The effect of calcium ions and the calcium ionophore A23187 on choline uptake and phosphatidylcholine biosynthesis in chick embryo hearts

Summary The effect of increases in extracellular calcium [Ca]₀ and the calcium ionophore A23187 on choline uptake and phosphatidylcholine biosynthesis was assessed in isolated cardiac myocytes. The cells were obtained from 7-day old chick embryos and were maintained in culture. Choline uptake was examined using [methyl 3H] choline. A23187 was found to increase choline uptake through the saturable choline uptake process. Pulse chase experiments using [methyl 3H] choline showed that after a 2 h incubation with choline, about 85% of the label was recovered in phosphocholine with most of the rest in phospholipid and a small amount in CDP-choline and glycerol phosphocholine. Increases in [Ca]₀ up to 10 mM did not affect the amount of label in phosphocholine or phospholipid, the rate of disappearance of label from phosphocholine, or the rate of appearance of labelled choline in phospholipid. In contrast, A23187, at concentrations up to 10^–4 M, was associated with a significant (p<0.05) increase in choline in the phosphocholine and phospholipid pool compared to control cells. The time course of the disappearance of choline from the phosphocholine pool and appearance in phospholipid pool was not significantly different between control cells and those treated with A23187. A23187 increased choline uptake via the specific uptake process. The effect on choline uptake may be attributed to the action of A23187 to facilitate the release of calcium from specific intracellular calcium storage sites rather than a nonspecific increase in [Ca]₁ that may have resulted from the increase in [Ca]₀.Supported in part by a grant from Canadian (British Columbia) Heart Foundation     

Rest- and stimulation-dependent changes in exchangeable calcium content in rabbit ventricular myocardium

Summary Ca²⁺ shifts in the isolated, perfused ventricular muscle of rabbit hearts were investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with⁴⁵Ca containing solution. The cellular⁴⁵Ca content was calculated by subtraction of⁴⁵Ca²⁺ dissolved in the free water of extracellular space from the total tissue⁴⁵Ca²⁺ content. The cellular content of⁴⁵Ca in stimulated (60 per min) preparation was 0.887±0.067 mmol/kg wet weight (w.w.). Six minutes of rest resulted in the drop of this content to 0.503±0.054 mmol/kg w.w. despite continued perfusion with⁴⁵Ca containing solution. Contractile force (CF) decreased at that time to 23% of control. The first post-rest contraction (RSC) resulted in a gain of 0.073 mmol⁴⁵Ca/kg w.w. Both the content of⁴⁵Ca and CF returned to the pre-rest values when stimulation was resumed.The difference between the⁴⁵Ca content of post-rest stimulated and rested preparations (0.384 mmol/kg w.w.) is equivalent to Ca₂ fraction (Ca₂), previously described in guinea pig ventricular myocardium (17, 12). However, the volume of rabbit Ca₂ is only about 42% of that in guinea pig. Caffeine in concentration 12.5 mM, which did not displace Ca₂ from guinea pig ventricular muscle, decreased Ca₂ in the rabbit ventricle by 44%. CCCP, a protonofore destroying the mitochondrial protone gradient essential for Ca²⁺ uptake and maintainability, displaced Ca₂ completely from rabbit ventricles. These results, although far from conclusive, do suggest that both the mitochondrial and sarcoplasmic reticulum might be the site of the rate-dependent Ca₂ fraction. The physiological meaning of differences in Ca₂ content between rabbit, guinea pig, and rat ventricular myocardium is discussed.This study was supported by the grant CPBR, 11.6     

Voltage-dependent calcium release in guinea-pig cardiac ventricular muscle as antagonized by magnesium and calcium

Summary An increase in extracellular potassium concentration from 4 to 16 mmol/l caused a decrease in membrane potential from –92 to –59 mV and selectively diminished the earlier of two contraction components of guinea-pig papillary muscles at 0.2 Hz stimulation frequency in the presence of noradrenaline. The influence on the early contraction component had a threshold of 8 mmol/l K⁺, corresponding to a membrane potential of –77 mV. However, test contractions elicited 800 ms after the 5 s stimulation interval exhibited an unimpaired early component. Since the activator calcium responsible for the early contraction component is derived, in mammalian ventricular muscle, from the junctional sarcoplasmic reticulum (20), it is assumed that the release site of the reticulum was filled with calcium shortly (800 ms) after a regular contraction, and lost its calcium at 16 mmol/l extracellular K⁺ during the 5 s stimulation interval. The potassium-induced depolarization determined the rate of calcium leakage during rest from the intracellular store. The depolarization-induced decline of the early contraction component was equally well antagonized by Mg²⁺ or Ca²⁺ without influencing the measured transmembrane potential. Both divalent cations shifted the relation between potassium concentration or membrane potential and the strength of the early contraction component to less negative membrane potentials. In order to reduce the early contraction component by 25% in the presence of 9.6 instead of 1.2 mmol/l Mg²⁺, the potassium concentration had to be increased from 9.6 to 22.0 mmol/l, with a respective decrease in resting membrane potential from –72.6 to –51.1 mV. The antagonistic effect of both divalent cations is thought to result from the neutralization of negative charges outside the sarcolemma with a respective decrease in the outside surface potential.     

Positive inotropic and chronotropic effects of the calcium ionophore A23187 (calimycin) on guinea-pig atria

Summary The inotropic and chronotropic effects of the calcium ionophore A23187 (calimycin=CA) in isolated, superfused, electrically driven, auxotonically contracting left and spontaneously beating right guinea-pig atria were examined at different Ca²⁺ concentrations. 10^–6 to 10^–5 mol/l CA shows significantly positive inotropic and chronotropic effects. The inotropic effect of CA can be significantly diminished by relatively high concentrations of Ca²⁺ channel antagonists and relatively low concentrations of ryanodine. The positive chronotropic effect of CA can be reduced slightly, but significantly, by beta-adrenoceptor antagonists and histamine H₂-receptor antagonists. From this we infer that the positive inotropic effect of CA is mainly due to the release of Ca²⁺ from cardiac sarcoplasmic reticulum, whereas catecholamine and histamine release appear to contribute to the positive chronotropic effect of the ionophore.Preliminary results were presented at the 28th Spring Meeting of the Deutsche Gesellschaft für Pharmakologie und Toxikologie, March 10–13, 1987, Mainz, and at the Herbsttagung der Deutschen Gesellschaft für Herz- und Kreislaufforschung, October 8–10, 1987, Freiburg.     

Increased calcium sensitivity of chemically skinned human atria by myosin light chain kinase

Summary We investigated the influence of myosin P-LC phosphorylation catalysed by calcium/calmodulin-dependent myosin light chain kinase (MLCK) on the tension-pCa relation of chemically skinned human atrial fibres. MLCK-induced increased myosin P-LC phosphorylation sensitized human atrial skinned fibres for calcium by 0.11 pCa-units in patients with valvular heart disease, and by 0.05 to 0.07 pCa-units in patients with coronary heart disease. The MLCK effect could be antagonized by a light chain phosphatase. The protein phosphatase ocadaic acid (OA) had no influence on the tension-pCa relation of skinned human atrial fibres and had no potentiating effect together with MLCK. The MLCK preparation used in this study was from bovine ventricle and revealed a K_M of 1.8×10^–5 M and a V_max of 822 nmol P_i/min/mg using purified bovine ventricular myosin-LCs as substrate.     

Binding of calcium to myoplasmic buffers contributes to the frequency-dependent inotropy in heart ventricular cells

Summary In guinea-pig ventricular cells, the Ca²⁺ buffer capacity of the myoplasm was estimated from the ratio of ionized calcium (from Indo-1 fluorescence) through total calcium (ionized plus bound calcium, fron x-ray microprobe analysis). During post-rest potentiation (1 Hz paired-pulses in voltage-clamp), where diastolic sarcomere length remained nearly constant, Ca²⁺ buffer capacity slowly fell from 55001 to 7001 suggesting that slow Ca²⁺ binding sites became saturated. We discuss that frequency-inotropy depends not only on the replenishment of intracellular stores with Ca²⁺, but also on binding of Ca²⁺ to these slow sites; the slow Ca²⁺ sites could complete with the fast activator sites on troponin C for systolic Ca²⁺, or they could enhance the Ca²⁺ affinity of the fast Ca²⁺ sites on troponin C by cooperative interaction.     

The reciprocal protective effects of magnesium and calcium in hyperkalemic cardioplegic solutions on ischemic myocardium

Summary The cardioprotective effects of magnesium and calcium in hyperkalemic cardioplegic solutions were investigated in isolated rat hearts. Isolated rat hearts were arrested for 30 min at 37°C in cardioplegic solutions containing magnesium and calcium in varying concentrations. In St. Thomas' Hospital cardioplegic solution, the magnesium and calcium concentrations were varied as follows: Mg 0; Ca 0.1, 0.3, 0.6, 1.2 mmol/l (mM)./ Mg 4; Ca 0.1, 0.3, 0.6, 1.2 mM./ Mg 8; Ca 0.1, 0.3, 0.6, 0.9, 1.2 mM./ Mg 12; Ca 0.1, 0.6, 0.9, 1.2 mM./ Mg 16; Ca 0.1, 0.6, 1.2, 1.5 mM. For each magnesium concentration, the percentage recovery of aortic flow generated dose-response curves depending on calcium concentration. The maximum percentage recovery of aortic flow was 76.0%±2.7% (mean ±SEM) in the Mg0–Ca0.1 mM group, 77.1%±2.0% in the Mg4–Ca0.3 mM group, 78.5%±2.3% in the Mg8–Ca0.6 mM group, 79.8%±2.4% in the Mg12–Ca0.9 mM group and 80.0%±3.4% in the Mg16–Ca1.2 mM (ST solution) group. Significant difference in the recovery of aortic flow has not been observed among these groups, and furthermore, significant differences in the recovery of other parameters of cardiac function and Ck leakage have not been observed among these groups. These results suggest that the cardioprotective effects depend on the relative combination of magnesium and calcium concentrations, and that it is important to maintain an appropriate balance of magnesium and calcium in hyperkalemic cardioplegic solutions.     

Effects of the calcium antagonist gallopamil on the increase of myocardial extracellular potassium activity during LAD occlusion in dogs

Summary It has been implied that the increase of myocardial extracellular potassium activity ([K⁺]_c) in the early stage of acute myocardial ischemia is a major cause of the increased likelihood of arrhythmia after acute coronary artery occlusion. There is also experimental evidence that some calcium antagonists reduce the occurrence of ischemia-induced early ventricular arrhythmias. In order to clarify the antiarrhythmic effect of gallopamil during the early phase of acute LAD occlusion, the influence of this calcium antagonist on the time course of [K⁺]_c during acute ischemia was measured in open-chest anesthetized dogs using a K⁺-selective surface multielectrode. The regional myocardial blood flow was determined with 9 m radioactive tracer microspheres.After application of gallopamil (bolus, 25 g/kg and infusion 2.5 g/kg·min for 30 min) the maximal and mean rate of rise of [K⁺]_c as well as the plateau of [K⁺]_c reached during ischemia were significantly diminished compared with the control occlusions. 90 min after gallopamil, the rate of rise of [K⁺]_c as well as the plateau of [K⁺]_c reached were still significantly reduced, but 180 min after the gallopamil application, no significant differences between the time course of [K⁺]_c and that of the two control occlusions could be found. Gallopamil significantly elevated, myocardial blood flow in the non-ischemic area, but did not influence blood flow in the ischemic region.While collateral perfusion remains unchanged, the slowed and reduced increase of myocardial [K⁺]_c during acute coronary artery occlusion may be an important component of the antiarrhythmic effect of gallopamil during early ischemia.Preliminary results were presented at the International Symposium on Calcium entry blockers and tissue protection, Rome, 1984, at the 59th Meeting of the Deutsche Physiologische Gesellschaft, Dortmund, 1984, at the 50th meeting of the Deutsche Gesellschaft für Herz- und Kreislaufforschung, Mannheim, 1984, and at the IXth European Congress of Cardiology, Düsseldorf, 1984.     

Signal transduction by cGMP in heart

Summary Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. However, the regulation of cGMP levels by a variety of agents was not always consistent with their effects on contractility. It is now clear that at least two major cell types in whole heart, cardiac myocytes and vascular smooth muscle cells, differ markedly in their mechanisms of cGMP regulation and response to cGMP. Furthermore, experiments on isolated cardiac myocytes indicate that the mechanism of cGMP action even in this single cell type can be multifaceted. Cyclic GMP inhibits the L-type calcium channel current (I_Ca), which is the major source of Ca⁺⁺ entry into heart cells, and which plays a predominant role in the initiation and regulation of cardiac electrical and contractile activities. Patch-clamp measurements of I_Ca indicate that in isolated frog myocytes cGMP inhibits I_Ca by stimulation of cAMP phosphodiesterase (cGS-PDE), whereas in purified rat ventricular myocytes, cGMP predominantly inhibits I_Ca via a mechanism involving cGMP-dependent protein kinase (cGMP-PK). Under certain conditions, cGMP can also inhibit a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) and thereby produce a stimulatory effect on I_Ca. Biochemical characterization of the endogenous PDEs and cGMP-PK in purified cardiac myocytes provided further evidence in support of these mechanisms of cGMP action on I_Ca.     

Sensitivity to oxidants of mitochondrial and sarcoplasmic reticular calcium uptake in saponin-treated cardiac myocytes

Summary Calcium transport functions of mitochondria and sarcoplasmic reticulum (SR) were studied without prior extraction using isolated rat heart myocytes permeabilized with saponin. Calcium uptake by SR was rapid and its affinity was high in comparison to calcium uptake by mitochondria, which had a higher capacity. The sensitivity of uptake to two oxidants, H₂O₂ and HOCl (hypochlorous acid), depended on the cytosolic calcium concentration; when this was similar to the concentration in diastole (180 nM), HOCl inhibited calcium uptake by mitochondria and SR, whereas when the calcium concentration was 750 nM, mitochondrial calcium uptake showed relatively high resistance, although SR uptake was still markedly inhibited by HOCl. Calcium uptake of both mitochondria and SR was less sensitive to the action of H₂O₂ than to HOCl, and the H₂O₂ effect was less dependent on the cytosolic calcium concentration. Therefore, HOCl, when produced by activated leukocytes and supplied to the heart cells, may seriously impair the excitation-contraction coupling function of SR, whereas H₂O₂, possibly generated directly by mitochondria or generated from superoxide anions, may be tolerated relatively well by heart SR and mitochondria.Preliminary accounts of this work were presented as FASEB 1988. Dr. Kaminishi was a Canadian Heart Foundation Post-Doctoral fellow; his present address is Yamagata University, Japan.     

Glutathione alters calcium responsiveness of cardiac skinned fibers

Summary The glutathione status of cardiac muscle, that is the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) changes in certain forms of cardiomyopathy and during reperfusion of ischemic heart. Here we show that it also affects the sensitivity of contractile proteins to calcium. GSH (4 mM) increased the pCa₅₀ for force development in skinned fibers by 0.2 pCa units and increased force ba 44%±5.4% at pCa 5.6 whereas GSSG (4 mM) decreased it by 54%±17.8% at pCa 5.6. Half maximal activations and inhibitions were seen with 4 mM GSH or GSSG, respectively.In contrast to GSH, the reducing agent dithiotreitol at 5 mM had no activating effect. Our results suggest that the loss of contractility observed after a reperfusion of the ischemic heart my, at least in part, be due to a decreased responsiveness of the contractile proteins due to an altered glutathione status.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320).     

Effects of in vivo manganese administration on calcium exchange and contractile force of rat ventricular myocardium

Summary The purpose of this study was to investigate the effect of prolonged (14 days) intragastric administration of Mn²⁺ (0.25 mmol/kg daily) on Ca²⁺ exchange and contractility of rat ventricular myocardium. Left-ventricular pressure and its first derivative (dP/dt) were recorded by means of a balloon catheter inserted via the left atrium into the left ventricle of the rat heart perfused by Langendorff method. Ca²⁺ exchange in the stimulated and rested ventricular myocardium was investigated with the aid of⁴⁵Ca under the conditions of complete equilibration of preparations with a solution containing⁴⁵Ca²⁺. The cellular⁴⁵Ca²⁺ content was calculated by subtraction of⁴⁵Ca²⁺ dissolved in the free water of extracellular space from the total tissue⁴⁵Ca²⁺ content. The cellular⁴⁵Ca²⁺ content in the stimulated (60/min) ventricles of control rats (without Mn²⁺) was 0.83 ± 0.09 mmol/kg wet weight (w.w.). Ten minutes of rest resulted in a gain of 0.06 mmol⁴⁵Ca/kg w.w. (not statistically significant). Fourteen days' exposure to Mn²⁺ resulted in an increase of the mean⁴⁵Ca content to 1.61 ± 0.09 mmol/kg w.w. in the stimulated preparations and to 1.35 ± 0.06 mmol/kg w.w. in the rested ones (p < 0.001). Thus, the control rest preparations did not change their Ca²⁺ content, while in the rats treated with Mn²⁺ the rest resulted in an increase at exchangeable Ca by 52 %. The maximal ventricular developed pressure (P_max) after 14 days of Mn²⁺ administration was increased by 35 % and dP/dt_max was 228 % of the value in the control group. The mean time from the maximal value of the first derivative to P_max (t-dP/dt_max) was reduced by one-half and mean time to peak developed pressure (TPT) was shortened to one-third of these in the control group. These results, although far from conclusive, do suggest that the long-lasting exposition to Mn²⁺ resulted in increased myocardial contractility caused, most probably, by the inhibition of Ca²⁺ efflux from the cell. Perhaps this is a compensation for the inhibitory effect of Mn²⁺ on the slow calcium channels.     

The role of calcium in the enhanced myocardial contractility of the hyperthyroid rat heart

Summary The hyperthyroid rat myocardium exhibits enhanced contractility. There is evidence that altered calcium handling by the myocardium may be responsible for this enhanced state. To investigate this, isolated hyperthyroid and cuthyroid hearts were perfused in the working mode and exposed to alterations in external calcium concentration. Heart rate was not significantly different in either group of hearts, nor was it altered by the change in calcium. The concentration of calcium needed to elicit half-maximal contractility (dP/dt_max) was lower in the hyperthyroid (0.81±0.07 mM) than in the cuthyroid hearts (1.12±0.09 mM, p<0.05). This increase in calcium sensitivity was unlikely to be at the site of the sarcolemma as verapamil exerted equal negative inotropic effects on both groups of hearts. Dantrolene, which blocks calcium release from the sarcoplasmic reticulum, exerted a significantly greater (p<0.01) depression in dP/dt_max after 12 min in the hyperthyroid (50±7%) than in the cuthyroid heart (15±2%). We conclude from our results that the enhanced contractile state of the hyperthyroid rat heart is likely to involve an altered mechanical response to calcium which is possibly at the level of enhanced calcium release from the sarcoplasmic reticulum.     

Mitochondrial calcium in hearts subjected to lipid peroxidation with contracture development

Summary In the present study we have investigated isolated rat hearts perfused with oxygen radicals generated by xanthine oxidase and hypoxanthine. The influence of verapamil (1 mg·l^–1) pretreatment on oxygen radical-induced contracture development and decrease in contractility was examined. In addition, we have measured mitochondrial calcium and magnesium levels in control hearts and hearts perfused with oxygen radicals with and without addition of superoxide dismutase (SOD) and catalase. The presence of oxygen radical-induced lipid peroxidation was confirmed by the increased level of conjugated diens in lipid extracts from oxygen radical-perfused hearts. Verapamil prevented contracture development in hearts perfused with oxygen radicals. Diastolic pressure measured with a left ventricle balloon was at the end of the experiments, 18±3 mm Hg (mean±SEM) with verapamil and 66±9 mm Hg without (p<0.001). Perfusion with oxygen radicals resulted in a reduction in mitochondrial calcium from 14.63±0.93 to 8.26±0.61 nmol·mg^–1 (p<0.001) which was partly reversed by superoxide dismutase and catalase. Mitochondrial magnesium levels were unchanged in all groups.     

Differential effect of nicotinic acid derivatives on smooth muscle and endothelial cell proliferation

Summary The pathogenesis of atherosclerosis is a multifactorial process. A possible anti-atherosclerotic drug should therefore interfere with different targets that are important during the development of an atherosclerotic lesion. Two of the early events are the activated migration and proliferation of arterial smooth muscle cells. Here we investigated in several in vivo and in vitro experiments the effect of two nicotinic acid derivatives L44 and L44-0, on smooth muscle cell migration and proliferation. Balloon catheter de-endothelialization was used as an animal model for intimal lesion formation. Migration was subsequently quantified in vitro using the explant outgrowth technique. Subcultured smooth muscle and endothelial cells were used to test the effect of the drugs on proliferation. Time-lapse video microscopy was applied to differentiate between smooth muscle cell migration and proliferation on the level of individual cells. We showed that L44 and L44-0 are very effective in decreasing smooth muscle cell proliferation and migration. Endothelial cell proliferation, important to re-establish endothelial integrity was, however, not affected.     

Binding of diltiazem and verapamil to isolated rat heart mitochondria

Summary The occurrence of arrhythmias was studied in the calcium-paradox model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period.The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60–90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias.     

Calcium repletion-induced arrhythmias after short periods of calcium-free perfusion in the isolated rat heart

Summary The occurrence of arrhythmias was studied in the calcium-paradox model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period.The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60–90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias.     

Morphological and smooth muscle cell phenotypic changes in fetal rabbit aorta during early development

Summary Previous studies from our laboratory noted a) high aortic cholesterol esterification activity in the fetal rabbit, and b) increased susceptibility of fetal aortic explants to smooth muscle cell proliferation in culture, two features commonly associated with atherogenesis. This prompted us to examine in detail morphological development of fetal aorta and its relationship to fetal plasma cholesterol levels. Our studies made three important observations: 1) plasma cholesterol levels were high in early fetus which decreased at term; 2) in early fetus aortic endothelial cells appear to protrude into the lumen, whereas at birth the cells become flat, forming a continuous endothelium sheet; 3) in early fetus, smooth muscle cells exist predominantly in synthetic phenotype, while at birth the cells appear contractile. Despite the presence of synthetic smooth muscle cells and hypercholesterolemia in early fetal life, no accumulation of lipids was evident under transmission electron microscopy.     

Vasomotor coronary oscillations: A model to evaluate autoregulation

Summary A simple model was proposed to characterise the oscillatory and nonoscillatory pattern of canine coronary circulation responses induced by a small dose of a vasodilator adenosine or the Ca²⁺-channel blocker diltiazem. This model consists of two differential equations describing the interaction of dilating (D) and constricting (C) resistance components. With the assumption that the rate constants associated with (D) were dependent on adenosine concentration and those associated with (C) were a function of Ca²⁺ channels, the model predicted: a) a damped oscillation of resistance to flow at low dose of adenosine, b) a predominant vasodilation at high dose of adenosine, and c) a sustained vasodilation in response to diltiazem. Parameters characterising the coronary resistance were evaluated by fitting the model results to calculated resistance from measured coronary flow and aortic pressure. As well, the model predicted accurately the peak resistance to great cardiac and coronary sinus venous flow in patients. This study indicates that the oscillation frequency of coronary resistance induced by a low dose of adenosine (0.01 mg/kg) is indicative of the uptake rate of adenosine by the heart and the coronary resistance provides considerable information on vasomotor control of the coronary circulation.This work was supported by grants from the Heart and Stroke Foundation of Nova Scotia.     

Response of cultured endothelial cells to mechanical stimulation

Summary Endothelial cells covering the luminal surface of vessels are exposed to at least two different mechanical forces: 1) fluid shear stress produced by the circulation of blood, and 2) periodic stretching and relaxing as a result of the diameter oscillations caused by blood pulsation.In this study we present an apparatus which was constructed to imitate the volume pulse with its typical incisura of the abdominal aorta. Using this apparatus, we exposed cultured endothelial cells to continuously produced cyclic and directional stretching and relaxation for three days. In all experiments cells remained attached and viable when subjected to mechanical stimulation. The vast majority of endothelial cells which underwent mechanical stimulation became elongated and oriented with their longer axis perpendicular to the direction of stretching (angle of cell orientation: =88.7°±12°; ±SD), whereas cells on unstretched membranes had a cobblestone-like appearance and remained in random orientation. In the stretched cells, the factor of elongation was f=6.8±1.3; ±SD; unstretched cells which exhibited a polygonal shape had a factor of elongation of f=1.8±0.8; ±SD. In addition, the behavior of cytoskeletal components such as microfilaments and microtubules was examined in the process of cell orientation as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented in parallel alignment perpendicular to the stretch direction (angle of actin filament orientation: =90.4°±9°; ±SD). A distinct orientation of microtubules was not observed, althougn a noticeable number of microtubules was observed to be in parallel alignment. Furthermore, microtubules of cells which underwent mechanical stimulation exhibited a pronounced asymmetric intracellular distribution with strongly fluorescent cytoplasmic areas in which microtubules seemed to be accumulated.The results indicate that endothelial cell elongation and orientation in vitro can be induced by periodic stretching and relaxation comparable to the periodic oscillations of the vessel wall due to blood pulsation in vivo.