Adult Guinea Pig Ventricular Myocytes Research Articles

Alkali cation permeability and caesium blockade of cromakalim‐activated current in guinea‐pig ventricular myocytes

1. The sensitivity of cromakalim-activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole-cell voltage clamp recordings from freshly-dispersed, adult guinea-pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mM) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2. Replacement of 12mM K+ with 12mM Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12mM Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3. Addition of Rb+ (1-30 mM) to 2.5 mM K(+)-containing medium produced a concentration-dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4. CS+ (0.01-30 mM), added to medium containing 12 mM Rb+, induced a potent, voltage-dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2-3 mM). Voltage-dependent inhibition of inward Icrom was observed only at considerably higher CS+ concentrations (IC50 = 4-30 mM). Extracellular Rb+ and CS+ did not substantially alter the amplitude of outward Icrom. 5. The results support the contention that the ATP-sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes.

Evidence against the presence of A 2 adenosine receptors on guinea pig ventricular myocytes

The effects of different adenosine agonists on cAMP levels in isolated adult guinea pig ventricular myocytes were investigated. 2-Chloro-N 6-cyclopentyladenosine (CCPA), R-N 6-phenylisopropyl-adenosine (R-PIA), S-N 6-phenylisopropyladenosine (S-PIA) and 5′-N-ethylcarboxolamidoadenosine (NECA) reduced isoprenaline-stimulated cAMP levels. Both the nonselective adenosine antagonist 8-{4-[([{(2-aminoethyl)amino}carbonyl]methyl)oxy]-phenyl}-1,3-dipropylxanthine (‘xanthine amine congener’; XAC) and the A 1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the inhibitory effect of NECA. Basal cAMP levels were not altered by NECA or the highly A 2-selective 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), either alone or in the presence of DPCPX or XAC. These results provide evidence against the existence of A 2 receptors on the ventricular myocyte but support the presence of A 1 receptors.

Effects of intracellular acidosis on [Ca2+]i transients, transsarcolemmal Ca2+ fluxes, and contraction in ventricular myocytes.

We examined the effects of intracellular acidosis produced by washout of NH4Cl on [Ca2+]i transients (indo-1 fluorescence), cell contraction (video motion detector), and 45Ca and 24Na fluxes in cultured chick embryo ventricular myocytes. Exposure of cells to 10 mM NH4Cl produced intracellular alkalosis (pH 7.6), and subsequent washout resulted in a transient acidosis (pH 6.5). Exposure to 10 mM NH4Cl slightly decreased [Ca2+]i transients but increased the amplitude of cell contraction. Subsequent washout of NH4Cl initially increased diastolic [Ca2+]i and decreased the peak positive and negative d[Ca2+]i/dt, while the amplitude of cell contraction was markedly decreased. Subsequently, peak systolic [Ca2+]i increased with partial recovery of contraction. A similar increase in [Ca2+]i and decrease in contraction after washout of NH4Cl was observed in single paced adult guinea pig ventricular cells. Acidosis decreased 45Ca uptake by sarcoplasmic reticulum vesicles isolated from chick embryo ventricle. However, the [Ca2+]i increase caused by intracellular acidosis was also observed in the presence of 10 mM caffeine, suggesting that altered sarcoplasmic reticulum handling of calcium is not the only mechanism involved. Intracellular acidosis only slightly increased total 24Na uptake under these conditions, an effect resulting from the combination of a stimulation of amiloride-sensitive sodium influx (Na(+)-H+ exchange) and inhibition of sodium influx via Na(+)-Ca2+ exchange, manifested by a significant decrease in 45Ca efflux. Further support for a lack of involvement of an increased [Na+]i in the observed increase in [Ca2+]i during acidosis was low-sodium, nominal 0-calcium extracellular solution, an experimental condition that minimizes the possible effects of Na(+)-H+ exchange and Na(+)-Ca2+ exchange. We conclude that the [Ca2+]i increase caused by intracellular acidosis in cultured ventricular cells is primarily due to changes in [Ca2+]i buffering and [Ca2+]i extrusion, rather than to an increase in transsarcolemmal calcium influx. Intracellular acidosis also markedly decreases the sensitivity of the contractile elements to [Ca2+]i in cultured chick embryonic and adult guinea pig ventricular myocytes.

Calcium binding to cardiac myocytes protected from proteolytic enzyme activity

Excitation-contraction coupling in cardiac muscle is dependent on extracellular calcium and calcium bound to the surface of the myocardial cell. In this study, we examined the physical characteristics of calcium binding to adult guinea pig ventricular myocytes disaggregated mechanically in oxygenated tissue culture medium containing a proteinase (aprotinin), and separated from cellular debris by Cytodex beads. Cells prepared in this manner excluded Trypan blue and showed no evidence of spontaneous contraction or contracture. Scatchard plots of calcium binding determined by continuous flow equilibrium dialysis revealed a high-affinity, low-capacity pool, K a=65·10 3 M −1 and B t=1.3 nmol·mg −1 and a low-affinity, high-capacity pool, K a=141 M −1 and B t=138 nmol·mg −1. The low-affinity pool was not detectable after lanthanum, trypsin or collagenase treatment or in cells prepared without aprotinin in the isolation medium. Both neuraminidase and phospholipase C reduced B t of the low-affinity pool by one half, but only neuraminidase affected the affinity constant of this pool. K a was increased to 516.7 M −1, similar to the apparent affinity constant for calcium binding estimated from d P/d t max measured at several extracellular calcium concentrations (470 M −1). The results suggest that calcium bound to sarcolemmal phospholipids represents the superficial calcium in olved in excitation-contraction coupling in the heart.