The role of sarcolemma calcium pump in regulating tonic smooth muscle contraction

Vanadate (10(-4)-10(-3) M) effectively blocks Mg2+, ATP-dependent Ca2+ transport in sarcolemmal vesicles and induces a slowly tonic contraction of the smooth muscle. This contraction was observed both with and without nifedipine (10(-5) M) evoking complete inhibition of hyperpotassium contracture, the Ca2+ removal from the solution washing the muscular preparation stimulating the tone decrease. There is a close correlation between the dose-dependent effects of vanadate on the Ca pump activity and tension. It is concluded that in smooth muscles, at least in myometrium, the sarcolemmal Ca-pump is involved into the control of the tonic tension.     

The role of sarcolemma and mitochondria in calcium-dependent control of myometrium relaxation

Using atomic absorption spectroscopy, it was shown that the amount of firmly bound Ca2+ in cattle mitochondria and myometrium sarcolemma is 160 +/- 10 and 30 +/- 10 mumol/kg of wet tissue, respectively. The Ca2+ 1 accumulating capacity of mitochondria (350 nmol per mg of protein) markedly exceeds that of sarcolemmal vesicles (30 nmol per mg of protein). Using a Ca2+-EGTA buffer, it was found that the affinity of ionized Ca for the mitochondrial transport system (Km = 5.69 microM) is higher than that for the Na+-Ca2+ system of sarcolemma exchange (Km = 30 microM), but is markedly lower than that for the Mg2+, ATP-dependent Ca2+ efflux (Km = 0.35 microM). A kinetic analysis demonstrated that the sarcolemmal Ca2+ pump is incapable of causing complete relaxation of the smooth muscle within the physiologically significant time, whereas the Ca2+ transport system of mitochondria evokes this process within 21 s. However, the contribution of the Ca2+ pump to the regulation of the Ca2+ content in myocytes is paralleled with the accumulation of Ca2+ in mitochondria and is realized at low concentrations of this cation in the myoplasm, i.e., at late steps of relaxation. A mechanism of Ca2+ control over myometrium relaxation is proposed. The system of non-electrogenic Na+-Ca2+ exchange maintains Ca2+ concentration in the myoplasm as high as 10(-5) M. Mitochondria which accumulate the bulk of Ca2+ rapidly decrease its concentration in the cytoplasm down to 10(-6)-10(-7) M; at these values, the activity of the sarcolemmal Ca2+ pump with a high affinity for the transfer substrate is manifested. In this way, the Ca2+ pump accomplishes fine regulation of Ca2+ concentration in the myocytes.     

Cyclic GMP- and AMP-modified calcium binding by the myocardial sarcolemma in circulatory hypoxia

10(-6) M cAMP were shown to induce a 61% and 21% increase in 45Ca binding to sarcolemma proteins in intact and injured (circulatory hypoxia) hearts, respectively. The addition of exogenous protein kinase equalized 45Ca-binding levels in normal and impaired sarcolemma. The decrease in 45Ca-binding capacity by 16 and 36% was detected in the presence of 10(-7) and 10(-6) M of cGMP, respectively. In impaired hearts, cAMP and Ca-ATPase activity levels remain constant, while cGMP content increases, as compared to normal myocardial level.     

The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.     

The role of intracellular calcium in the ischemic myocardial damage

The isolated, perfused ventricles of guinea-pig and rat hearts stimulated at the rate of 60/min were equilibrated for 60 min with 45Ca containing solution. Thereafter some of them were perfused for the last 10 min of experiment with deoxygenated (pO2 = 35 not equal to 7 mm Hg) radioactive solution. Hypoxia resulted in decrease of exchangeable calcium (45Ca) content by 0.90 mmol/kg w.w. in guinea-pig and by 0.26 mmol/kg w.w. in the rat. The amount of 15Ca lost by guinea-pig ventricles is equal to the content of rate-dependent fraction Ca2 described in the previous papers [Pytkowski et al., 1983; Lewartowski et al., 1984]. The isolated papillary muscles of the right ventricles of guinea-pig and rat hearts were subjected to 90 min of ischemia simulated by immersion in the warm, deoxygenated paraffin oil. Some of the guinea-pig muscles were deprived of Ca2 fraction by means of prolonged rest (20 min) immediately prior to ischemia. All the preparations were quiescent during ischemia. The guinea-pig muscles deprived of fraction Ca2 and the rat muscles developed much weaker contracture during ischemia and showed better recovery of phasic contraction upon reperfusion than the guinea-pig muscles containing Ca2 fraction prior to ischemia. We propose that Ca2 fraction is released from its binding sites at the early phases of ischemia contributing to the disturbances in Ca homeostasis and to mechanism of damage of ischemic cardiac muscle.     

Relation between the passive transport of calcium into vesicles of the myocardial sarcolemma and membrane potential

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.     

The role of calcium in the pH-dependent control of Photosystem II

pH-dependent inactivation of Photosystem (PS) II and related quenching of chlorophyll-a-fluorescence have been investigated in isolated thylakoids and PS II-particles and related to calcium release at the donor side of PS II. The capacity of oxygen evolution (measured under light saturation) decreases when the pH is high and the pH in the thylakoid lumen decreases below 5.5. Oxygen evolution recovers upon uncoupling. The pH-response of inactivation can be described by a 1 H⁺-transition with an apparent pK-value of about 4.7. The yield of variable fluorescence decreases in parallel to the inactivation of oxygen evolution. pH-dependent quenching requires light and can be inhibited by DCMU. In PS II-particles, inactivation is accompanied by a reversible release of Ca²⁺-ions (one Ca²⁺ released per 200 Chl). In isolated thylakoids, where a pH was created by ATP-hydrolysis, both inactivation of oxygen evolution (and related fluorescence quenching) by internal acidification and the recovery of that inactivation can be suppressed by calcium-channel blockers. In the presence of the Ca²⁺-ionophore A23187, recovery of Chl-fluorescence (after relaxation of the pH) is stimulated by external Ca²⁺ and retarded by EGTA. As shown previously (Krieger and Weis 1993), inactivation of oxygen evolution at low pH is accompanied by an upward shift of the midpoint redox-potential, E_m, of Q_A. Here, we show that in isolated PS II particles the pH-dependent redox-shift (about 160 mV, as measured from redox titration of Chl-fluorescence) is suppressed by Ca²⁺-channel blockers and DCMU. When a redox potential of –80 to –120mV was established in a suspension of isolated thylakoids, the primary quinone acceptor, Q_A, was largely reduced in presence of a pH (created by ATP-hydrolysis) but oxidized in presence of an uncoupler. Ca²⁺-binding at the lumen side seems to control redox processes at the lumen- and stroma-side of PS II. We discuss Ca²⁺-release to be involved in the physiological process of high energy quenching.     

HIP-55 negatively regulates myocardial contractility at the single-cell level

Myocardial contractility is crucial for cardiac output and heart function. But the detailed mechanisms of regulation remain unclear. In the present study, we found that HIP-55, an actin binding protein, negatively regulates myocardial contractility at the single-cell level. HIP-55 was overexpressed and knocked down in cardiomyocytes with an adenovirus infection. The traction forces exerted by single cardiomyocyte were measured using cell traction force microscopy. The results showed that HIP-55 knockdown significantly increased the contractility of the cardiomyocytes and HIP-55 overexpression could markedly reverse this process. Furthermore, HIP-55 was obviously co-localized with F-actin in cardiomyocytes, suggesting that HIP-55 regulated cardiac contractile function through the interaction between HIP-55 and F-actin. This study reveals the regulatory mechanisms of myocardial contractility and provides a new target for preventing and treating cardiovascular disease.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

Intracellular Ca2+ homeostasis in the smooth muscle and functional role of calcium pump in the sarcolemma

It has been found that Ca-pump of the smooth muscle sarcolemma has much greater affinity to Ca2+ (Km = 0.5 M) than the system Na-Ca2+ of the exchanger (Km = 40-60 M). The maximal rate of Mg2+, ATP-dependent translocation of Ca2+ is 2-3 times higher than that of Na-dependent. The results of kinetic analysis show that Ca-pump of the smooth muscle sarcolemma is able to compensate the basal diffusion flow of this cation entering into unexcited cells of smooth muscle (5 x 10(-15) mol Ca2+ per 1 cm2 for 1 sec). It can also stationary support the value of Ca2+ concentration in relaxed myocytes on a physiologically significant level (10(-7)-10(-6) M).     

Calcium-binding sites in rat myocardial sarcolemma.

Myocardial sarcolemmal preparations have the ability to bind Ca²⁺; this binding shows saturation kinetics, has a pH optimum of 7.4 to 7.8, and is stimulated by ATP. Scatchard analysis reveals both high- and low-affinity binding sites. Both classes are inhibited by ruthenium red, whereas only the high-affinity sites are affected by lanthanum and hydroxylamine. Electrophoresis in the presence of sodium dodecyl sulfate suggests that the high-affinity sites are associated with a protein peak of molecular weight of about 100,000. Enzyme treatment of the membranes suggested that proteins accounted for most of the ⁴⁵Ca binding with phospholipids and sialic acids residues playing a secondary role. Interaction with different function group reagents indicated that the carboxyl residues were necessary for the calcium binding, whereas thiol, amino, and sulfhydryl groups were unimportant.     

Effect of oxytocin on the calcium pump in myometrium sarcolemma

Oxytocin (10(-7) M) administered inside the myometrium sarcolemma vesicles closed outward by the cytoplasmic side is shown to inhibit Mg2+, ATP-dependent Ca2+ accumulation in these structures having no effect on the passive release of cation out of them. According to these results and to the data available in literature on the inhibitory action of the peptide hormone on Mg2+, Ca2+-ATPase of myometrium sarcolemma a conclusion is drawn that oxytocin inhibits the Ca pump activity in plasma membranes of the myometrium cells.     

Anaerobic rat heart: mitochondrial role in calcium uptake and contractility.

In order to evaluate the manner by which fumarate enhances contractility in the anaerobic heart, we examined Ca++ movements in isolated heart mitochondria and in the isolated perfused heart. Our experiments showed that in isolated antimycin A plus cyanide treated mitochondria: (a) Ca++ uptake was promoted by electron transport generated by fumarate-dependent oxidation of NADH, (b) Ca++ stimulated fumarate-dependent oxidation of NADH, (c) the ratio of Ca++ uptake:NADH oxidized was 1.7 and (d) the Ca++ sequestered is transiently highly mobile and is rapidly released upon collapse of the membrane potential. In anaerobic hearts perfused with glucose plus fumarate, malate and glutamate Ca++ levels were the same as those in oxygenated hearts while in anaerobic organs perfused with or without glucose Ca++ content was appreciably lower. Succinate production in anaerobic heart perfusions was related to: (a) an increased retention of Ca++ by the heart, (b) a diminution in peak aortic pressure generated by cardiac contractions and (c) an increase in heart rate. The information obtained indicates that mitochondria have a capability for Ca++ movement which be used physiologically, particularly in fumarate perfused anaerobic hearts, to assist the mechanism for contraction and relaxation of the heart.     

Sodium Exchange in Dog Ventricular Muscle : Relation to frequency of contraction and its possible role in the control of myocardial contractility

Sodium exchange was studied in the arterially perfused papillary muscle of the dog. Three kinetically defined phases accounted for all the myocardial sodium: phase 0 (vascular)-λ_o (exchange constant) = 3.6 min^-1 phase 1 (interstitial)-λ₁ = 0.62 min^-1; phase 2 (intracellular)-λ₂ < 0.020 min^-1 in quiescent muscles. The phase 2 exchange rate was proportional to frequency of contraction and increased by approximately 0.004 min^-1 for each 1 beat/min increment in rate in muscles demonstrating stable function. A sudden increase in frequency of contraction was followed by a marked increase in phase 2 sodium exchange if muscle function did not deteriorate. This increased exchange required 14 min to achieve a steady state. During this time active tension increased (positive staircase) and then declined to become stable as the sodium exchange stabilized. In muscles in which increased frequency of contraction produced a progressive decrease in active tension and contracture, sodium exchange failed to increase. The characteristics of sodium exchange are compared to those previously defined for calcium and potassium in the perfused dog papillary muscle. It is proposed that alteration in sodium exchange is a primary determinant of calcium and potassium movements and thereby plays a significant role in the control of myocardial contractility.     

Na,K-ATPase activity in the myocardial sarcolemma in ischemia

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.     

Potential-dependent passive transport of calcium in myocardium sarcolemma vesicles

The effect of membrane potential on passive Ca2+ transport in isolated cardiac sarcolemmal vesicles was investigated. The membrane potentials were induced by creating potassium gradients across the vesicular membranes in the presence of valinomycin. The fluorescence changes in the voltage-sensitive dye, dis-C3(5), were consistent with the induction of potassium equilibrium potentials. The rate of 45Ca2+ efflux from inside-out vesicles was considerably greater at 0 than at -80 or +55 mV; prepolarization of the membrane to +90 mV did not enhance the 45Ca2+ efflux upon subsequent depolarization. The voltage-dependent 45Ca2+ efflux increased with a rise in internal Ca2+ concentration and exhibited a saturation effect. Furthermore, evaluation of the rate of 45Ca2+ efflux over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. It is concluded that the voltage-dependent Ca2+ efflux from the vesicles occurs via Ca2+-channels.