Roles of Ca2+ on the inositol 1,4,5-trisphosphate-induced release of Ca2+ from saponin-permeabilized single cells of the porcine coronary artery

The release of Ca2+ from the intracellular store site, as induced by inositol 1,4,5-trisphosphate, was studied in relation to free Ca2+ concentrations or amounts of stored Ca2+ in smooth muscle cells. The maximal Ca2+ release induced by inositol 1,4,5-trisphosphate was observed when the amount of Ca2+ in the store site was about 50% of the maximal capacity of the Ca2+ storage, and when the extravesicular free Ca2+ concentration was less than 1.5 X 10(-6) M. The Ca2+ release induced by inositol 1,4,5-trisphosphate was accelerated by ATP and 5'-adenylylimidodiphosphate (AMPPNP), but not by ADP and AMP. This inositol 1,4,5-trisphosphate-induced Ca2+ release appeared to be specific for intracellular Ca2+ store sites (mainly sarcoplasmic reticulum), and this Ca2+ release was not apparent in the sarcolemmal fraction.     

Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca(2+)] (27). This Ca(2+)-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca(2+) sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used alpha-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca(2+)-desensitizing hypoxic relaxation.     

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.     

Effects of nitrates and calcium channel blockers on Ca2+-ATPase in the microsomal fraction of porcine coronary artery smooth muscle cells

The effects of the antianginal drugs nitroglycerin, nicorandil, diltiazem, verapamil and nicardipine on the activity of calcium-stimulated magnesium-dependent ATPase (Ca2+-ATPase) were investigated in the microsomal fraction from porcine coronary artery smooth muscle cells. Two discrete Ca2+-dependent ATPase components were observed: [1] a high affinity component, which was a specific Ca2+-ATPase, [with a half saturation constant for Ca2+ (Km) of 0.44 microM, and maximum velocity (Vmax) of 124.3 pmol of phosphate (Pi) released/micrograms of protein/30 min]: [2] a low affinity component in which Ca2+ could be replaced by Mg2+ without loss of its activity. Nitroglycerin and nicorandil (1 microM and 10 microM) both stimulated the activity of the Ca2+-ATPase significantly [142 +/- 12 (mean +/- standard error), and 137 +/- 10% of the control with nitroglycerin, and 152 +/- 17 and 135 +/- 20% with nicorandil] at a Ca2+ concentration of 0.3 microM. Diltiazem, verapamil and nicardipine did not cause significant stimulation. Nitroglycerin and nicorandil (1 microM), significantly decreased the Km for Ca2+ from the control value of 0.44 +/- 0.06 microM to 0.26 +/- 0.03 and 0.22 +/- 0.03 microM, respectively. Nitroglycerin and nicorandil may dilate coronary arteries by stimulating this Ca2+ extrusion pump enzyme through reduction of intracellular Ca2+ in smooth muscle cells.     

Sarcoplasmic reticulum Ca2+-pump density is higher in distal than in proximal segments of porcine left coronary artery

Densities of sarcoplasmic reticulum (SR) Ca²⁺-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca²⁺-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca²⁺-pump acylphosphate was 7.1 ± 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca²⁺-pump antibody was 5.3 ± 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca²⁺-pump inhibitors Cyclopiazonic acid (CPA, EC₅₀ = 0.19 ± 0.06 M) and thapsigargin (EC₅₀ = 0.0095 ± 0.0035 M). With 10 M CPA, the force of contraction per tissue wet weight was 4.2 ± 0.5-fold greater in distal than in proximal rings, and with 1 M thapsigargin it was 4.0 ± 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca²⁺-pump than the proximal segments.Abbreviations CPA cyclopiazonic acid - DTT dithiothreitol - SR sarcoplasmic reticulum     

Sarcoplasmic reticulum Ca2+-pump density is higher in distal than in proximal segments of porcine left coronary artery

Molecular and Cellular Biochemistry - Densities of sarcoplasmic reticulum (SR) Ca2+-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods:...     

Intercellular Ca2+ signalling: the artery wall

The fact that many cells in the cardiovascular system are coupled via gap junctions enables direct intercellular Ca2+ signalling. Ca2+ ions and/or inositol trisphosphate (IP3) can pass through these aqueous pores. Cell-cell coupling can occur between cells of the same type, or via special junctions between adjacent cells of different types. Homocellular coupling acts to amplify and prolong Ca2+ signals, whereas heterocellular coupling allows complex interactions between the cells, including the modulation of their respective responses. This review will focus on the interactions between cells that form the blood vessel wall, illustrating how cell-cell communication defines important physiological functions.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.     

Constriction of pulmonary artery by peroxide: role of Ca2+ release and PKC

Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca2+ using Fura-PE3. Ca2+ sensitization was examined in alpha-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC(20) was assayed by Western blot. Peroxide (30 microM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca2+ was affected by removal of extracellular Ca2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCalpha, but had no significant effect on MYPT-1 or MLC(20) phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC(20)-independent constrictor mechanism.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Cytosolic Ca2+ concentration during Ca2+ depletion of isolated rat hearts

Reperfusion of isolated mammalian hearts with a Ca²⁺-containing solution after a short Ca²⁺-free period at 37°:C results in massive influx of Ca²⁺ into the cells and irreversible cell damage: the Ca²⁺paradox. Information about the free intracellular, cytosolic [Ca²⁺] ([Ca²⁺]_i) during Ca²⁺ depletion is essential to assess the possibility of Ca²⁺ influx through reversed Na⁺/Ca²⁺ exchange upon Ca²⁺ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca²⁺-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca²⁺ from intracellular stores. Therefore, in this study, we measured [Ca²⁺]i during Ca²⁺- free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca²⁺-transients were recorded. Ca²⁺-transients disappeared within 1 min of Ca²⁺ depletion. Systolic [Ca²⁺]i during control perfusion was 268±54 nM. Diastolic [Ca²⁺]i during control perfusion was 114±34 nM and decreased to 53±19 nM after 10 min of Ca²⁺ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13±4 mmHg during control perfusion after Indo-1 AM loading to 31±5 mmHg after 10 min Ca²⁺ depletion. Left ventricular developed pressure did not recover during Ca²⁺ repletion, indicating a full Ca²⁺ paradox. These results show that LVEDP increased during Ca²⁺ depletion despite a decrease in [Ca²⁺]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na⁺/Ca²⁺ exchanger showed that reversed Na⁺/Ca²⁺ exchange during Ca²⁺ repletion is not able to increase [Ca²⁺]i to cytotoxic levels.     

Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery.

The activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE) and inositol 1,4,5,-trisphosphate (IP3) phosphatase in the particulate and cytosol fractions prepared from porcine coronary artery smooth muscles were examined using 32P-labelled PIP2 and IP3 as substrates, respectively. The activity of PIP2 PDE, as assessed from the production of IP3, in the cytosol fraction was about 10-fold higher than that in the particulate fraction. In the absence of MgCl2, the activity of PIP2 PDE in both fractions showed no causal relation to the free Ca2+ concentration in the physiological range of 10(-7)-10(-5) M, but was enhanced remarkably by 10(-4) M free Ca2+. The addition of 1 mM-MgCl2 to the assay medium markedly inhibited the activity of PIP2 PDE in both fractions in the presence of free Ca2+ (10(-8)-10(-5) M). In the absence of MgCl2, 10(-5)M-acetylcholine (ACh) produced IP3, and this action was blocked by 3 X 10(-6) M-atropine. The ACh-induced activation of PIP2 PDE ceased in the presence of 1 mM-MgCl2; however, the reactivation occurring on the addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate did not depend on the free Ca2+ concentrations (10(-7)-10(-5)M). The activities of IP3 phosphatase, determined from decrease in the amount of IP3 in the particulate and cytosol fractions, had much the same potency in both fractions. The activity of IP3 phosphatase in the cytosol fraction was enhanced by MgCl2 in a concentration-dependent manner, the maximal value occurring at 1 mM-MgCl2, and was also enhanced in the presence of physiological concentrations of free Ca2+ (10(-7)-10(-6) M). These findings suggest that the activation of PIP2 PDE which occurs with application of ACh in the presence of guanine nucleotides and 1 mM-MgCl2 is independent of the free Ca2+ concentration, and that the hydrolysis of IP3 by phosphatase increases, depending on the concentration of free Ca2+.     

The role of Ca2+ and hemodynamics in the action of diltiazem on hepatic energy metabolism

Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca²⁺-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic effects. In the absence of Ca²⁺, the hemodynamic effects are no longer present and, consequently, Ca²⁺-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent effects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca²⁺. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The effects of diltiazem can be divided into three groups: (1) Effects that are strictly dependent on the Ca²⁺-mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50–500 mol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) Effects that are independent of the hemodynamic action. The most relevant effect of this type is inhibition of gluconeogenesis. (3) Effects that are influenced by Ca²⁺ but are independent of the hemodynamic effects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca²⁺ than in its absence.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

Effect of Ca2+ on the cryoprotective action of trehalose

The competitive effect of Ca2+ on the cryoprotective action of carbohydrates has been investigated during freeze-thaw processes of unilamellar egg phosphatidylcholine vesicles. Ca2+ inhibits the cryoprotection achieved by trehalose to a greater extent than other sugars such as galactose, sucrose, and fructose. The cryoprotection by trehalose is also dependent on the Ca2+ concentration in the inside solution of the vesicle, even in the absence of external Ca2+. The competitive effect of Ca2+/trehalose is interpreted as a consequence of the different amount of interfacial water displaced by each compound in their adsorption on the water/lipid interface.