Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation.

It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+).

Coding data from child health records: the relationship between interrater agreement and interpretive burden.

The relationship between interrater reliability and interpretive burden when coding information from Swedish Child Health records was studied. Information on preschool children's living conditions, health, and different aspects of care was sorted into one of four groups according to degree of interpretive burden. Two interrater assessments were conducted and compared. The results showed that a low degree of interpretive burden correlated to high interrater agreement. It was possible to increase concordance by coder training, clarifying definitions of variables, and coding instructions, but not so as to eliminate totally the general differences between groups of variables that differed regarding interpretive burden.

Equal changes in L-type calcium channel density after 60 min of ischaemia in normal and ischaemically preconditioned porcine myocardium.

Long-lasting myocardial ischaemia reduces the density of sarcolemmal L-type calcium channels (LCC). Ischaemic preconditioning protects the myocardium against development of infarction. The aim of this study was to investigate if ischaemia-induced loss in LCC is affected by ischaemic preconditioning. Specific (+) - [3H]isradipine binding to LCC was compared in membranes and homogenates from control and ischaemic regions of non-preconditioned and ischaemically preconditioned hearts [two 10 min left anterior descending coronary artery (LAD) occlusions, each followed by 30 min reperfusion]. Biopsies were sampled after 60 min mid LAD occlusion from ischaemic and control (supplied by circumflex artery) regions. Sixty min ischaemia reduced binding density of specific (+) - [3H]isradipine in membranes by 23 +/- 11% (n = 7, P < 0.05) in the non-preconditioned group and by 20 +/- 8% (n = 6, P < 0.05) in the preconditioned group. Binding density in homogenates was reduced by 36 +/- 5% (n = 5. P < 0.05) in the non-preconditioned group and by 21 +/- 5% (n = 5. P < 0.05) in the preconditioned group. The reductions in the two groups and reductions in membranes and homogenates were not statistically different. The dissociation constant of binding was similar in the groups. In conclusion, 60 min of ischaemia reduced the binding density of (+)-[3H]isradipine in membranes and homogenates by 20-36%. The reduction in density of binding sites was not caused by redistribution of sarcolemmal LCC to an intracellular compartment. Ischaemic preconditioning did not affect the decline in binding density as hypothesized.

Effects of ATP on specific [3H](+)-isradipine binding in rat ventricular cardiomyocytes.

In heart membranes, specific [3H](+)-isradipine binding is reduced in membranes from ischemic hearts and by adding 1 mM ATP at low Ca2+ concentrations (1 microM). We investigated if ATP affected specific [3H](+)-isradipine binding in intact rat ventricular cardiomyocytes. Reducing intracellular ATP by 2 h hypoxia (N2 gas) and glucose-free buffer with 1 mM CN-, did not affect density or dissociation constant of [3H](+)-isradipine binding in cardiomyocytes at extracellular 30 mM K+. Extracellular 10 mM ATP inhibited binding in cardiomyocytes by 90% and 50%, respectively, in 30 mM and 120 mM K+ buffer with Ca2+ and Mg2+. Omitting Ca2+ and Mg2+ from the buffer had no effect on the binding inhibition of ATP. Hence, in cardiomyocytes, reducing intracellular ATP has no effect on specific [3H](+)-isradipine binding, whereas high extracellular ATP in the presence of Ca2+ and Mg2+ inhibits binding. Apparently, ATP effects on binding differ in cardiomyocytes and membranes.

Stable guanosine 5'-triphosphate-analogues inhibit specific (+)-[3H]isradipine binding in rat hearts by a Ca(2+)-lowering, G protein-independent mechanism.

We investigated if and how stable guanosine 5'-triphosphate-analogues affect (+)-[3H]isradipine binding in rat hearts. Gpp(NH)p and GTP-gamma-S inhibit specific (+)-[3H]isradipine binding in membranes and cell-homogenates by reducing the binding density without changing the Kd of the k-1. Inhibition by Gpp(NH)p was less in crude tissue homogenates than in membranes apparently due to a soluble factor. Pretreatment of cardiomyocytes with cholera toxin or the presence of the protein kinase A inhibitor, PKI6-22, did not influence the effect of 10(-3) M Gpp(NH)p on binding. The inhibitory effect of 10(-3) M Gpp(NH)p was not significantly altered in membranes from in vivo pertussis toxin treated rats. The addition of 10(-3) M Ca2+ or Mg2+ abolished the inhibitions. Gpp(NH)p in the concentration that inhibits binding, reduced the free concentration of Ca2+. The Ca(2+)-lowering effect of 10(-3) M Gpp(NH)p produced 70%, 60% and 100% of the inhibition in membranes, sonicated and unsonicated cell homogenates. Thus, Gpp(NH)p inhibited specific (+)-[3H] isradipine binding mainly by lowering the free concentration of Ca2+ by chelation and not by activation of cholera toxin or pertussis toxin-sensitive G proteins or protein kinase A.

Density of L-type calcium channels in ischaemically preconditioned porcine heart regions.

Ischaemic preconditioning by brief ischaemic episodes could be explained by reduced cellular calcium ion (Ca2+) influx, reduced cytosolic Ca2+ overload and delayed cell-injury during subsequent long-lasting ischaemia. L-type calcium channels (LCC) regulate sarcolemmal Ca2+ influx in myocardial cells. The aim of this study was to investigate if preconditioning was associated with reduced density or altered state of LCC in the preconditioned region of the heart. To test this we compared the density and the dissociation constant of (+)-[3H]isradipine binding to LCC in membranes from preconditioned and control regions of porcine hearts. Eight porcine hearts were regionally preconditioned by two 10-min occlusions of the mid left anterior descending artery, and each occlusion was followed by 30 min of reperfusion. Biopsies were taken from the preconditioned regions and control regions supplied by the circumflex artery at the end of the last reperfusion, and (+)-[3H]isradipine binding to membranes made from the biopsies was measured. The differences in density and dissociation constant of (+)-[3H]isradipine binding to LCC in membranes from preconditioned and control regions were not significant. In conclusion, the proposed effect of ischaemic preconditioning to reduce Ca2+ influx, does not involve local changes in density or state of LCC that could be detected by (+)-[3H]isradipine binding.

Ca2+ modulates the inhibition of (+)-[3H]isradipine binding by amiloride and quinacrine.

Binding studies were performed to characterize the inhibition by amiloride, 3,4-dichlorobenzamil and quinacrine of specific binding of (+)-[3H]isradipine to L-type voltage-operated calcium ion channels in rat cardiac membranes at 37 degrees C with and without 10(-3) M calcium added. By analysis of saturation, inhibition and dissociation curves we find that without the addition of calcium, amiloride (constant of inhibitor producing 50% inhibition (K0.5) = 6.9 x 10(-4) M, Hill coefficient (nH) = 1.99, k-1 increased) and 3,4-dichlorobenzamil (K0.5 = 7.7 x 10(-7) M, nH = 1.13, k-1 increased) inhibit (+)-[3H]isradipine binding by complex, allosteric interactions, suggesting positive cooperativity between sites for the inhibitors. Quinacrine (K0.5 = 6.7 x 10(-6) M, nH = 0.84, k-1 increased) inhibits the binding allosterically by an action compatible with binding to one site. Addition of 10(-3) M calcium affected the inhibition by amiloride (K0.5 = 1.02 x 10(-3) M, nH = 1.41) and quinacrine (K0.5 = 3.3 x 10(-5) M, nH = 0.65). With calcium added the mechanisms of inhibitions were complex, allosteric, and could be explained by positive cooperativity between sites for amiloride and negative cooperativity between sites for guinacrine. We conclude that calcium addition modulates the inhibitions by amiloride and quinacrine by increasing the inhibition constants and changing the cooperativity.

Inhibition by amiloride and quinacrine of specific [3H]nitrendipine binding to rat cardiac membranes.

Binding studies were designed to test if and how amiloride and quinacrine affected the specific binding of [3H]nitrendipine (NIT) to rat cardiac membranes. Specific binding of NIT was inhibited in a dose-dependent manner by amiloride [Hill coefficient (nH), 1.46; concentration of inhibitor producing 50% inhibition (K0.5) = 9.2 x 10(-4) M] and quinacrine (nH = 0.54, K0.5 = 7.7 x 10(-6) M). The inhibitions were incomplete in the presence of 10(-3) M Ca ions. The Hofstee plot was convex upwards for amiloride and concave upwards for quinacrine. Amiloride increased the Kd, decreased the maximum specific binding and increased the k-1. Quinacrine increased the Kd without changing the maximum specific binding and increased the k-1. The effects of amiloride and quinacrine on k-1 were nonadditive. We conclude that amiloride and quinacrine bind to or close to the L-type Ca channel, and inhibit the specific binding of NIT by allosteric, complex interactions influenced by the free concentration of Ca++. The nonadditive allosteric effects suggest a shared mechanism of interaction for amiloride and quinacrine with the site(s) of NIT. Several mechanisms are discussed to explain how amiloride and quinacrine can produce such inhibition of NIT binding.