Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.     

Pharmacological characterization of the receptors involved in the beta-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes

1. The whole-cell patch-clamp was used for studying the effects of various beta1- and beta2-adrenoceptor agonists and antagonists on the L-type Ca current (Ica) in frog ventricular myocytes. 2. Dose-response curves for the effects of isoprenaline (non selective beta-agonist), salbutamol (beta2-agonist), dobutamine (beta1-agonist) on ICa were obtained in the absence and presence of various concentrations of ICI 118551 (beta2-antagonist), metoprolol (beta1-antagonist) and xamoterol (partial beta1-agonist) to derive EC50 (i.e. the concentration of beta-agonist at which the response was 50% of the maximum) and Emax (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K(B)) for the antagonist-receptor complex. 3. Isoprenaline increased ICa with an EC50 of 20.0 nM and an Emax of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K(B) of 3.80 nM and 207 nM, respectively. 4. Salbutamol increased ICa with an EC50 of 290 nM and an Emax of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K(B) of 1.77 nM and 456 nM, respectively. 5. Dobutamine increased ICa with an EC50 of 2.40 microM and an Emax of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K(B) of 2.84 nM and 609 nM, respectively. 6. Xamoterol had no stimulating effect on ICa. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on ICa with a K(B) of 58-64 nM. 7. We conclude that a single population of receptors is involved in the beta-adrenoceptor-mediated regulation of ICa in frog ventricular myocytes. The pharmacological pattern of the response of ICa to the different beta-adrenoceptor agonists and antagonists tested suggests that these receptors are of the beta2-subtype.     

Actions of taurine on the L-type Ca2+ channel current in guinea pig ventricular cardiomyocytes

This article presents a new method for analyzing the spreading of skin erythemas. These occur as a result of the cutaneous vascular axon reflex which can be evoked by a noxious stimulation of the skin. Series of true-color images of the observed skin patch were recorded using a video camera. The images were digitized and stored on computer disk. The delineation of the reddening was segmented for every image of the sequence by a newly developed image processing method. Each image taken after the noxious stimulation was compared with the baseline before the stimulation and each image point was classified as: "unchanged" or "changed skin color." To improve the classification the CIE L*a*b* color space was used. The boundaries of the erythema were extracted from the resulting binary images. Every image of a sequence was analyzed in the same way in order to follow the time course of the flare response. The erythema reaction could be determined in an objective way using this methods. The automatically detected flare sizes were independent of human observers and had a high spatial and temporal resolution. It was used for a crossover study to assess the power of new drugs which modify the blood flow of the skin induced by an intradermal histamine application.     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degreesC. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.     

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.     

Acetaldehyde depresses shortening and intracellular Ca2+ transients in adult rat ventricular myocytes

Numerous studies have shown that the developing tip of a neurite, the growth cone, can respond to environmental cues with behaviors such as guidance or collapse. To assess whether a given cell type can use more than one second-messenger pathway for a single behavior, we compared the influence of two well-characterized guidance cues on growth cones of chick temporal retinal ganglion cells. The first cue was the repulsive activity derived from the posterior optic tectum (p-membranes), and the second was the collapse-inducing activity derived from oligodendrocytes known as NI35/NI250. p-Membranes caused permanent growth cone collapse with no recovery after several hours, while NI35 caused transient collapse followed by recovery after about 10 min. The p-membrane-induced collapse was found to be Ca2+ independent, as shown using the Ca2+-sensitive dye Fura-2 and by the persistence of collapse in Ca2+-free medium. Dantrolene, a blocker of the ryanodine receptor, had only a minor effect on the collapse frequency caused by p-membranes. In contrast, the NI35-induced collapse was clearly Ca2+ dependent. [Ca2+]i increased sevenfold preceding collapse, and both dantrolene and antibodies against NI35 significantly reduced both the Ca2+ increase and the collapse frequency. Thus, even in a single cell type, growth cone collapse induced by two different signals can be mediated by two different second-messenger systems.     

Activation of Ca2+-sensitive Cl- current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36 degreesC. In cells dialysed with Na+-free solutions, the application of nicardipine (5 microM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i>/=5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 microM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Measurements of Ca2+ entry and sarcoplasmic reticulum Ca2+ content during the cardiac cycle in guinea pig and rat ventricular myocytes

This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species.     

Cell cycle-related changes in the voltage-gated Ca2+ currents in cultured newborn rat ventricular myocytes

The expression of T-type Ca2+ current (ICa,T) has been reported to change during postnatal heart development and myocardial hypertrophy, which are characterized respectively by the arrest of the cell cycle soon after birth and a switching on of DNA synthesis in the terminally differentiated cardiac myocytes. The hypothesis that there are cell cycle-related changes in cardiac Ca2+ channel expression was tested by performing whole-cell voltage-clamp recording and BromodeoxyUridine (BrdU) immunolabeling to determine the S phase of the cell cycle in the same single cultured newborn rat ventricular cells. Myocytes were isolated from 1-day-old Wistar rats and cultured for 15 days. ICa,T was detected in 27% of the 5-day cultured myocytes. The progressive loss of ICa,T during the period of 15-day incubation, which resembles the developmental changes in vivo, paralleled the decrease in the percentage of cells showing BrdU labeling. At day 5 of cell culture, the fraction of myocytes expressing ICa,T was significantly higher in the BrdU-labeled population (95%) as compared with the non-labeled cells (19%). In addition, a 72-h treatment with 20 microM nickel, an ICa,T blocker, revealed no effect on the percentage of BrdU-positive cells. L-type Ca2+ current (ICa,L) was constantly expressed throughout the 15-day cell culture. The frequency of ICa,L expression was identical between the BrdU-labeled and the non-labeled myocytes, although the latter cell population demonstrated a relatively greater current density. No differences in the inactivating kinetics of ICa,L and their reaction to beta-adrenoceptor stimulation were observed between the two groups. These findings provide convincing evidence for the cell cycle-related expression of cardiac Ca2+ channel. Cardiomyocytes at the S phase of the cell cycle predominantly express ICa,T, while the major properties of ICa,L' are unchanged during the cell cycle. Such a cell cycle-related channel expression may play a critical role in regulating the cardiac electrophysiological properties during heart development and myocardial remodeling.     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

After a brief presentation of the development of free walking interpreted as learning dynamical equilibrium, the problem of sensory integration in the process of walking development is discussed. A critical review of the role of vision in the development of posturo-locomotor task is presented, along with recent test results on the development of the vestibular system. A final section presents the development of head stabilization and coordination as a necessary means to assist sensory integration. It is suggested that if sensory information is necessary to enhance posturo-locomotor skills, a good mastery of walking is in turn necessary to increase the efficiency of sensory integration.     

Differential effects of fentanyl and morphine on intracellular Ca2+ transients and contraction in rat ventricular myocytes

BACKGROUND: Our objective was to elucidate the direct effects of fentanyl and morphine on cardiac excitation-contraction coupling using individual, field-stimulated rat ventricular myocytes. METHODS: Freshly isolated myocytes were loaded with fura-2 and field stimulated (0.3 Hz) at 28 degrees C. Amplitude and timing of intracellular Ca2+ concentration (at a 340:380 ratio) and myocyte shortening (video edge detection) were monitored simultaneously in individual cells. Real time Ca2+ uptake into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. RESULTS: The authors studied 120 cells from 30 rat hearts. Fentanyl (30-1,000 nM) caused dose-dependent decreases in peak intracellular Ca2+ concentration and shortening, whereas morphine (3-100 microM) decreased shortening without a concomitant decrease in the Ca2+ transient. Fentanyl prolonged the time to peak and to 50% recovery for shortening and the Ca2+ transient, whereas morphine only prolonged the timing parameters for shortening. Morphine (100 microM), but not fentanyl (1 microM), decreased the amount of Ca2+ released from intracellular stores in response to caffeine in intact cells, and it inhibited the rate of Ca2+ uptake in isolated sarcoplasmic reticulum vesicles. Fentanyl and morphine both caused a downward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on the Ca2+ transient. CONCLUSIONS: Fentanyl and morphine directly depress cardiac excitation-contraction coupling at the cellular level. Fentanyl depresses myocardial contractility by decreasing the availability of intracellular Ca2+ and myofilament Ca2+ sensitivity. In contrast, morphine depresses myocardial contractility primarily by decreasing myofilament Ca2+ sensitivity.     

Sarcoplasmic reticulum Ca2+ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes

Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were measured simultaneously with indo 1 and a Ca(2+)-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes (approximately 10 mg/mL protein). In the presence of 25 mumol/L ruthenium red and 10 mmol/L oxalate, the Km for Ca2+ uptake by the SR was approximately 250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary to inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 mumol/kg wet wt for rabbit and 54.6 mumol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to < 150 nmol/L from a peak value > 10 mumol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in < 15 seconds and reduced the uptake rate by 95% in 5 seconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amiodarone blocks L-type calcium current in single myocytes isolated from the rabbit atrioventricular node

CD22 antibodies (Abs) bound to B-cell lymphomas are known to be internalized and catabolized rapidly. Therefore, it would be expected that use of CD22 as a target for radioimmunotherapy should be enhanced by the use of "residualizing" radiolabels, which are trapped within the cell after catabolism of the Ab to which they had been conjugated. Our study was intended to evaluate this hypothesis using Ab LL2. In initial experiments, we found that LL2 binding was strongly temperature dependent, with approximately 15-fold greater binding at 37 degrees C than at 0 degrees C. A series of experiments suggested that this difference is due to a conformational change in the antigen at low temperature, so that the LL2 epitope is partially blocked. In vitro, residualizing labels-including 125I-dilactitol tyramine and 111In-DTPA-were retained by cells much longer than a conventional iodine label. In vivo, residualizing labels also showed a marked advantage in terms of uptake by Ramos B-cell lymphoma xenografts in nude mice. However, the absolute Ab uptake by xenografts was quite low, in comparison with results obtained with many carcinoma xenografts, which appears to be due in part to vascular properties of the B-cell lymphoma xenografts.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

Propofol and ketamine only inhibit intracellular Ca2+ transients and contraction in rat ventricular myocytes at supraclinical concentrations

BACKGROUND: The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation-contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field-stimulated ventricular myocytes. METHODS: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field-stimulated at 0.3 Hz. RESULTS: Baseline [Ca2+]i (mean +/- SEM) was 80 +/- 12 nM, and resting cell length was 112 +/- 2 microm. Field stimulation increased [Ca2+]i to 350 +/- 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose-dependent decreases in peak [Ca2+]i and shortening. At 300 microM, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. CONCLUSIONS: These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.     

Withdrawal of acetylcholine elicits Ca2+-induced delayed afterdepolarizations in cat atrial myocytes

BACKGROUND: Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS: A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS: Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

Thiopental alters contraction, intracellular Ca2+, and pH in rat ventricular myocytes

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.     

Temperature dependence of macroscopic L-type calcium channel currents in single guinea pig ventricular myocytes

INTRODUCTION: Lowering temperature greatly reduces calcium influx through calcium channels. Studies on a number of tissues demonstrate that the peak inward current, ICa, exhibits Q10 values ranging from 1.8 to 3.5; however, it remains unclear which component(s) of calcium channel gating may give rise to this large temperature sensitivity. Components of gating that may affect channel availability include phosphorylation and changes in [Ca2+]i, processes that vary in pertinence depending on the channel examined. This study addresses this problem by examining the temperature sensitivity (from 34 degrees to 14 degrees C) of cardiac ICa under control conditions, during attenuation or activation of protein kinase A (PKA) activity, and when intracellular [Ca2+] has been elevated. METHODS AND RESULTS: ICa was studied using the whole cell configuration of the patch champ technique. In control, lowering temperature from 34 degrees to 24 degrees C resulted in a shift in the potential for maximum slope (Va) and the peak current (Ymax) toward more positive membrane potentials. The Q10 values for the decrease in Ymax and the macroscopic slope conductance (Gmax), which reflects the number of available channels, were 3.15 +/- 0.19 and 2.57 +/- 0.13, respectively. At 0 mV the Ca2+ current decayed biexponentially, and the two time constants (tau 1 and tau 2) showed Q10 values of 1.79 +/- 0.21 and 2.06 +/- 0.38, while their contribution to the total current (I1 and I2) showed a Q10 of 5.99 +/- 0.83 and 1.61 +/- 0.22. In myocytes loaded with inhibitors of the PKA cycle sufficient to inhibit the increase of ICa to 1 microM isoprenaline, the Q10 values for some of the kinetic parameters were increased with the Q10 for I1 increasing to 17.06 +/- 3.48. Stimulation of ICa by exposing myocytes to 1 microM isoprenaline reduced the temperature sensitivity of Ymax, Gmax and I1, yielding respective values of 2.00 +/- 0.18, 1.85 +/- 0.07, and 2.04 +/- 0.15. Raising [Ca2+]i to enhance Ca2+i-dependent inactivation, while affecting inactivation and activation kinetics, affected temperature sensitivity little compared to control. The Q10 for time to peak changed little under experimental conditions (2.3 to 2.4) CONCLUSIONS: Increasing the phosphorylated states of calcium channels, but not Ca2+i-dependent inactivation, reduces temperature sensitivity of certain gating parameters. The data suggest that the rate of the transitions between the unavailable and also between the various closed states are changed in the opposite direction to that induced by PKA-dependent phosphorylation. Processes, e.g., inhibitory mechanisms, may be involved to maintain channels in unavailable or "unphosphorylated" states, and it may be these that contribute to the high Q10 of macroscopic channel currents.