The non-neuronal heart's acetylcholine in health and disease.

In this review we describe the history of almost one century lasting investigations which eventually provided evidence convincing that cardiac myocytes possess all elements of the system of synthesis, intracellular transport and release of acetylcholine (ACh) independent of parasympathetic cholinergic innervation. The myocytes synthesis and release of ACh is tightly connected with their contractile activity. Moreover, it is necessary for maintaining the balance of autonomic control of the heart, particularly important in the heart failure. It has an antyhypertrophic activity and protects myocardium against ischemic/reperfusion injury which shows that it is involved in the regulation of the important cellular signaling pathways. These properties of the non-neuronal ACh system of the heart also rise the hope that it might be used for the therapeutic measures.

The effect of sarcoplasmic reticulum Ca2+ leak on contractile activity of guinea pig heart myocytes depends in activity of sarcoplasmic reticulum Ca2+-ATPase and Na+/Ca2+ exchanger.

Decreased expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), overexpression of Na(+)/Ca(2+) exchanger (NCX) and diastolic SR Ca(2+) leak from the ryanodine receptors (RyRs) are believed to contribute to the decrease of myocyte contraction in failing heart. In this work we induced Ca(2+) leak through RyRs in isolated myocytes of guinea pig hearts by 20 microM FK-506. The SR Ca(2+) leak resulted in (1) decreased amplitude of cell shortening and of Ca(2+) transients, (2) decreased rate of Ca(2+) transients decay (3) enhanced diastolic Ca(2+) loss. The effect of FK-506 on amplitude of cell shortening was reversed and that on diastolic Ca(2+) loss blocked by partial inhibition of NCX due to lowering Na(+) concentration in superfusion solution from 144 mM to 100 mM. The amplitude of cell shortening and Ca(2+) transients decreased by FK-506 was significantly increased by 10(-7) M thapsigargin. In conclusion, the effect of SR Ca(2+) leak induced by FK-506 on myocyte contraction is strictly dependent on activity of SERCA and NCX.

Temperature dependent contribution of Ca2+ transporters to relaxation in cardiac myocytes: important role of sarcolemmal Ca2+-ATPase.

UNLABELLED: Activities of Ca(2+) -ATPase of sarcoplasmic reticulum (SERCA) and Na(+)/Ca(2+) exchanger (NCX) involved in cellular Ca(2+) turnover greatly change in hypertrophied and failing hearts. Unfortunately, contribution of these proteins as well as of the sarcolemmal Ca(2+)-ATPase (PMCA) to cellular Ca(2+) turnover has been investigated almost exclusively at room temperature. PMCA is of particular interest since it may affect activity of calcineurin and nNOS. Therefore the objective of this study was to reinvestigate contribution of SERCA, NCX and PMCA to cell relaxation and the effect of PMCA on cell contraction at 37 degrees C. Myocytes isolated from the ventricles of guinea pig and rat hearts and incubated with Indo-1 were field stimulated at the rate of 60/min. Contribution of SERCA, NCX and PMCA was calculated from the rate constants of the decaying components of electrically stimulated Ca(2+) transients or of the transients initiated by caffeine dissolved in normal Tyrode or in 0Na, 0Ca Tyrode. Increase in temperature from 24 to 37 degrees C increased the relative contribution of NCX from 6.1% to 7.5% in rat and from 21.3 to 51.9% in guinea pig at the expense of SERCA. The contribution of the PMCA to relaxation in both species increased upon rise in temperature from 24% to 37 degrees C from negligible values to 3.7%. In both species amplitude of Ca(2+) transients was at 24 degrees C nearly twice as high as at 37 degrees C. It was nearly doubled by carboxyeosine (CE), a PMCA blocker at 37 degrees C but was hardly affected at 24 degrees C. The effects of CE were concentration-dependent and conformed with the degree of inhibition of activity of PMCA. CONCLUSIONS: PMCA plays an important role in regulation of myocardial contraction despite its small contribution to relaxation. In guinea pig but not in rat relative contribution of SERCA and NCX to relaxation is highly temperature dependent.

Voltage dependent activation of tonic contraction in cardiac myocytes.

UNLABELLED: Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.

Effects of thapsigargin on stimulation frequency--dependent changes in mitochondrial calcium in rat cardiac myocytes.

We investigated in the single myocytes of rat heart the effect of blocking of ATP-ase of sarcoplasmic reticulum (SR) on mitochondrial Ca2+ uptake and release. Mitochondrial Ca2+ content was investigated as Mn(2+) - resistant fluorescence of Indo 1 - AM loaded into cells. SR ATP-ase was blocked with 10(-6) M thapsigargin (Tg). Tg blocked almost completely stimulation -dependent mitochondrial Ca2+ uptake and slowed down its release despite that the maximal cytosolic Ca2+ concentration was not decreased. We propose that mitochondrial stimulation -dependent Ca2+ uptake is greatly enhanced by [Ca2+] built by SR in microdomains adjacent to these organelle.

Agonist of dihydropyridine receptors, BayK8644 depresses excitation-contraction coupling in myocytes of guinea pig heart.

BayK8644(-)(BayK), an agonist of L-type Ca2+ channels has been recently shown to impair excitation-contraction coupling in cardiac myocytes by increasing Ca2+ leak from the sarcoplasmic reticulum (SR) and by decreasing the gain factor of calcium induced release of calcium. It has been proposed that BayK affects the properties of ryanodine receptors (RyRs) of SR by binding to the sarcolemmal dihydropyridine receptors (DHPRs). This would suggest that the linkage between these receptors is more direct than currently sought. However, it has been recently found that BayK may also directly affect the RyRs increasing their open probability. In this paper we tested the effect of BayK on excitation-contraction coupling in single ventricular myocytes of guinea-pig heart superfused with 5 mM Ni2+ which blocks the L-type Ca2+ current and Na+/Ca2+ exchange. We have previously shown that it is possible to activate in these cells nearly normal Ca2+ transients and contractions despite total inhibition of ICa. This eliminated the effect of ICa increased by BayK on excitation contraction coupling thus simplifying the studied system. 0.5 microM BayK increased the diastolic [Ca2+]i and decreased the diastolic length in stimulated or rested cells superfused with Ni2+, decreased by approximately 50% amplitude of Ca2+ transients and contractions and decreased by approximately 70% the responses of cells to rapid superfusion of 15mM caffeine used as an indirect index of the SR Ca2+ content. The effects on diastolic length and [Ca2+]i in rested cells were not affected by 20 microM nifedipine. We conclude that under our experimental conditions the dominating mechanism of suppression of excitation-contraction coupling by BayK was depletion of the SR Ca2+ by the direct effect on the RyRs.

On the source of Ca(2+) activating the tonic component of contraction of myocytes of guinea pig heart.

OBJECTIVE: Contractions of isolated, single myocytes of guinea pig heart stimulated at 37 degrees C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca(2+) activating the tonic component. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. RESULTS: Superfusion of 5 mmol/l Ni(2+) during 30 s pause did not inhibit subsequent biphasic Ca(2+) transients and contractions despite inhibition of Ca(2+) current and Na(+)/Ca(2+) exchange. KB-R7943 (5 micromol/l) or intracellular dialysis with 0 Na(+) solution, both of which inhibit reversed Na(+)/Ca(2+) exchange, decreased amplitude of Ca(2+) transients and contractions by approximately 40%. The ratio of amplitudes of tonic to phasic component was increased by Ni(2+) and was not changed by KB-R7943 or 0 Na(+)(i). Ryanodine (200 micromol/l) inhibited both components of contractions in cells superfused with Ni(2+). The phasic component but not the tonic component was inhibited by 20 micromol/l nifedipine in cells superfused with Ni(2+). CONCLUSIONS: Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca(2+) current or by the reverse mode Na(+)/Ca(2+) exchange as currently proposed in literature. Rather, it is activated by Ca(2+) released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca(2+) fraction activating the phasic component of contraction.

Excitation-contraction coupling in cardiac muscle revisited.

According to the current views the direct and indispensable source of Ca2+ activating contraction is sarcoplasmic reticulum (SR). Ca2+ is released from the SR when its release channels (ryanodine receptors) are activated by Ca2+ influx through the L-type Ca2+ channels (dihydropyridine receptors). In contrast, ryanodine receptors of skeletal muscles are activated by conformational changes in dihydropyridine receptors induced by sarcolemmal voltage. Ca2+ influx is not necessary for their activation. In this review the papers not quite conforming with the current views are referred to and discussed. Their results suggest that SR is not an indispensable source of contractile Ca2+ at least in some mammalian species, and that cardiac ryanodine receptors may be activated by conformational changes in dihydropyridine receptors without Ca2+ influx (like in skeletal muscle). This may be a mechanism parallel to or accessory to the Ca2+ induced release of Ca2+ (CIRC).

Dihydropyridine receptors functioning as voltage sensors in cardiac myocytes.

Isolated, superfused with [Ca2+]0 = 2.5-3 mM (T = 37 degrees C) and voltage clamped ventricular myocytes of guinea-pig hearts were stimulated by pulses from a holding potential -40 mV to +5 mV (duration 300 ms). They activated L-type Ca2+ current and a biphasic contractile response: a phasic component of amplitude approximately 7% of resting cell length (duration approximately 150 ms) and a tonic component of amplitude approximately 3% of resting cell length. The phasic component was inhibited by 10(-6) M thapsigargin (Tg). Pulses from -40 mV to +5 mV stimulated a similar bi-phasic contractile response in 74% of cells (n = 126) superfused from the beginning of a 30 s period of rest with 5-10 mM Ni2+ which blocked the Ca2+ current and Na+ -Ca2+ exchanger (Ni2+-contractions). Thus, the Ni2+-contractions could be activated only by intracellular Ca2+ release. The phasic component of control contractions showed the bell-shaped voltage relation at [Ca]0 = 2 mM and sigmoid relation at [Ca]0 = 3 mM. The phasic component o Ni2+-contractions showed a sigmoid relation at voltages from -40mV to +100 mV and could not be activated at [Ca]0 = 2 mM. It was inhibited by 20 microM nifedipine, a blocker of dihydropyridine receptors, even when activated by the pulses to +70 mV, during which the Ca2+ current does not flow. We proved that nifedipine does not affect Na+-Ca2+ exchange. The phasic component of Ni2+-contractions was also inhibited by 2 nM indolizinesulphone SR33557, another dihydropyridine receptor blocker, which halved the phasic component of contractions in sontrol cells without any significant effect on the Ca2+ current. Stimulation did not activate contraction in any of 19 cells in which 20 microM nifedipine was superfused from the beginning of 30 s rest instead of 5 mM Ni2+. These cells were depolarized to +5 mV over the rest period in order to prevent intracellular Ca2+ loss by Na+ -Ca2+ exchange. Residual Ca2+ currents were much stronger in cells superfused with nifedipine than residual currents in cells superfused with Ni2+ (hardly visible in the records). Our results suggest that a vestigial remnant of a voltage-sensing mechanism similar to that in the skeletal muscle may trigger the Ca2+ release from the SR of cardiac myocytes under specific experimental conditions. In normal cells it may be complementary to calcium induced calcium release (CICR).

Properties of ventricular myocytes isolated from the hypertrophied and failing hearts of spontaneously hypertensive rats.

OBJECTIVE: To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles. METHOD: Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin. RESULTS: The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes. CONCLUSION: Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.

Dibucaine displaceable sarcolemmal Ca2+ fraction in guinea-pig cardiac myocytes.

In previous work we found that Ca2+ bound to the internal leaflet of sarcolemma of the neonatal cultured rat cardiomyocytes may be displaced by a local anesthetic, dibucaine (DBC). This resulted in inhibition of Na/Ca exchange. Now we found that the DBC displaceable Ca2+ fraction may be demonstrated also in single, enzymatically isolated cardiomyocytes of adult guinea pigs. Only cell length was used as an index of the free sarcoplasmic Ca2+ concentration since we found that DBC is fluorescent when irradiated with UV light, the wave length overlapping that of Indo 1 fluorescence. DBC (0.5 mM) induced a contracture whose amplitude ranged from 30%-100% of that of the electrically stimulated twitch. Blocking of Na/Ca exchange with 0.5 mM Ni2+, or 0Na+, 0Ca2+ solution did not affect the amplitude nor the time course of the contracture. Caffeine (15 mM) superfused during exposure of the cell to DBC initiated contraction which relaxed despite continued caffeine superfusion. DBC also initiated contracture in cells pretreated with thapsigargin, a blocker of sarcoplasmic reticulum Ca2+ uptake. DBC did not initiate contracture in skinned myocytes and decreased their sensitivity to Ca2+. These results suggest that DBC displaces Ca2+ from sarcolemma and that the displacement results in inhibition of Na/Ca exchange. Inhibition may however, be overcome by high subsarcolemmal Ca2+ concentration induced by its SR release with caffeine. DBC rapidly blocked sodium and calcium currents in voltage clamped cells. Calcium current recovered within about a minute upon washout of DBC, whereas recovery of sodium current started after about 8 min and was completed within about 12 min. Rapid depolarisation from a holding potential of -80 mV to 0 or +20 mV potentiated the response of the cell to DBC. This suggests that the Ca2+ fraction which is displaced by DBC is distinct from that released by depolarisation and therefore does not contribute to activation of a normal, electrically stimulated contraction.

Effects of mibefradil, a blocker of T-type Ca2+ channels, in single myocytes and intact muscle of guinea-pig heart.

We investigated the effects of a relatively selective blocker of the T-type Ca2+ channels, mibefradil (MBF), in the isovolumic left ventricles of the isolated, perfused hearts of guinea-pigs and single myocytes isolated from the ventricles of this species. In the myocytes superfused with 0 Na+ solution containing 200 microM lidocaine and pulsed from -90 mV to -40 mV to +5 mV, MBF proved to be about 3 times more potent inhibitor of the T-type than of the L-type Ca2+ current. The effect on the L-type current was strongly voltage and use dependent. In the ventricles and in the myocytes contraction was reduced by 50% by about 1 microM MBF, the concentration 12 times higher than this increasing the coronary flow by 50%. In myocytes the decrease in unloaded shortening paralleled inhibition of the T-type rather, than of the L-type Ca2+ current. Inhibition of electrically stimulated contraction of the myocytes was three times stronger than inhibition of the caffeine contractures regarded as an index of sarcoplasmic reticulum (SR) Ca2+ content. These findings are consistent with the hypothesis that the T-type Ca2+ channels may contribute to release of Ca2+ from the SR. It is concluded that MBF has a definite negative inotropic effect in the ventricular myocardium of guinea-pig heart at the concentrations found in the blood of the patients submitted to the clinical trials.

Effect of Na+ current on excitation-contraction coupling in ventricular myocytes of guinea pig heart.

We investigated the effect of Na+ current on the Ca2+ current and Ca2+ transients in cardiac myocytes. Myocytes were isolated from the ventricles of guinea-pig hearts by enzymatic dispersion. The membrane currents were recorded by the whole-cell voltage clamping. The Ca2+ current was activated by depolarisation from -80 to +5 mV preceded by the prepulses to -40 mV. Cellular action potentials (APs) were recorded by current clamping. Intracellular [Ca2+] was assessed by recording of fluorescence of Indo-1 loaded into cells. In current clamped cells (APs recorded) 20 microM tetrodotoxin (TTX) reduced the time to 75% of amplitude of Ca2+ transients from 50 +/- 6.6 ms to 32 +/- 5 ms (n = 7). In voltage clamped cells prepulses from the holding potential of -80 mV to -40 mV 50-100 ms long activated the Na+ current and initiated step increase in [Ca2+] reaching 30-50% of the total amplitude of the transient. Prepulses 10-20 ms long initiated increase in [Ca2+] merging with that elicited by Ca2+ current into smooth rising phase. Blocking of Na+ current with TTX or by switching the holding potential from -80 to -40 mV increased the amplitude of the Ca2+ current by 38 +/- 3.2% (n = 8) and 43 +/- 9% (n = 7), respectively, and eliminated the initial step increase in [Ca2+]. When 10-20 ms prepulses were used, blocking of Na+ current with TTX or switching of the holding potential decreased the time to 75% of amplitude of Ca2+ transients from 27 +/- 3.7 ms to 12 +/- 1.2 ms (n = 5) and from 25 +/- 3.1 ms to 14 +/- 1.1 ms (n = 9), respectively. 100 microM Cd2+ inhibited the initial rise in [Ca2+], however, the inhibition did not correlate with degree of inhibition of Ca2+ current. The Na+ current activated prior to Ca2+ current reduces its amplitude and decreases the rate of release of Ca2+ from sarcoplasmic reticulum. In voltage clamped cells this could result from Ca2+ influx prior to onset of Ca2+ current initiated by Na+ current escaping from the voltage control and/or reversal of Na/Ca exchange due to increase in subsarcolemmal [Na+]. In cells in which Ca2+ transients were initiated by APs only the second mechanism is conceivable.

Effect of sarcoplasmic reticulum Ca release into diadic region on Na/Ca exchange in cardiac myocytes.

The hypothesis that sarcoplasmic reticulum (SR) generates subsarcolemmal [Ca] higher than that in the bulk of sarcoplasm and that it affects the rate of Na+Ca exchange were tested. Voltage clamped cardiomyocytes of guinea-pigs and rats were stimulated by pre-pulses from a holding potential of -80 mV to -40 mV (20 ms) followed by 200 ms depolarizations to +5 mV. Single 5, 10, 20, 30, 50, 100 and 300 ms depolarizations were interposed between 200 ms pulses. The amplitude of the tail (Na/Ca exchange) currents recorded upon repolarization were compared with instantaneous fluorescence on Indo 1 loaded into cells. In both species amplitude of the tail currents were higher during the ascending phase of the Ca transient than during the descending phase, although the fluorescence was lower. The dissociation was abolished by thapsigargin (TG), the blocker of the SR Ca-ATPase. The results suggest that over the initial < 50 ms of the transient the Na/Ca exchangers are exposed to [Ca] higher than that in the bulk sarcoplasm and that it is generated by the SR.

Early after-depolarisations induced by noradrenaline may be initiated by calcium released from sarcoplasmic reticulum.

We investigated the effect of 10(-8) M noradrenaline (NA) on [Ca2+]i and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATs caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADs). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 microM BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by 70% and appearance of DATs. Also, the frequent multiple oscillations of Ca2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 microM Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 x 10(-7) M thapsigargin, a selective blocker of Ca(2+)-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance.

The source of contracticle calcium in guinea-pig cardiac myocytes treated with thapsigargin.

We investigated the source of activator Ca2+ in the cells deprived of sarcoplasmic reticulum (SR) Ca2+ by pretreatment with 10(-6) mM thapsigargin (TG). These cells show Ca2+ transients of nearly normal amplitude, albeit with the slowed kinetics. We found in the voltage clamped and loaded with Indo 1-AM cells that at depolarizing potentials from -30 to +70mV, blocking of sarcolemmal Ca2+ channels with 20 microM nifedipine or 20 microM Cd2+ reduced Ca2+ transients and contractions as much in the cells treated with TG as in the normal cells. The residual Ca2+ transients were mostly subthreshold for the contractile system. The result suggests that in the cells treated with TG, Ca2+ influx by the reversed Na/Ca exchange is not more important for activation of contraction than in the normal cells. In the normal cells shortening of one of the depolarizing pulses (+ 5mV) applied at a steady rate of 30/min from 200 ms to 5, 10, 20, 30, 50, or 100 ms little affected amplitude of the respective Ca2+ transients, although their duration was decreased proportionally to the decrease of the duration of the pulse. In the cells pretreated with TG, 20 ms pulses initiated Ca2+ transients which were hardly visible in the records of fluorescence. Their amplitude increased with increase in the duration of the pulses linearly correlating with the charge transfered with the Ca2+ current. We propose that the direct source of Ca2+ activating contraction in the guinea-pig ventricular myocytes is sarcolemmal Ca2+ influx mostly through the sarcolemmal Ca2+ channels. The alternative hypothesis is that there is some yet unidentified cellular source of activator Ca2+ (internal leaflet of sarcolemma?) from which it may be released by sarcolemmal Ca2+ influx.

Effect of cholinergic stimulation on free intracellular Ca2+ concentration in human lymphocytes.

Binding of ligand to receptor, in various types of cells, results in changes in calcium concentration, which is an important factor in cellular signal transduction. Lymphocytes receive signals from the parasympathetic nervous system through the cholinergic receptors. Cholinergic receptors mediate response to stimuli through changes of the IP3, or cAMP level and Ca2+ mobilization in various types of cells. The aim of this work was to measure changes in calcium concentration in cytosol of lymphocytes stimulated with acetylcholine agonists - carbachol (analogue of acetylcholine) or nicotine. Human peripheral blood mononuclear cells (PMBC) and lymphocytes from the leukemic cell lines Jurkat and Raji were used. Immune cells were preactivated with mitogen phytohemagglutinin (PHA) for PBMC and Jurkat cells or lipopolysaccharide for Raji cells. Two methods of measurement of calcium concentration were used. With the first, calcium concentration was measured in the suspension of cells loaded with the fluorescent dye Fura-2AM. With the other method, calcium concentration was assessed in single cells loaded with the fluorescent dye Indo-1AM. Using the method of single cell investigation, we observed an increase in the level of calcium concentration induced by carbachol and nicotine. The method of measuring the Ca2+ concentration in a cell suspension was found to be not sensitive enough for this purpose. The increase in calcium concentration resulted both from the stimulation of Ca2+ influx and from the release of Ca2+ from intracellular stores, most likely due to the increase in the concentration of IP3. In conclusion, we suggest that the lymphocytes activated with PHA respond to cholinergic stimulation with an increase in their free cytoplasmic Ca2+ concentration.

Functional coupling between sarcoplasmic reticulum and Na/Ca exchange in single myocytes of guinea-pig and rat heart.

It has been proposed that the cardiac Na/Ca exchanger is primed by high Ca2+ concentrations derived from the sarcoplasmic reticulum (SR) in a recently identified subsarcolemmal, "Na/Ca exchange-dependent Ca2+ compartment". We tested this hypothesis by investigating the effect on Na/Ca exchange of interventions affecting Ca2+ flux through SR. Experiments were performed in single, isolated myocytes of guinea-pig and rat hearts loaded with Indo 1-AM and free Ca2+ concentration was assessed by measuring the ratio of fluorescence at 405 and 495 nm wavelength. In guinea-pig 1.0 microM ryanodine (Ry), expected to increase Ca2+ flux through the SR, decreased the amplitude of electrically stimulated Ca2+ transients by 35% and inhibited their initial, rapid phase. Responses of these cells to brief superfusions with 15 mM caffeine were inhibited which suggests that 1.0 microM Ry depleted the SR Ca2+ stores. Diastolic Ca2+ concentration was slightly increased, but it dropped below control during prolonged rest. Decrease of the amplitude of the transients in these ryanodine-treated cells were reversed by 2 x 10(-7) M thapsigargin (TG), an inhibitor of the SR Ca(2+)-ATPase, by 1.0 mM Ry, a blocker of the SR Ca2+ release channels and by low Na+ (50.0 mM) superfusion. This suggests that the decreased transients in 1.0 microM Ry result from uptake of Ca2+ by the SR and its rapid release to the subsarcolemmal (cleft) space where a fraction is diverted out of the cell via Na/Ca exchange before it can diffuse to the myofilaments. Removal of the SR from the pathway (addition of TG on 1.0 mM Ry) or reversal of Na/Ca exchange diverts more transsarcolemmal Ca2+ influx to the myofilaments and increases the Ca2+ transient. Decrease of the resting Ca2+ concentration was blocked by 2 x 10(-7) M TG, 1.0 mM Ry, and by 5.0 mM Ni2+, a blocker of Na/Ca exchange. This result suggests that the effect of 1.0 microM Ry on resting Ca2+ concentration also resulted from increase of flux of Ca2+ through the SR and out of the cell by Na/Ca exchange. In rat 1.0 microM Ry decreased amplitude of the transients by approximately 75% but did not affect their kinetics. TG and 1.0 mM Ry decreased the rate of rise of the transients and greatly delayed their decay. This result suggests that normal kinetics of the transients in the cells treated with 1.0 microM Ry depended on the preserved Ca2+ flux through the SR. As SR was not able to retain Ca2+ in these cells, decay of the transients must have depended on stimulated Na/Ca exchange. The results in guinea-pig and rat taken together are compatible with the proposal that Ca2+ released from the SR interacts with the cell's Na/Ca exchanger most probably within the newly defined subsarcolemmal "Na/Ca exchange-dependent Ca2+ compartment".

A protective role of nitric oxide in isolated ischaemic/reperfused rat heart.

OBJECTIVES: The importance of NO-induced vasodilator tone in maintaining adequate coronary flow to sustain hemodynamic function in aerobically perfused heart and the role of NO in the injury development in ischaemic/reperfused heart was studied. METHODS: Effect of NO synthesis inhibitor (N omega-nitro-L-arginine, L-NOARG) on isolated working rat hearts subjected to either 90 min of aerobic perfusion or to a global ischaemia (27.5 to 42.5 min) followed by 40 min reperfusion was studied. To overcome coronary flow reducing effect of L-NOARG either perfusion pressure was raised from 75 to 120 cm H2O or adenosine (400 nM) was administered. RESULTS: In the hearts perfused at coronary pressure of 75 and 120 cm H2O, L-NOARG (10 microM) reduced coronary flow by 30% and 17%, respectively, while cardiac output was not affected. Only a transient increase in adenosine and lactate outflow occurred in L-NOARG-treated hearts. The post-ischaemic recovery of functions was impaired in L-NOARG-treated hearts, an effect not correlating with L-NOARG-induced reduction in coronary flow. Although the pre-ischaemic coronary flow was similar in the untreated hearts perfused at 75 cm H2O and in L-NOARG-treated hearts perfused at 120 cm H2O, the post-ischaemic recovery in the latter group was still impaired as compared to that in the untreated hearts. Likewise, coronary flow was similar in the untreated hearts and in those treated with L-NOARG plus adenosine, nevertheless, the post-ischaemic recovery in the latter group was impaired as compared to that in the untreated hearts. CONCLUSIONS: While the inhibition of NO synthesis resulted in coronary flow reduction it did not induce a state of permanent ischaemia in isolated rat heart. L-NOARG-induced augmentation of the ischaemia/reperfusion injury was related to the deficit of NO, itself, rather than to the reduction in myocardial perfusion.

Thapsigargin inhibits the effects of noradrenaline and high [Ca2+]o on kinetics but not on amplitude of contraction in the single myocytes of guinea-pig heart.

We investigated the effect of selective blocker of Ca(2+)-ATPase of sarcoplasmic reticulum (SR), thapsigargin (TG), on the responses of single myocytes of guinea-pig heart to high [Ca2+]o and noradrenaline (NA). The cells were loaded with acetomethylester of Indo 1 and free [Ca2+]i monitored as the ratio of fluorescence at 405 and 495 nm. The changes in cell length were monitored by a TV displacement system. In the normal cells increase in [Ca2+]o from 2.0 to 5.0 mM increased amplitude of contraction by 154 +/- 8%, decreased the time to peak contraction from 343 +/- 25 ms to 328 +/- 20 ms and decreased the total duration of contraction from 813 +/- 81 ms to 800 +/- 47 ms (not significant). The rate of rise of rapid phase of Ca2+ transients and their amplitude was increased and decay accelerated. The 10(-9) mM NA increased the amplitude of contraction by 154 +/- 9%, decreased time to peak contraction from 343 +/- 25 ms to 273 +/- 17 ms and the total duration of contraction from 813 +/- 81 ms to 603 +/- 28 ms. The rate of rise of the rapid phase of Ca2+ transients was greatly accelerated and their amplitude was increased. The 10(-7) M(TG) blocked completely the rapid phase of Ca2+ transients, increased time to peak [Ca2+]i and delayed decay. Total amplitude of the transients was hardly affected. Accordingly, time to peak contraction was prolonged to 583 +/- 50 ms but the amplitude of contraction was only slightly decreased. Calcium stores in the SR were completely depleted as assessed by caffeine Ca2+ transients and contractures. In the cells pretreated with TG 10(-9) M NA but not 5.0 mM Ca2+ increased amplitude of the transients. Amplitude of contractions was increased by both agents more than in the control cells. The 5.0 mM Ca2+ slightly decreased time to peak contraction and NA tended to increase it. Both inotropic agents accelerated relaxation. It is concluded that amplitude of contractions may be increased by stimulation of Ca2+ influx, however, the control of kinetics of contraction results from altered handling of the influx by the functioning SR.