Total Tissue Ca Research Articles

Mitochondria-targeted Cardioprotection in Aldosteronism

Chronic aldosterone/salt treatment (ALDOST) is accompanied by an adverse structural remodeling of myocardium that includes multiple foci of microscopic scarring representing morphologic footprints of cardiomyocyte necrosis. Our previous studies suggested that signal-transducer-effector pathway leading to necrotic cell death during ALDOST includes intramitochondrial Ca overloading, together with an induction of oxidative stress and opening of the mitochondrial permeability transition pore (mPTP). To further validate this concept, we hypothesized that mitochondria-targeted interventions will prove to be cardioprotective. Accordingly, 8-week-old male Sprague-Dawley rats receiving 4 weeks ALDOST were cotreated with either quercetin, a flavonoid with mitochondrial antioxidant properties, or cyclosporine A (CsA), an mPTP inhibitor, and compared with ALDOST alone or untreated, age/sex-matched controls. We monitored mitochondrial free Ca and biomarkers of oxidative stress, including 8-isoprostane and H2O2 production; mPTP opening; total Ca in cardiac tissue; and collagen volume fraction to quantify replacement fibrosis, a biomarker of cardiomyocyte necrosis, and employed terminal deoxynucleotidyl transferase dUTP nick end labeling assay to address apoptosis in coronal sections of ventricular myocardium. Compared with controls, at 4 weeks ALDOST we found a marked increase in mitochondrial H2O2 production and 8-isoprostane levels, an increased propensity for mPTP opening, and greater concentrations of mitochondrial free [Ca]m and total tissue Ca, coupled with a 5-fold rise in collagen volume fraction without any terminal deoxynucleotidyl transferase dUTP nick end labeling-based evidence of cardiomyocyte apoptosis. Each of these pathophysiologic responses to ALDOST was prevented by quercetin or cyclosporine A cotreatment. Thus, mitochondria play a central role in initiating the cellular-subcellular mechanisms that lead to necrotic cell death and myocardial scarring. This destructive cycle can be interrupted and myocardium salvaged with its structure preserved by mitochondria-targeted cardioprotective strategies.

Calcium accumulation and regulation in Daphnia magna: Links with feeding, growth and reproduction

Calcium is involved in a wide variety of biological processes and has an important structural role in crustaceans. The present study aimed at exploring the possible link between Ca body concentrations and the ingestion rate and the role of soft tissue vs. total tissue Ca accumulation in Daphnia magna. D. magna was cultured for 21 days at different water Ca concentrations ranging from 3.4 to 32.5 mg/L. Every week Ca body concentrations (soft and total tissues), ingestion rate, growth, survival and reproduction were measured. Daily, algal food that was not deficient in Ca was supplied. Ca in the soft tissues represented 8 to 26% of the total Ca body concentrations. The ratio Ca in soft tissue/Ca in total tissue was generally not influenced by the Ca exposure concentration but decreased with time, i.e., age (from an average of 0.24 at day 7 to 0.09 at day 21). During week 1, a 54% decrease in Ca body concentrations was observed in daphnids exposed in medium with 3.4 mg/L Ca compared to those exposed to 32.5 mg/L. The concurrent decrease in ingestion rate was 14%. No significant differences among Ca treatments were observed during week 2 for ingestion rate and week 3 for calcium body concentrations. Also, no effects on growth and reproduction were observed, although these were expected at the lowest Ca concentration tested. It is hypothesised that Ca absorption from food in combination with an increased ingestion rate is used to maintain Ca homeostasis under Ca limiting conditions.

The molecular basis for the use of calcium antagonists in ischaemic heart disease.

Calcium antagonists are useful for the management of patients with ischaemic heart disease, particularly when used prophylactically. At the cellular level, these drugs act primarily by limiting calcium ion (Ca++) entry through the voltage-sensitive Ca(++)-selective channels, an effect that contributes markedly to their 'energy sparing' properties. However, the long term use of these drugs has additional advantages, particularly with respect to their ability to slow Ca(++)-dependent processes involved in the formation of atherogenic lesions, partially antagonise the effects of the raised levels of circulating endothelin-1 encountered during ischaemia-induced heart failure and hypertension, and trap and immobilise oxyradicals. Prolonged episodes of ischaemia result in an irreversible loss of homeostasis with respect to Ca++. However, the increase in myocardial cytosolic Ca++ caused by relatively short periods of ischaemia is small, reversible, and markedly attenuated by the prophylactic use of calcium antagonists. In the isolated, perfused rat heart, verapamil pretreatment produces statistically significant inhibition of the increase in cytosolic Ca++ during 20-minute global ischaemia. This stereospecific effect is associated with a decrease in the rise in total tissue Ca++ during reperfusion and amelioration of the adenosine triphosphate depletion caused by ischaemia. In general, discussion relating to the molecular basis of the use of calcium antagonists in the management of patients with ischaemic heart disease needs to take into account the duration of the ischaemic event, the workload on the myocardium, the need for prophylactic therapy, and the presence of exacerbating factors such as atherosclerosis and tobacco smoking. The early rise in cytosolic Ca++, the source of which remains uncertain, appears to be an important focus for anti-ischaemic drug therapy.

Reoxygenation-dependent decrease in mitochondrial NADH:CoQ reductase (Complex I) activity in the hypoxic/reoxygenated rat heart

Reoxygenation of the hypoxic myocardium results in a number of processes, including an O2-dependent increase in total tissue Ca2+ and cell lysis in which mitochondrial electron transport plays a key role. In the present study we have isolated mitochondria from perfused rat hearts subjected to hypoxia and found no change in their respiratory function relative to controls. In contrast, mitochondria isolated immediately after reoxygenation of hypoxic-perfused hearts exhibited a specific and significant decrease in NADH:CoQ reductase (Complex I; EC 1.6.5.3) activity, as measured both polarographically and spectrophotometrically. Isolated cardiomyocytes subjected to a similar protocol of hypoxia/reoxygenation also exhibited a specific decrease in Complex I activity. Myocardial perfusion with media containing Ruthenium Red protected against the reoxygenation-dependent loss of Complex I activity. These observations taken together suggest that mitochondrial Ca2+ uptake on reoxygenation is implicated in the mechanism of the specific loss of Complex I activity.

Calcium handling by renal tubules during oxygen deprivation injury to the kidney prior to reoxygenation

Efforts to more precisely define the mechanism(s) of ischemic injury to renal epithelial tissue, both during O2 deprivation and reflow, have led to the expanding use of freshly isolated renal tubules. This tissue is prepared in a manner that eliminates the impact of changes in vascular resistance and influences of hormones, as well as temporal changes in pH and in regional pO2, and has permitted investigators to focus on the definitive cellular responses to O2 deprivation itself. When such studies are evaluated it becomes clear that in vitro anoxia, for up to 60 minutes, is not associated with any increase in total tissue Ca2+ as measured by atomic absorption techniques, whereas severe hypoxia is attended by a time-dependent increase in total tissue Ca2+. In both severe hypoxia and anoxia, however, the Ca2+ uptake rate is increased, but during hypoxia the less severe acidosis, as well as the continued, albeit modest, mitochondrial energization, appears to facilitate mitochondrial (and thus total tissue) Ca2+ accumulation. In vivo and in vitro, the administration of calcium channel blockers (CCB) attenuates renal oxygen-deprivation-induced injury and one, often overlooked, effect of verapamil, a prototypical CCB, is to reduce K+ loss from treated tissue via inhibition of Ca2(+)-mediated K+ efflux pathways. This may be the cause of the higher levels of K+ reported for several tissues, including kidney tubules, during CCB treatment. In addition, reduced rates of Ca2+ uptake effected by CCB may modify cytosolic free Ca2+ levels such that activation of phospholipases is impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of calcium in the brain following head trauma

Previous studies have reported accumulation of calcium (Ca) in brain tissue of injured or ischaemic experimental animals. In the present study, head trauma (HT) was induced in the left hemisphere of rats which were subsequently sacrificed 15 min, 1, 2, 4, 24 or 48 h later. Their brains were analysed for oedema formation by the determination of specific gravity (SG), using linear gradient columns, and water content, by dry to wet weight ratio. Total tissue Ca content was measured by atomic absorption spectroscopy. These values, in both the injured and contralateral hemispheres were compared with values obtained from sham-operated rats. Specific gravity of the contused hemisphere was lower than that of the contralateral hemisphere or sham and its water content was higher, at all time points studied. Calcium content was significantly higher in the contused grey matter at 1 h, and in the grey and white matter of both hemispheres at 24 and 48 h after HT. Statistical analysis revealed excellent correlation (cc = 0.65, p less than 0.001) between Ca levels and water content in the grey matter, whenever Ca concentrations were elevated (1, 24 and 48 h). These findings suggest that in the late phase of the post-HT period, Ca accumulation might play a role, along with other mediators, in the development of brain oedema after HT.

Alleviation of cyclosporine nephrotoxicity with verapamil and ATP-MgCl2. Mitochondrial respiratory and calcium studies.

Although recent studies have shown that combined treatment with verapamil and ATP-MgCl2 (ATP) prevents cyclosporine (CyA)-induced nephrotoxicity, the mechanism of these effects remains unknown. To study this, rat kidneys were perfused at 100 mmHg for 100 minutes with Krebs buffer containing 7.5 g/dL of albumin and substrates. After an equilibration period of 30 minutes, 500 ng/mL CyA was added. In some experiments 1 microgram/mL verapamil was added 10 minutes prior to CyA and in others 2 mM ATP was added to CyA. At the end of the perfusion, cortical mitochondria (mito) were isolated and mito Ca2+ and Mg2+ (mumoles/g protein) and respiratory control ratios (RCR) were measured. In addition, total tissue Ca2+ and Mg2+ levels were measured. The results indicate that CyA treatment leads to an accumulation of mito Ca2+ and a decrease in ADP/O ratio. Simultaneous administration of ATP with CyA led to an increased mito Ca2+ accumulation and depressed RCR, which were corrected by verapamil pretreatment. The combination of verapamil pretreatment and ATP cotreatment with CyA increased tissue ATP levels from 0.8 +/- 0.4 (control) to 1.4 +/- 0.1 mumol/g. This pharmacologic regimen may prevent CyA-induced nephrotoxicity by preventing mito Ca2+ accumulation and by preserving mitochondrial respiratory function. This allows a more efficient generation of ATP and consequently preservation of renal function.

Protective effect of coenzyme Q10 on thyrotoxic heart in rabbits

An excess of thyroid hormone is known to produce cardiac dysfunction and failure, i.e., thyrotoxic heart. We studied the protective effect of coenzyme Q10 (CoQ10) on the thyrotoxic heart in 29 rabbits. A group treated with 1-thyroxine sodium salt (T4; 167 micrograms/kg) for 3 weeks showed marked decreases in the myocardial content of norepinephrine (NE) and ATP (0.5 +/- 0.2 microgram/g wet weight, P less than 0.05 and 31.1 +/- 2.6 nmol/mg protein, P less than 0.05, respectively) as compared with a group treated with CoQ10 solvent (2 ml/kg) for 3 weeks (1.1 +/- 0.1 microgram/g wet weight and 45.7 +/- 4.7 nmol/mg protein). The mitochondrial Ca2+ content of the T4 group showed significant increases (21.3 +/- 0.6 nmol/mg protein, P less than 0.05) compared with the solvent group (18.2 +/- 0.8 nmol/mg protein), while the total tissue Ca2+ content of the T4 group was unchanged compared with the solvent group. These biochemical derangements suggest that T4-treated rabbits were in a state of cardiac dysfunction. In contrast, a group which was assigned to concomitant treatment of T4 and CoQ10 (5 mg/kg) for 3 weeks showed no reductions in NE and ATP (0.9 +/- 0.2 micrograms/g wet weight and 44.6 +/- 1.9 nmol/mg protein, respectively) and protected an increase in the mitochondrial Ca2+ content (18.2 +/- 1.2 nmol/mg protein). A group treated with CoQ10 (5 mg/kg) for 3 weeks showed no changes in myocardial NE, ATP, and Ca2+ content in the mitochondria. These results suggest that exogenously administered CoQ10 may protect against biochemical derangements in the thyrotoxic heart.

Effect of chronic digoxin treatment on cardiac function, electrolytes and ATPase in failing heart due to pressure overload

A decrease in the cardiac function and intracellular calcium, and an increase in the cardiac sarcolemmal ATPase have been reported in aortic stenosis of 6 to 9 months duration in dogs. The present investigation deals with the effect of 3 months of digoxin treatment on cardiac function, electrolytes and ATPase in dogs with 3 months of aortic stenosis in order to determine whether digoxin treatment gives only haemodynamic improvement or if it also improves the condition of the myocardium in terms of contractility and biochemical changes. There were no significant changes in any of the haemodynamic parameters of left and right ventricles except the left ventricular end-diastolic pressure which increased significantly at 3 months of aortic stenosis. All the hearts developed left ventricular hypertrophy. Concomitant with these changes, there was a tendency for a decrease in the total tissue Ca2+, intracellular Ca2+ and K+, and a tendency for an increase in the sarcolemmal ATPase. There were no significant differences in any of the haemodynamic parameters between the aortic stenotic and digoxin treated aortic stenotic dogs indicating that digoxin was at least maintaining the haemodynamics close to the pretreatment level. Although digoxin treatment prevented the changes in the sarcolemmal ATPase and extracellular space, it did not prevent the further decrease in the total or intracellular Ca2+. The total and intracellular Ca2+ was still significantly higher than previously observed after 6 to 9 months of aortic stenosis. These results suggest that digoxin treatment not only tended to prevent further deterioration of cardiac function but also tended to prevent further changes in the sarcolemmal ATPase and electrolytes.

Effect of prolonged prazosin treatment on hemodynamic and biochemical changes in the dog heart due to chronic pressure overload.

A decrease in cardiac function and intracellular calcium, and an increase in cardiac sarcolemmal ATPase have been reported in experimentally induced aortic stenosis of 6 to 9 months duration. Prazosin has been used in the treatment of heart failure due to mechanical ventricular overload. It is, however, not known whether prazosin treatment gives only hemodynamic benefit with accompanying subjective improvement or if it also improves the condition of the myocardium in terms of contractility and biochemical changes. The present investigation deals with the effects of 3 months of prazosin treatment on cardiac function, electrolytes, and ATPase in dogs with aortic stenosis of 3 months duration. Although there were no significant changes in most of the left and right ventricular hemodynamic parameters, the left ventricular end-diastolic pressure increased significantly after 3 months of aortic stenosis. Prazosin prevented further deterioration of cardiac function. All the dogs developed left ventricular hypertrophy and all chest X-rays showed cardiomegaly. Concomitant with these changes, there was a tendency towards a decrease in total tissue Ca++ and intracellular Ca++ and K+ and a tendency towards an increase in sarcolemmal Na+-K+-ATPase. Prazosin treatment, although it markedly reduced left ventricular end-diastolic pressure, did not reduce the cardiomegaly. There were no significant changes in any of the other hemodynamic parameters. Prazosin treatment decreased sarcolemmal ATPase and tended to increase intracellular Ca++. It appears therefore that prazosin not only tends to bring the cardiac function towards control values but also tends to correct the ATPase and intracellular Ca++ levels of the failing heart.

Acid and alkaline phosphatase activities and calcium transport in aortic smooth muscle from DOCA hypertensive rats

The activities of acid and alkaline phosphatases were significantly increased in subcellular fractions isolated from the aorta of deoxycorticosterone-acetate induced hypertensive rats as compared to normotensive Sprague-Dawley rats. Calcium uptake by the microsomal vesicles of deoxycorticosterone-acetate induced hypertensive rats was slightly reduced as compared to normotensive Sprague-Dawley rats, particularly in the presence of 10 μ m free CaCl 2. Total tissue Ca 2+ content in the aortae was significantly increased in deoxycorticosterone-acetate induced hypertensive rats as compared to normotensive Sprague-Dawley rats. These results suggest that increased activities of phosphatases in the vascular smooth muscle may be representative of biochemical alterations occurring in the deoxycorticosterone-acetate-hypertensive rats.

Ca2+ binding, ATP-dependent Ca2+ transport, and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis.

Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition. Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca2+. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 A particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport. Our studies confirm earlier observations of a decreased rate of Ca2+ uptake and Ca2+ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca:ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of 32Pi liberation from gamma-ATP32 during active calcium transport by the isolated sarcoplasmic reticulum SR. In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring 35S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.

THE EFFECT OF MAGNESIUM ON THE RESPONSE OF SMOOTH MUSCLE TO 5‐HYDROXYTRYPTAMINE

1 The responses to 5-hydroxytryptamine (5-HT) of rabbit isolated mesenteric artery and vein and longitudinal smooth muscle of guinea-pig ileum were examined in Krebs solution containing 0, 1.2 or 2.4 mM Mg2+. 2 When the concentration of Mg2+ was raised the spontaneous contractile activity of rabbit mesenteric vein was depressed. The responses to 5-HT in rabbit mesenteric artery and vein and guinea-pig ileum were greater in the absence of Mg2+. The initial fast component of 5-HT-induced contractions in rabbit mesenteric vein was reduced more consistently than the subsequent slow component by increasing the Mg2+ concentration. 3 Exposure of mesenteric vein to Ca-free solution containing ethyleneglycoltetra-acetic acid (EGTA) promptly abolished 5-HT contraction in normal-Mg but not in low-Mg Krebs solution. 4 In mesenteric veins, no difference was observed in either the 'lanthanum-resistant' uptake of 45Ca or total tissue Ca, measured by atomic absorption spectrophotometry, after 60 min exposure to either low-Mg or normal-Mg Krebs solution. On the other hand, after 5 min exposure, the 'lanthanum-resistant' uptake of 45Ca was greater in the absence of Mg2+ than in the presence of higher Mg2+ concentrations. 5 It is suggested that Mg2+ depressed the 5-HT response at least partly by reducing the availability of Ca2+ from a rapidly equilibrating intracellular pool.

High-resolution radioisotopic measurements of calcium, strontium, and barium in heart.

A gamma-ray detector system used in conjunction with isolated vascularly perfused rabbit septa labeled with the Ca-like cations 85Sr and 133Ba has been developed to test the feasibility of constructing a considerably more efficient detector system capable of measuring the highly energetic (Emax congruent to 1.3 MeV) 47Ca radionuclide. In addition an experimental design has been developed to quantify, statistically, perturbations of total tissue Ca, Sr, or Ba should they occur. This project was undertaken because the severe attenuation by living tissue of the beta-emitting radionuclide 45Ca (Emax congruent to 250 keV) limits its usefulness as a Ca tracer during experiments in which total tissue Ca is being measured minute-by-minute by an external detector. Analyses of over 100 experiments conducted on 35Sr- and 133Ba-labeled rabbit septa indicate that a tissue-detector system can be developed having the capacity to resolve perturbations of as little as 2 mumol Ca/kg wet tissue at 1-min sampling times or 6 mumol Ca/kg wet tissue at 0.1-min sampling times.

Ca++ and pancreatic amylase release.

The effects of Ca'++ on release of amylase by mouse pancreas in vitri was studied. The cholinergic agonist bethanechol was depressed about 50% in O Ca'++ medium. When pancreatic fragments were superfused with O Ca'++ medium, bethanechol still produced a normal stimulation of amylase release after half of the total tissue Ca'++ was washed out. This Ca'++ loss included both extracellular and some intracellularCa'++. Uptake and equilibration of '45Ca'++ into pancreatic fragments was multiphasic, with total equilibration with stable Ca'++ still not reached after 3 h. Addition of bethanechol had no effect on the rate of '45Ca uptake and equilibration. '45Ca'++ efflux was not influenced by superfision in O Ca'++ medium, while thestimulation of '45Ca'++ efflux by bethanechol was enhanced. It is concluded that extracellular Ca'++ and/or Ca'++ influx is not of major importance in triggering pancreatic enzyme release, but that extracellular Ca'++ may regulate the release process thus accounting for the parallel changes in unstimulated and stimulated amylase release.

The significance of a fast exchanging superficial calcium fraction for the regulation of contractile force in heart muscle

The force of contraction of isolated guinea-pig atria decreased with time during 5 h of incubation in Tyrode solution containing 0.9 m m-CaCl 2. The relative effect of ouabain strongly depended on the duration of incubation (“age”) of the tissue. Freshly dissected atria showed a 12% increase in contractile force, whereas aged atria (5 h of incubation) produced a 240% increase in tension in the presence of ouabain (1.5 × 10 −7 m). In freshly dissected atrial preparations, after an equilibration period of 30 mins, ouabain had no influence on Ca metabolism (total tissue Ca, rate of exchange, and size of exchangeable fraction). After aging of the tissue the rate of Ca exchange was significantly decreased, and ouabain considerably accelerated the Ca exchange rate. The presence of pentobarbital also slowed the Ca exchange rate. Ouabain was able completely to restore the rate of Ca exchange to control valves. Analysis of the Ca exchange curves yielded a fast and a slowly exchanging Ca compartment. The size of the fast exchanging fraction was significantly reduced by aging and by the treatment with pentobarbital. Under these conditions ouabain enhanced the size of this particular Ca compartment. It is suggested that the fast exchanging Ca compartment is superficially located and is responsible for rapid changes in contractile force. This Ca fraction may provide a Ca concentration of about 1.5 × 10 −5 m in the intracellular aqueous space, which is generally believed to be sufficient for the activation of contraction.

Increased uptake and efflux of calcium with acetylcholine stimulation in smooth muscle of toad stomach.

SummaryThe effect of acetylcholine stimulation on Ca movements in depolarized smooth muscle from toad stomach muscle was investigated.No difference in the content of Ca45 due to ACh stimulation could be detected in muscle containing its full complement of Ca. However, in Ca depleted muscle exposed to Ca 0·05 mM, solution, uptake of Ca was accelerated and the equilibrium content of total and exchangeable Ca was increased by some 5% of the total tissue Ca. This amount was similar to the extra Ca lost when muscle was depleted into Ca‐free solution with intermittent ACh stimulation.The efflux of Ca45 from normal and replcted muscle was studied, using a constant flow system. ACh stimulation of both normal and repleted muscle resulted in a transient increase in the efflux of Ca45. The slow component of the efflux curve occupied a larger proportion of the cellular Ca in the repleted than in the normal muscle. The increment of Ca uptake due to ACh stimulation during repletion was found to be wholly in this fraction.The significance of these results in relation to an excitation‐contraction‐coupling fraction are discussed.

Calcium metabolism in atrial tissue during frequency potentiation and paired stimulation

In order to study whether the application of paired stimuli and also the occurrence of frequency potentiation were accompanied by changes in calcium metabolism, the uptake and release of45Ca and also the total tissue Ca were studied in guinea pig isolated left auricles, subjected to either single or paired stimulation at different basic frequencies.

The Ca metabolism of intestinal smooth muscle during forced electrical stimulation.

1. The Ca content and Ca fluxes were determined in longitudinal muscle strips of guinea pig small intestine at rest and during forced electrical stimulation. 2. Total tissue Ca was measured by a spectrofluorometric method and45Ca by means of liquid scintillation counting. 3. During stimulation a large net uptake of Ca occurred, whereas the relative influx and the efflux were not altered. 4. The amount of Ca taken up per stimulus amounted to about 5×106 Ca atoms per cell resp. 10−12 Mol Ca/cm2 cell surface. If the cell volume of the smooth muscle cells is taken into account these cells take up much more Ca per stimulus than skeletal muscle cells under comparable circumstances. 5. The smooth muscle cell needs a rather long time to extrude the Ca taken up during a stimulus. The stimulation frequency of 0.1 resp. 0.2 Hz as applied in the present study is so high that it prevents an equilibrium stage of Ca metabolism in the smooth muscle cell. 6. The result does not contradict the assumption that Ca plays an important role in the excitation contraction coupling process of smooth muscle.