ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated rat liver nuclei.

Addition of ATP to a highly purified fraction of rat liver nuclei incubated with submicromolar concentrations of Ca2+ and trace amounts of 45Ca2+ resulted in the rapid accumulation of 45Ca2+ in the nuclei. This was associated with an increase in intranuclear free Ca2+ concentration as measured with the fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-a mino-5'- methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (fura-2). Inhibitors of microsomal and mitochondrial Ca2+ translocases had no effect on nuclear Ca2+ sequestration, indicating that it was distinct from previously known intracellular Ca2+-transporting systems. Ca2+ uptake and the associated increase in intranuclear free Ca2+ concentration were prevented by calmidazolium, a potent calmodulin antagonist. Partial characterization of the ATP-stimulated nuclear Ca2+ uptake showed that maximal rates of Ca2+ uptake and increase in intranuclear free Ca2+ level occurred at concentrations of Ca2+ normally present in the cytosol of mammalian cells. Together, these results show that a distinct, ATP- and calmodulin-dependent Ca2+ uptake system exists in liver nuclei. This system may play an important role in the regulation of intranuclear Ca2+-dependent processes.     

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.     

Plasma membrane Ca2+ release-activated Ca2+ channels with a high selectivity for Ca2+ identified by patch-clamp recording in rat liver cells

Repetitive waves of increased cytoplasmic Ca2+ concentration play a central role in the process by which hormones regulate liver function. Maintenance of these Ca2+ waves requires Ca2+ inflow through store-operated Ca2+ channels. The properties and mechanism(s) of activation of these channels are not well understood. Store-operated Ca2+ channels (SOCs) in the H4-IIE rat liver cell line were studied by whole-cell patch clamping. Depletion of Ca2+ in intracellular stores by intracellular perfusion with either inositol 1,4,5-trisphosphate (InsP(3)) or thapsigargin in the presence of 10 mmol/L ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), or with 10 mmol/L EGTA alone, activated an inward current that reversed at a membrane potential above +40 mV. In physiologic extracellular medium, this inward current was carried exclusively by Ca2+ and was blocked by a variety of di- and trivalent cations. In the absence of extracellular Ca2+ and Mg2+, the inward current was carried by monovalent cations. This current was 10 to 30 times larger than that observed in the presence of extracellular Ca2+, and permitted the detection of single-channel events that corresponded to a single-channel conductance of about 40 pS. Both the Ca2+ and Na+ inward currents were blocked by the calmodulin antagonist, N-(6-amino hexyl)-5-chloro-1-naphthalenesulphonamide (W7), but not by calmidazolium or calmodulin-dependent protein kinase II fragment 290-309. It is concluded that liver cells possess plasma membrane Ca2+ channels that have a high selectivity for Ca2+, are activated by a decrease in the concentration of Ca2+ in intracellular stores through a mechanism that is unlikely to involve calmodulin, and are involved in re-filling intracellular Ca2+ stores during Ca2+ signaling.     

Role of extracellular Ca2+ in ischemia-reperfusion injury in the isolated perfused rat liver.

The influx of extracellular Ca2+ has been postulated to be one of the mediators of ischemia-reperfusion injury. A possible link between Ca2+ influx and oxygen radical generation has also been suggested. In the present study, using the isolated perfused rat liver, we evaluated the role of extracellular Ca2+ on oxygen radical generation, liver damage, and lipid peroxidation during 30 min ischemia and 60 min of reperfusion. Oxygen radical generation in the liver was continuously monitored by lucigenin-enhanced chemiluminescence. Liver damage and lipid peroxidation were evaluated by measuring lactate dehydrogenase (LDH) and thiobarbituric acid reactive substances (TBARS) release into the effusate, respectively. In the absence of extracellular Ca2+ (much less than 30 microM) oxygen radical generation from the liver increased gradually over 2 hr and there were concomitant increases in LDH and TBARS release. When livers were made ischemic and then reperfused, oxygen radical generation increased at the onset of reperfusion and then decreased over 30 min of reperfusion. After 30 min of reperfusion, livers reperfused with low Ca2+ buffer showed a linear increase in oxygen radical generation as well as progressive increases in LDH and TBARS release. On the other hand, livers reperfused with Ca2+ containing (1.25 mM) buffer showed no further increase in oxygen radical generation and no evidence of progressive liver damage and lipid peroxidation. These results suggest that Ca2+ overload is not a primary cause of liver ischemia-reperfusion injury and that the presence of extracellular Ca2+ during reperfusion is necessary to maintain normal liver function.     

Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver.

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+)-sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterogeneous responses of cell Ca2+ in human airway epithelium.

The Ca(2+)-mobilizing actions of adenosine 5'-triphosphate (ATP), bradykinin, and histamine were compared in phenotypically distinct human nasal epithelial (HNE) cell types and as a function of time in cell culture. Single-cell measurements of intracellular free Ca2+ (Ca2+i, Fura-2 fluorescence) were recorded in ciliated cells 1-2 days in primary culture, and in nonciliated cells 1-2 days (keratin 14-positive) or 4-5 days (keratin 18-positive) after seeding. No difference in basal Ca2+i was noted between ciliated and nonciliated cell preparations. For ciliated and nonciliated cells studied 1-2 days in culture, ATP, bradykinin, and histamine elicited a cytosolic Ca2+ response in 100% of the cells examined. For nonciliated HNE cells maintained 4-5 days in culture, ATP (10(-4) M) increased cytosolic Ca2+ in all cells tested, but only 85% of the cells responded to bradykinin (10(-5) M) addition, and 65% to histamine (10(-4) M) stimulation. In terms of the absolute change of Ca2+i (delta Ca2+i, peak-basal value), the efficacy was ATP > bradykinin > histamine for the 3 HNE cell preparations. However, the delta Ca2+i in response to agonists was smaller in nonciliated HNE cells studied 1-2 days or 4-5 days in culture as compared to the ciliated cell preparation. Thapsigargin (300 nM), an agent that mobilizes Ca2+i, was equally effective in raising cytosolic Ca2+ in nonciliated (1-2 days and 4-5 days in culture) and ciliated HNE cells. These data show that ciliated cells consistently respond to all agonists, whereas the cytosolic Ca2+ response to ATP, bradykinin, and histamine in nonciliated cells was quantitatively reduced at a comparable time period (1-2 days) and became smaller and less frequent in nonciliated cell preparations maintained 4-5 days in culture. These results demonstrate time-dependent differences in the magnitude and frequency of cytosolic Ca2+ responses to certain agonists, strongly indicating that measurements of Ca2+i in HNE cells must account for the heterogeneity of the cell types and the time cells are maintained in primary culture.     

Stimulus interval-dependent differences in Ca2+ transients and contractile responses of diabetic rat cardiomyocytes

OBJECTIVE: The aim of this study was to gain further insights into the consequences of insulin-dependent diabetes mellitus on cardiomyocyte calcium handling. METHODS: The effects of steady state and transient changes in stimulus frequency on the intracellular Ca2+ transient and cell shortening were examined in left ventricular cardiomyocytes isolated from the hearts of control and streptozotocin-induced diabetic rats. RESULTS: During steady state stimulation diabetic rat cardiomyocytes displayed a slower decay of the Ca2+ transient and longer times for maximum cell shortening and re-lengthening. At 1.5 mM extracellular [Ca2+], increasing stimulus frequency over the range 0.2-1.0 Hz led to an increase in resting and peak [Ca2+]i as well as the amplitude of the transient in both the control and diabetic groups. At frequencies greater than 0.4 Hz the amplitude of the transient was significantly depressed in diabetic rat cells and this was not normalized by increasing extracellular [Ca2+] to 2.5 mM. Recovery of sarcoplasmic reticulum (SR) Ca2+ release was measured from the time course of restitution of the intracellular Ca2+ transient. In both control and diabetic rat cardiomyocytes recovery of the transient occurred in two phases. In diabetic rat myocytes, the initial rapid phase of restitution at intervals <1 s was markedly slowed. The fraction of Ca2+ recirculating between the SR and the cytosol was estimated from the decline in amplitude of transients following post-rest potentiation. There was no difference in this fraction between control and diabetic rat cells either at 1.5 or 2.5 mM extracellular [Ca2+]. CONCLUSION: The blunted frequency response of diabetic rat cardiomyocytes at frequencies greater than 0.4 Hz is consistent with reduced SR Ca2+ uptake leading to reduced SR Ca2+ content and subsequent release. At stimulus intervals greater than 1 Hz this is likely to be exacerbated by slower recovery of SR Ca2+ release. Despite the evidence for depressed SR Ca2+ uptake, the relative amount of Ca2+ recirculating within diabetic rat cardiomyocytes remains unaltered. This is most likely due to an accompanying reduction in Ca2+ efflux from the cell due either to depressed Na+/Ca2+ exchanger activity, or an elevation in intracellular Na+ levels.     

Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons.

The role of intracellular Ca2+ concentration ([Ca2+]i) in the muscarinic suppression of Ca2+ current and M-type K+ current has been investigated in isolated rat sympathetic neurons using the whole-cell patch-clamp technique and fura-2 fluorescence measurements. Muscarinic stimulation suppressed currents without raising [Ca2+]i. Nonetheless, intracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2(+)-current suppression from 82 to 15%. For the latter, we explain the BAPTA action by a requirement for a certain minimum [Ca2+]i for continued operation of the pathway coupling muscarinic receptors to M-type K+ channels. The pathway coupling muscarinic receptors to Ca channels also showed some dependence on [Ca2+]i, but there may also be a blocking action of BAPTA that is independent of Ca2+ chelation.     

Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

OBJECTIVE: The aim of this study was to characterize the spatio-temporal dynamics of [Ca(2+)](i) in rat heart in the fetal and neonatal periods. METHODS: Using confocal scanning laser microscopy and the Ca(2+) indicator fluo-3, we investigated Ca(2+) transients and Ca(2+) sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. RESULTS: Ca(2+) transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca(2+)](i) compared to those in adult myocytes. The magnitude of fetal Ca(2+) transients was decreased after application of ryanodine (1 microM) or thapsigargin (1 microM). However, Ca(2+) sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca(2+) sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca(2+) sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca(2+) sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). CONCLUSIONS: The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca(2+) sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.     

An inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in liver nuclei.

Recent studies in our laboratory have revealed the existence of an ATP- and calmodulin-dependent Ca2+ uptake system in rat liver nuclei that can promote increases in the free Ca2+ concentration in the nuclear matrix. In the present investigation we show that liver nuclei possess a marked ability to sequester and buffer Ca2+, suggesting a potential role for the nucleus in the regulation of the cytosolic free Ca2+ concentration. In addition, we demonstrate that the intracellular messenger, inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3], stimulates the release of a fraction of the nuclear Ca2+ and transiently lowers the intranuclear free Ca2+ concentration. The Ins(1,4,5)P3-stimulated Ca2+ release is followed by Ca2+ reuptake into an inositol phosphate-insensitive nuclear compartment. Together, these results demonstrate that liver nuclei contain, at least, two Ca2+ pools, one of which is releasable by Ins(1,4,5)P3. These findings are consistent with a role for the nucleus in the modulation of the cytosolic free Ca2+ level by agonists and suggest that the control of the nuclear Ca2+ load by second messengers may participate in the regulation of intranuclear Ca2(+)-dependent processes by hormones and other agents.     

Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels

It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+. With 2 mmol/L Ca2+ in the external solution, we observed sparks at the beginning of every evoked AP at numerous locations. Each spark was observed repeatedly at a fixed location and began during a limited interval after the AP peak. These sparks occurred with a probability of approximately unity. However, the chance that an LCC does not open during the interval when a spark is triggered is quite high ( approximately 0.13). Therefore, because single channels open with a probability significantly lower than 1, more than one LCC must be available to ensure that sparks are triggered with a probability of approximately unity. We conclude that it is likely that a cluster of LCCs is involved in gating a cluster of ryanodine receptors at the beginning of an AP.     

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.     

The hemodynamic effects of diltiazem in the isolated perfused rat liver are Ca2+-dependent

Abstract: Aims/Background: Diltiazem reduces systemic blood pressure by decreasing the vascular smooth muscle tone. In the liver however, diltiazem seems to cause vasoconstriction, as evidenced by increases in portal pressure. The questions raised by this observation are concerned with a) the site of action of diltiazem (large vessels or sinusoids), b) the formation of permeability barriers and c) the role of Ca²⁺. The experiments in the present study should provide an answer to these questions. Methods: The experimental System was the hemoglobin-free perfused rat liver. The multiple-indicator dilution technique was employed with simultaneous injection of [¹⁴C]sucrose and [³H]water. Mean transit times and distribution spaces were calculated from the normalized outflow profiles. Results: Calcium alone did not affect the hemodynamics of the liver. Diltiazem, however, changed several hemodynamic parameters when Ca²⁺ was present, but it was inactive in the absence of this cation. The hemodynamic effects of 500 μM diltiazem were: a) diminution of the transit time through the large vessels (t_o) and, consequently, of the accessible vascular space (66.9%); b) diminution of the mean transit time of [¹⁴C]sucrose (t_suc) and, consequently, of the accessible sinusoidal space (28.1%); c) diminution of the mean transit time of tritiated water (t_water) and, consequently, of the accessible cellular space (68.9%); d) diminution of the cellular to extracellular space ratio (θ) from 1.42±0.05 to 0.46±0.11. Conclusions: The linear superposition of the tritiated water and labeled sucrose curves, predicted by Goresky's model, could be optimized even when the curves were obtained with diltiazem+Ca²⁺, indicating that the distribution of both tracer s was still flow-limited. The hemodynamic effects of diltiazem seem to be restricted to a vasoconstriction of the great vessels, an action which was strictly dependent on Ca²⁺. At the concentration of 500 μM, the effects of diltiazem were pronounced to the point of excluding completely about 2/3 of the liver parenchyma from the microcirculation, as indicated by the observed reduction in the accessible cell space. The sinusoids that were still supplied with perfusion fluid suffered considerable distension (2.19 fold) because the whole perfusate flow was deviated into the remaining 1/3 microcirculatory units. Diltiazem did not seem to induce the formation of intrahepatic shunts or diffusion barriers in the liver.     

Linoleic acid induces Ca2+-induced inactivation of voltage-dependent Ca2+ currents in rat pancreatic beta-cells

Free fatty acids (FFAs) regulate insulin secretion in a complex pattern and induce pancreatic beta-cell dysfunction in type 2 diabetes. Voltage-dependent Ca2+ channels (VDCC) in beta-cells play a major role in regulating insulin secretion. The aim of present study is to clarify the action of the FFA, linoleic acid, on VDCC in beta-cells. The VDCC current in primary cultured rat beta-cells were recorded under nystatin-perforated whole-cell recording configuration. The VDCC was identified as high-voltage-gated Ca2+ channels due to there being no difference in current amplitude under holding potential between -70 and -40 mV. Linoleic acid (10 microM) significantly inhibited VDCC currents in beta-cells, an effect which was fully reversible upon washout. Methyl-linoleic acid, which does not activate G protein coupled receptor (GPR)40, neither did alter VDCC current in rat beta-cells nor did influence linoleic acid-induced inhibition of VDCC currents. Linoleic acid-induced inhibition of VDCC current was not blocked by preincubation of beta-cells with either the specific protein kinase A (PKA) inhibitor, H89, or the PKC inhibitor, chelerythrine. However, pretreatment of beta-cells with thapsigargin, which depletes intracellular Ca2+ stores, completely abolished linoleic acid-induced decrease in VDCC current. Measurement of intracellular Ca2+ concentration ([Ca2+](i)) illustrated that linoleic acid induced an increase in [Ca2+](i) and that thapsigargin pretreatment inhibited this increase. Methyl-linoleic acid neither did induce increase in [Ca2+](i) nor did it block linoleic acid-induced increase in [Ca2+](i). These results suggest that linoleic acid stimulates Ca2+ release from intracellular Ca2+ stores and inhibits VDCC currents in rat pancreatic beta-cells via Ca2+-induced inactivation of VDCC.     

Depletion of intracellular Ca2+ stores sensitizes the flow-induced Ca2+ influx in rat endothelial cells

Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 micromol/L) or cyclopiazonic acid (10 micromol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 micromol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.     

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.     

Different actions of cardioprotective agents on mitochondrial Ca2+ regulation in a Ca2+ paradox-induced Ca2+ overload.

BACKGROUND: Mitochondrial Ca2+ overload is a major cause of irreversible cell injury during various metabolic stresses. The protective effects of various agents that affect mitochondrial function against Ca2+ overload during Ca2+ paradox were investigated in rat ventricular myocytes. METHODS AND RESULTS: On Ca2+ repletion following Ca2+ depletion, [Ca2+]i increased rapidly, and 90 of 210 cells (43%) died. In viable cells, the increase in [Ca2+]i was lower than in dead cells. KB-R7943 prevented the increase in [Ca2+]i, and completely inhibited cell death. Ruthenium red (RuR), diazoxide (Dz) or cyclosporin A (CsA) prevented cell death (15%, 26% and 17%, respectively; p < 0.05), and the protective effect of Dz was abolished by 5-hydroxydecanoate. These agents did not reduce the increase in [Ca2+]i in viable cells or the rate of initial increase in [Ca2+]i in all cells. RuR and Dz decreased [Ca2+]m in skinned myocytes, but CsA did not affect [Ca2+]m. Dz reduced NADH fluorescence, whereas RuR and CsA did not. CONCLUSIONS: The protective effects of RuR and Dz could be ascribed to altered Ca2+ regulation by decreasing [Ca2+]m, and Dz could have an additional effect on oxidative phosphorylation. The protective effect of CsA could be directly associated with the mitochondrial permeability transition pore.     

Cytosolic Ca2+-dependent neutral proteinases from rabbit liver: activation of the proenzymes by Ca2+ and substrate.

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.