellular free [Ca(2+)] ([Ca(2+)](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO(-)). In this paper, chronic exposure to ONOO(-) has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO(-) during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca(2+)-ATPase). The plasma membrane Ca(2+)-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca(2+), and millimolar Mg(2+)-ATP concentrations), which plays a major role in the control of synaptic [Ca(2+)](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO(-) (e.g., to 100 pulses of 10 microM ONOO(-)). This inhibition is irreversible and mostly due to a decreased V(max), and to the 2-fold increase of the K(0.5) for Ca(2+) stimulation and about 5-fold increase of the K(M) for Mg(2+)-ATP. [Ca(2+)](i) increases to >400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO(-) pulses. Therefore, inhibition of the plasma membrane Ca(2+)-pump activity during chronic exposure to ONOO(-) may account by itself for a large and sustained increase of intracellular [Ca(2+)](i) in synaptic nerve terminals.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

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.     

NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli

In guinea pig taenia coli, the nitric oxide (NO) donor sodium nitroprusside (SNP, 1 microM) reduced the carbachol-stimulated increases in muscle force in parallel with a decrease in intracellular Ca(2+) concentration ([Ca(2+)](i)). A decrease in the myosin light chain phosphorylation was also observed that was closely correlated with the decrease in [Ca(2+)](i). With the patch-clamp technique, 10 microM SNP decreased the peak Ba(2+) current, and this effect was blocked by an inhibitor of soluble guanylate cyclase. Carbachol (10 microM) induced an inward current, and this effect was markedly inhibited by SNP. SNP markedly increased the depolarization-activated outward K(+) currents, and this current was completely blocked by 0.3 micorM iberiotoxin. SNP (1 microM) significantly increased cGMP content without changing cAMP content. Decreased Ca(2+) sensitivity by SNP of contractile elements was not prominent in the permeabilized taenia, which was consistent with the [Ca(2+)](i)-force relationship in the intact tissue. These results suggest that SNP inhibits myosin light chain phosphorylation and smooth muscle contraction stimulated by carbachol, mainly by decreasing [Ca(2+)](i), which resulted from the combination of the inhibition of voltage-dependent Ca(2+) channels, the inhibition of nonselective cation currents, and the activation of Ca(2+)-activated K(+) currents.     

Mg2+ blocks myogenic tone but not K+-induced constriction: role for SOCs in small arteries

The effects of Mg(2+) and nifedipine (Nif) on vasoconstriction and Ca(2+) transients were studied in intact, pressurized rat mesenteric arteries with myogenic tone. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) were measured with confocal microscopy in fluo 4-AM loaded, individual myocytes. Myogenic tone was abolished by 10 mM Mg(2+) or 0.3 microM Nif. Contractions induced by 75 mM K(+) depolarization were blocked by 0.3 microM Nif, but not by 10 mM Mg(2+). Phenylephrine (PE; 5 microM) evoked sustained [Ca(2+)](cyt) elevation and vasoconstriction with superimposed Ca(2+) oscillations and vasomotion. The subsequent addition of 10 mM Mg(2+) or 0.3 microM Nif reduced [Ca(2+)](cyt) and abolished plateau vasoconstriction. When added before PE, both Mg(2+) and Nif abolished the PE-evoked Ca(2+) oscillations and vasomotion. Mg(2+) dilated the PE-constricted arteries after a brief (< or =180-240 s) vasoconstriction, but Nif did not. Both agents also abolished the vasoconstriction attributed to Ca(2+) entry through store-operated channels (SOCs) during internal Ca(2+) store refilling that followed store depletion. The data suggest that Ca(2+) entry through SOCs helps maintain both myogenic tone and alpha(1)-adrenoceptor-induced tonic vasoconstriction.     

Effect of timosaponin A-III,from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.