Paola Galbani

Postischemic Changes in Cardiac Sarcoplasmic Reticulum Ca2+ Channels : A Possible Mechanism of Ischemic Preconditioning

We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca2+ release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [3H]ryanodine binding sites averaged 372 +/- 18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290 +/- 15 fmol/mg, P < .01), was still significantly reduced after 120 minutes (298 +/- 17 fmol/mg, P < .05), and recovered after 240 minutes (341 +/- 21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The Kd for ryanodine (1.5 +/- 0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with 45Ca, and Ca(2+)-induced Ca2+ release was studied by the quick filtration technique. In both preparations, the rate constant of Ca(2+)-induced Ca2+ release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7 +/- 1.4 and 18.9 +/- 0.9 s-1 vs a control value of 25.4 +/- 1.7 s-1, P < .05 in both cases) and recovered after 240 minutes (23.0 +/- 1.9 s-1). The Ca2+ dependence of Ca(2+)-induced Ca2+ release was not affected by preconditioning ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of creatine phosphate administration on the rat heart adenylate pool

Creatine phosphate (CP) has been shown to possess some pharmacological properties. When added to cardioplegic solutions it improves their myocardial protection. Furthermore exogenous creatine phosphate shows an anti-arrhythmic effect in the experimental animal and appears to decrease lactate formation following haemorrhagic shock. These properties have been ascribed to the conservation of the tissue stores of ATP. Recently Down et al. have observed higher rat heart levels of ATP and creatine phosphate after the intravenous administration of creatine phosphate. Since it is difficult to find the conditions for an increase of the adenylate pool, it seems of interest to establish whether the ATP increase is due to a rise of the total adenylate pool or of the energy charge or of both. Similarly the higher creatine phosphate concentration may be ascribed to a variation of the CP/creatine ratio or to an increase in the creatine plus CP pool. In the present paper we report that the administration of creatine phosphate raises both the adenylate pool and the energy charge. An increase of creatine and the creatine phosphate pool was also observed.

Effect of gallopamil on cardiac sarcoplasmic reticulum

We investigated the effect of gallopamil on cardiac sarcoplasmic reticulum (SR) function. Heavy SR was prepared from bovine ventricular muscle. Oxalate-supported calcium uptake was stimulated by gallopamil at concentrations ranging from 10 to 300 nM, whereas higher concentrations were ineffective. Peak stimulation averaged 25–30% of control calcium uptake and was observed at free calcium concentrations ranging from I to 6 μM Calcium uptake is actually the difference between active calcium transport by SR calcium-adenosine triphosphatase (calcium-ATPase), and passive efflux through SR calcium-release channels. In the presence of 300 μM of ryanodine, a blocker of SR channels, calcium uptake increased by 43% under control conditions, but no further stimulation was produced by gallopamil. SR calcium-ATPase was not affected by gallopamil. Similar results were obtained when oxalate-supported calcium uptake was determined with use of unfractionated homogenate obtained from rat hearts. We conclude that gallopamil acts on SR calcium-release channels and reduces the probability of channel opening and/or channel conductivity. The dose-response curve is bell shaped, and the maximum effect, which corresponds to 65% of the maximum effect of ryanodine, is achieved at therapeutic concentrations. Such action might contribute to the beneficial effect of gallopamil in the treatment of myocardial ischemia.

Are dihydropyridine receptors downregulated in the ischemic myocardium

OBJECTIVE We investigated the effect of ischemia on cardiac dihydropyridine receptors, which correspond to L-type sarcolemmal calcium channels. METHODS Isolated working rat hearts were perfused aerobically for 10 min, and then subjected to 10-60 min of global ischemia. Control hearts were perfused aerobically for 30 min. [3H]PN 200-110 binding was measured in the unfractionated homogenate, in a crude membrane preparation and in a microsomal fraction. RESULTS In the homogenate obtained from control hearts, the Kd and Bmax averaged 0.23 +/- 0.05 nM and 84 +/- 4 fmol/mg protein, respectively, and ischemia did not produce any significant change in these variables. Similar results were obtained in the crude membrane preparation (Kd = 0.29 +/- 0.08 nM, Bmax = 113 +/- 7 fmol/mg, yield of binding sites = 98 +/- 6%, no significant change in these variables during ischemia). On the contrary, in the microsomal fraction, the Bmax for [3H]PN 200-110 decreased after ischemia (115 +/- 15 fmol/mg after 20 min of ischemia vs. 190 +/- 34 fmol/mg in the control condition, P < 0.05), without any change in the Kd. In this fraction, the yield for PN 200-110 binding sites was 4.7 +/- 0.6% in the control condition and 2.8 +/- 0.5% after ischemia (P < 0.05). The yield of other sarcolemmal markers such as [3H]quinuclidinyl benzylate and [3H]ouabain binding sites was not reduced in the microsomal fraction obtained ischemic hearts. CONCLUSIONS The total number of cardiac dihydropyridine binding sites was not downregulated during ischemia, although their distribution after tissue fractionation was slightly modified, possibly reflecting receptor redistribution between different subcellular pools.

Interaction between gallopamil and cardiac ryanodine receptors

1 In a sarcoplasmic reticulum fraction obtained from rat hearts, the analysis of equilibrium [3H]‐ryanodine binding showed high and low affinity sites (KD = 1.3 nm and 2.8 μm, Bmax = 2.2 pmol mg−1 and 27.8 pmol mg−1). The dissociation rate constant increased at 1 μm VS 4 nm [3H]‐ryanodine concentration, and micromolar ryanodine slowed the dissociation of nanomolar ryanodine. 2 The binding of 4 nm [3H]‐ryanodine was not affected by gallopamil, while the binding of 100 nm to 18 μm [3H]‐ryanodine was partly displaced. Data analysis suggested that gallopamil inhibited low affinity [3H]‐ryanodine binding, with IC50 in the micromolar range. 3 Gallopamil decreased the dissociation rate constant of 1 μm [3H]‐ryanodine. While gallopamil alone did not affect the dissociation of 4 nm [3H]‐ryanodine, gallopamil and micromolar ryanodine slowed it to a greater extent than micromolar ryanodine alone. 4 Our results are consistent with the hypothesis that the ryanodine receptor is a negatively cooperative oligomer, which undergoes a sequential alteration after ryanodine binding. Gallopamil has complex actions: it inhibits ryanodine binding to its low affinity site(s), and probably modulates the cooperativity of ryanodine binding and/or the transition to a receptor state characterized by slow ryanodine dissociation. These molecular actions could account for the previously reported effect of gallopamil on the sarcoplasmic reticulum calcium release channel.

Effect of Ischaemia on Cardiac Adenosine Binding Sites

Adenosine produces different effects on the heart. It markedly increases coronary blood flow and exerts negative inotropic, chronotropic and dromotropic effects on atrial myocardium. Adenosine does not appear to have any direct action on ventricular contractility but significantly reduces the positive inotropic effect of catecholamines on both atrial and ventricular myocardium.1