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.     

Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium

Because glycolysis is thought to be important for maintenance of cellular ion homeostasis, the aim of the present study was to examine the role of glycolysis in the control of cytosolic calcium ([Ca2+]i) and cell shortening during conditions of increased calcium influx. Thus, [Ca2+]i and unloaded cell shortening were measured in fura-2/AM loaded rat ventricular myocytes. All cells were superfused with Tyrode's solution containing glucose and pyruvate (to preserve oxidative metabolism), and glycolysis was inhibited by iodoacetate (IAA, 100 microM). Calcium influx was increased, secondary to an increase in intracellular sodium, by addition of veratrine (1 microgram/ml), or directly by either elevating [Ca2+]o from 2 to 5 mM or by exposing the cells to isoproterenol (1 to 100 nm). Veratrine exposure caused a time-dependent increase in both diastolic and systolic [Ca2+]i that resulted in cellular calcium overload and hypercontraction. The rate of increase in [Ca2+]i was more rapid in IAA-treated than in untreated myocytes, leading to a 13+/-3 v 5+/-2% increase (P<0.05) in diastolic [Ca2+]i after 5 min of exposure. The corresponding increases in systolic [Ca2+]i were 43+/-6 and 24+/-5% (P<0.05). Elevated [Ca2+]o resulted in increased [Ca2+]i transient amplitudes and cell shortening. These responses were each attenuated by inhibiting glycolysis, so that the increase was 38+/-5 v 68+/-9% ([Ca2+]i transient amplitude, P<0.05) and 41+/-11 v 91+/-18% (cell shortening, P<0.05). Inhibition of glycolysis did not, however, affect the increase in calcium transient or cell shortening during addition of isoproterenol. We conclude that glycolysis plays an essential role in the maintenance of intracellular calcium homeostasis during severe calcium overload. Glycolysis was also essential for signalling the inotropic effect that accompanied elevation in extracellular calcium, while the changes in intracellular calcium following administration of isoproterenol were not influenced by glycolysis in the present model.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.     

New advances in sex preselection

The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.     

Dietary and physiological studies to investigate the relationship between calcium and magnesium signalling in the mammalian myocardium

This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca2+) and magnesium (Mg2+) signalling in the mammalian myocardium. Rats maintained on a low Mg2+ diet (LMD; 39 mg Kg-1 Mg2+ in food) consumed less food and grew more slowly than control rats fed on a control Mg2+ diet (CMD; 500 mg Kg-1 Mg2+ in food). The Mg2+ contents of the heart and plasma were 85 +/- 3% and 34 +/- 6.5%, respectively relative to the control group. In contrast, Ca2+ contents in the heart and plasma were 177 +/- 5% and 95 +/- 3%. The levels of potassium (K+) was raised in the plasma (129 +/- 16%) and slightly decreased in the heart (88 +/- 6%) compared to CMD. Similarly, sodium (Na+) contents were slightly higher in the heart and lowered in the plasma of low Mg2+ diet rats compared to control Mg2+ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg2+]o resulted in a small transient increase in the amplitude of contraction compared to control [Mg2+]o (1.2 mM). In contrast, elevated [Mg2+]o (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca2+ current (ICa,L) was significantly (p < 0.001) attenuated in cells dialysed with 7.1 mM Mg2+ compared to cells dialysed with 2.9 microM Mg2+. The results indicate that hypomagnesemia is associated with decreased levels of Mg2+ and elevated levels of Ca2+ in the heart and moreover, internal Mg2+ is able to modulate the Ca2+ current through the L-type Ca2+ channel which in turn may be involved with the regulation of contractile force in the heart.     

Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats

Effects on isometric tension generation and maximum velocity of unloaded shortening after exposure to cAMP-dependent protein kinase (PKA) were investigated in rat enzymatically isolated, tritonized ventricular myocytes. Exposure of myocytes to PKA in the presence of [32P]ATP resulted in phosphorylation of troponin I and C protein. Ca2+ sensitivity of isometric tension was assessed as pCa50, ie, the [Ca2+] at which tension was 50% of maximum, and was lower after PKA treatment (pCa50 5.58) than before PKA treatment (pCa50 5.74). This suggests beta-adrenergic stimulation of the heart and subsequent increases in PKA activity and phosphorylation of troponin I and C protein lead to a significant decrease in tension-generating ability at a given submaximum [Ca2+]. Unloaded shortening velocity was determined by measuring the time required to take up various amounts of slack imposed at one end of the cardiac myocyte preparation. Unloaded shortening velocity during maximum activation was 2.88 +/- 0.11 muscle lengths per second (mean +/- SEM) before PKA exposure and 2.86 +/- 0.13 muscle lengths per second after PKA exposure. Unloaded shortening velocity during 40% of maximum activation was 1.91 +/- 0.25 muscle lengths per second before PKA exposure and 2.17 +/- 0.15 muscle lengths per second after PKA exposure. The absence of an effect of PKA on unloaded shortening velocity in skinned ventricular myocytes suggests that beta-adrenergic stimulation of myocardium either does not affect myofilament velocity of shortening or alters velocity of shortening by a non-PKA-dependent process.     

Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure

Systolic [Ca2+]i-transients have been shown to be depressed in isolated ventricular myocytes from patients with terminal heart failure compared to controls. Experiments were performed in human ventricular cells to investigate whether this reduced systolic [Ca2+]i-transient may be due to a decreased Ca(2+)-content of the sarcoplasmic reticulum (SR). Single myocytes were isolated from left ventricular myocardium of patients with terminal heart failure undergoing cardiac transplantation. These results were compared to those obtained from cells of healthy donor hearts that were not suitable for transplantation for technical reasons. [Ca2+]i-transients were recorded from isolated cells under voltage clamp perfused internally with the Ca(2+)-indicator fura-2. The Ca(2+)-content of the SR was estimated by rapid extracellular application of caffeine (10 mM) to open the Ca(2+)-release channel of the SR and comparison of the caffeine-induced [Ca2+]i-transients in cells from patients with heart failure and from controls without heart failure. Upon steady-state depolarizations to +10 mV (maximum of the Ca(2+)-current), [Ca2+]i-transients in cells from patients with heart failure were significantly smaller than in myocytes from undiseased hearts (333 +/- 26 v 596 +/- 80 nM, P < 0.05). Application of caffeine caused a [Ca2+]i-transient that was always larger than during depolarization. Caffeine-induced [Ca2+]i-transients were significantly smaller in cells from diseased hearts compared with controls (970 +/- 129 v 2586 +/- 288 nM, P < 0.01). A positive correlation was found between left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients in these cells. It is concluded, that depressed [Ca2+]i-transients in myocytes from patients with heart failure may be caused by a decreased Ca(2+)-content of the SR possibly due to an altered Ca(2+)-ATPase activity in these hearts. It is not necessary to postulate an additional defect of the Ca(2+)-release function of the SR to account for the alterations of intracellular (Ca2+]i-handling.     

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase

OBJECTIVES: Human cardiac muscle from failing heart shows a decrease in active tension development and a rise in diastolic tension at stimulation frequencies above 50-60 beats/min due to both systolic and diastolic dysfunction. We have investigated underlying changes in cellular [Ca2+]i regulation. METHODS: Single ventricular myocytes were isolated enzymatically from the explanted hearts of transplant recipients with ischemic cardiomyopathy (nhearts = 5 ncells = 15) or dilated cardiomyopathy (nhearts = 6, ncells = 19). Cells were studied during whole-cell patch clamp with fluo-3 and fura-red as [Ca2+]i indicators (36 +/- 1 degrees C). RESULTS: In current clamp mode (action potential recording), the amplitude of Ca2+ release from the sarcoplasmic reticulum (SR) decreased at stimulation frequencies above 0.5 Hz; this decrease was more pronounced for cells from dilated cardiomyopathy. Diastolic [Ca2+]i increased at 1 and 2 Hz for both groups. Action potential duration (APD90) decreased with frequency in all cells; in addition there was a drop in plateau potential of 10 +/- 1 mV for cells from ischemic cardiomyopathy and of 13 +/- 2 mV for cells from dilated cardiomyopathy. In voltage clamp mode the L-type Ca2+ current showed reversible decrease during stimulation at 1 and 2 Hz. Recovery from inactivation during a double pulse protocol was slow (75 +/- 3% at 500 ms, 89 +/- 3% at 1000 ms) and followed the decay of the [Ca2+]i transient. CONCLUSIONS: The negative force-frequency relation of the failing human heart is due to a decrease in Ca2+ release of the cardiac myocytes at frequencies > or = 0.5 Hz, more pronounced in dilated than in ischemic cardiomyopathy. Inhibition of ICaL at higher frequencies, at least partially related to an increase in diastolic [Ca2+]i, will contribute to this negative staircase because of a decrease in the trigger for Ca2+ release, and of decreased loading of the SR.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Passive Ca2+ binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca2+ binding was determined in ventricular homogenates (VH) from neonatal (4-6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca2+ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca2+ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca2+] ([Ca2+]F) was measured with Indo-1 and bound Ca2+ ([Ca2+]B) was the difference between [Ca2+]F and total Ca2+. Apparent Ca2+ dissociation constants (Kd) for BAPTA and Indo-1 were increased by 10-20 mg VH protein/ml (from 0.35 to 0.92 microM for Indo-1 and from 0.20 to 0.76 microM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5-10 nmoles) yielded delta[Ca2+]B/delta[Ca2+]F for the sum of [Ca2+]B at all three classes of binding sites. From this function, the apparent number of endogenous sites (Ben) and their Kd (Ken) were determined. Similar Ken values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 +/- 0.14 microM, 0.69 +/- 0.13 microM and 0.53 +/- 0.10 microM, respectively). However, Ben was significantly higher in adult myocytes than in adult VH (1.73 +/- 0.35 versus 0.70 +/- 0.12 nmol/mg protein, P < 0.01), which correspond to approximately 300 and 213 mumol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that Ben determined in VH underestimates cellular Ben by 29%. Although Ben values in nmol/mg protein were similar in adult and neonatal VH (0.69 +/- 0.12), protein content was much higher in adult ventricle (125 +/- 7 versus 80 +/- 1 mg protein/g wet weight, P < 0.01). Expressing Ben per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca2+ binding capacity at high-affinity sites is approximately 300 and 176 mmol/l cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca2+ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.     

Expression of calcium channels in adult cardiac myocytes is regulated by calcium

In addition to playing a significant role in cardiac excitation-contraction coupling, intracellular Ca2+ ([Ca2+]i) can regulate gene expression. While the mechanisms regulating expression of Ca2+ channels are not entirely defined, some evidence exists for Ca2+-dependent regulation. Using an adult ventricular myocyte culture system, we determined the effects of Ca2+ on: (1) abundance of mRNA for L-type Ca2+ channel alpha1 subunit (DHP receptor); (2) amount of DHP receptors; and (3) whole-cell Ca2+ current (ICa). Rat ventricular myocytes were cultured for 1-3 days in serum-free medium containing either normal (1.8 mM) or high (4.8 mM) Ca2+. Exposing myocytes to high Ca2+ rapidly elevated [Ca2+]i as determined by fura-2. Northern blot analysis revealed that culturing cells in high Ca2+ produced 1.5-fold increase in mRNA levels for the DHP receptor. The abundance of DHP receptors, determined by ligand binding, was two-fold greater in myocytes after 3 days in high Ca2+. Moreover, peak ICa was larger in myocytes cultured for 3 days in high Ca2+ (-17.8+/-1.5 pA/pF, n=26) than in control cells (-11.0+/-1.0 pA/pF, n=23). Voltage-dependent activation and inactivation, rates of current decay, as well as percent increases in ICa elicited by Bay K8644 were similar in all groups. Therefore, larger ICa is likely to represent a greater number of functional channels with unchanged kinetics. Our data support the conclusion that transient changes in [Ca2+]i can modulate DHP receptor mRNA and protein abundance, producing a corresponding change in functional Ca2+ channels in adult ventricular myocytes.     

Effects of angiotensin II on inotropy and intracellular Ca2+ handling in normal and hypertrophied rat myocardium

The direct inotropic effect of angiotensin II on the myocardium is still controversial and little information exists as to its potential modification by heart disorders. Therefore, this study performed simultaneous measurements of isometric force and intracellular Ca2+ concentrations ([Ca2+]i) in left ventricular papillary muscles from sham-operated and aortic-banded rats at 10 weeks post-surgery. Angiotensin II (10(-6) M) induced a reduction of peak systolic [Ca2+]i (0.56 +/- 0.03 to 0.48 +/- 0.04 microM; P<0.05) and a parallel but insignificant diminution of developed tension (10.5 +/- 1.3 to 9.6 +/- 0.8 mN/mm2) in normal papillary muscles from sham-operated animals. Hypertrophied papillary muscles from aortic-banded rats demonstrated a significant decline in both peak systolic [Ca2+]i (0.51 +/- 0.02 to 0.44 +/- 0.01 microM; P<0.05) and developed tension (8.4 +/- 1.1 to 6.8 +/- 1.7 mN/mm2; P<0.05) after addition of angiotensin II. The time courses of the mechanical contraction and the intracellular Ca2+ signal were prolonged by angiotension II in both groups. Isoproterenol dose-dependently increased developed tension and peak systolic [Ca2+]i in papillary muscles from sham-operated rats. In contrast, the positive inotropic response to isoproterenol was markedly reduced in hypertrophied muscles despite a seemingly unimpaired increase in peak systolic [Ca2+]i. Pretreatment with angiotensin II (10(-6) M) resulted in a significant attenuation of the systolic [Ca2+]i response to isoproterenol stimulation in both normal and hypertrophied papillary muscles. Neither the bradykinin B2 antagonist icatibent (10(-6) M) nor the nitric oxide (NO) inhibitor L-NMMA (10(-6) M) abolished the depressant effects of angiotension II. Thus, ANG II induces a parallel decline of the mechanical performance and Ca2+ availability in rat myocardium. These effects are more distinct in hypertrophied than in normal muscle and become accentuated during beta-adrenergic stimulation. The underlying mechanism is not associated with the NO pathway but might involve a negative functional coupling between the angiotensin and beta-adrenergic-receptor complex.     

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.     

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.     

Effect of inhibition of sarcoplasmic Ca(2+)-ATPase on vasoconstriction and cytosolic Ca2+ in aortic smooth muscle from spontaneously hypertensive and normotensive rats

To evaluate the influence of the sarcoplasmic Ca(2+)-ATPase, isometric vasoconstrictions of aortic strips from spontaneously hypertensive rats from the Münster strain (SHR) and normotensive Wistar-Kyoto rats (WKY) were measured after inhibition of Ca(2+)-ATPase by thapsigargin. Inhibition of Ca(2+)-ATPase by thapsigargin caused a biphasic contractile response of the aorta in both SHR and WKY (maximum increase of tension: 1.7 +/- 0.3 x 10(-3) Newton and 2.1 +/- 0.3 x 10(-3) Newton, respectively; mean +/- SE). The second peak of the contractile response was abolished in the absence of external calcium or by inhibition of transplasmamembrane calcium influx by nifedipine, indicating that the second peak occurs as a consequence of calcium influx from the extracellular space. The initial peak of the contractile response after thapsigargin administration was abolished in the presence of an intracellular calcium antagonist, 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the initial response was due to calcium release from intracellular stores. Measurements using the fluorescent dye fura2 showed that thapsigargin increased the cytosolic free calcium concentration ([Ca2+]i) in SHR by 72.6 +/- 7.3 nmol/l (n = 34) and in WKY by 53.3 +/- 6.6 nmol/l (n = 39), showing no significant differences between the two strains. The inhibition of Ca(2+)-ATPase increases [Ca2+]i and causes vasoconstriction. The vasoconstriction produced by thapsigargin is not significantly different between SHR and WKY.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets

We investigated the effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) on intracellular Ca2+ concentration ([Ca2+]i) and cell length in isolated and field-stimulated rat cardiomyocytes. [Ca2+]i and cell length of field-stimulated cells were determined simultaneously by confocal laser scan microscopy by using the fluorescent Ca2+ dye Fluo-3. PAF (10(-12)-10(-8) M) inhibited systolic [Ca2+]i increase in a time- and concentration-dependent manner. Maximal effects were observed after an incubation time of 6-8 min, resulting in a 17% (10(-12) M), 41% (10(-10) M), and 52% (10(-8) M PAF) inhibition of systolic [Ca2+]i increase. A time- and concentration-dependent decrease in simultaneously measured cell shortening also was demonstrated. Cell shortening was inhibited by 10% (10(-12) M), 32% (10(-10) M), and 50% (10(-8) M) after an incubation time of 8 min. The effects of PAF could be antagonized by the PAF-receptor antagonist WEB 2170. These data demonstrate that PAF receptor-dependently induces a negative inotropic effect, which is correlated with a decrease in systolic [Ca2+]i and is most likely not due to a decrease in myofilament sensitivity.     

JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.     

Selectivity of felodipine for depolarized ventricular myocytes: a study at the single-cell level

We studied the effects of felodipine (a second-generation dihydropyridine Ca2+ channel blocker) on excitation-contraction coupling (E-C coupling) in single isolated guinea-pig ventricular myocytes, using the whole-cell perforated patch-clamp technique or the Ca indicator, indo-1. Felodipine inhibited both L-type Ca2+ channel currents (ICa) and cell contractions in a concentration-dependent manner (10 pM to 100 nM) when we used a holding potential of -80 mV or -40 mV. The potency of felodipine was sharply dependent on a holding potential. Namely, use of a more depolarized holding potential markedly increased the potency of felodipine for inhibition of ICa and cell contraction. Next we current-clamped cells and obtained the resting membrane potential of -82 +/- 8 mV. When cells were current-injected at 0.1 Hz, exposure to 10 nM felodipine slightly but significantly diminished the amplitude of cell contractions (7.2 +/- 1.6 to 6.7 +/- 1.7 microns, P < 0.05) within 10 min. When cells were field stimulated, exposure of cells to 10 nM felodipine also slightly diminished the amplitude of cell shortening (5.1 +/- 2.0 to 4.6 +/- 1.9 microns, P < 0.05) and [Ca2+]i transients. We observed clear voltage-dependent blockade of E-C coupling by felodipine in ventricular myocytes. Thus, therapeutic concentrations (1-10 nM) of felodipine could inhibit E-C coupling in depolarized ventricular myocytes, which might simulate an ischemic or failing heart.     

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.