Karen E. Anderson

Suppression of Ventricular Arrhythmias During Ischemia-Reperfusion by Agents Inhibiting Ins(1,4,5)P3 Release

BACKGROUNDnReperfusion following myocardial ischemia causes a rapid and transient release of inositol (1,4,5)triphosphate [Ins(1,4,5)P3]. The aim of this study was to test whether this increased Ins(1,4,5)P3 release was important for the development of ventricular arrhythmias and whether agents that inhibit this signal transduction pathway, such as aminoglycoside antibiotics, suppress arrhythmias.nnnMETHODS AND RESULTSnIn perfused rat hearts, ventricular tachycardia (VT), ventricular fibrillation (VF), and accumulation of Ins(1,4,5)P3 were measured during early reperfusion. A number of different compounds, including neomycin, gentamicin, streptomycin, spermine, reserpine, and prazosin, were effective in inhibiting the reperfusion-induced Ins(1,4,5)P3 release and the onset of VT and VF in parallel. A strong correlation existed between Ins(1,4,5)P3 content, measured at 2 minutes of reperfusion, and the incidence of reperfusion VT and VF. In addition, intravenous gentamicin suppressed the onset of arrhythmias under ischemic and reperfusion conditions in vivo.nnnCONCLUSIONSnOur results are consistent with the view that Ins(1,4,5)P3 release plays a pivotal role in mediating arrhythmias during early reperfusion. Agents inhibiting Ins(1,4,5)P3 release are antiarrhythmic and may have potential use clinically.

Inositol Phosphate Release and Metabolism During Myocardial Ischemia and Reperfusion in Rat Heart

A detailed study of the effects of global myocardial ischemia and reperfusion on inositol phosphate release and metabolism has been undertaken by using isolated perfused rat hearts. Ischemia for longer than 5 minutes caused a cessation of inositol phosphate production, with inositol phosphates initially present accumulating as isomers of inositol monophosphate. This inhibition was independent of norepinephrine. In contrast, 2-minute reperfusion following 20-minute ischemia produced a rapid and transient release of inositol phosphates that was dependent on the release of norepinephrine and mediated by alpha 1-adrenergic receptors. By a number of criteria, this reperfusion response was different from the norepinephrine response in normoxic tissue. First, total release of inositol phosphates was greater (466 +/- 37 compared with 345 +/- 29 cpm/mg protein, P < .05). Second, inositol 1,4,5-trisphosphate was released with postischemic reperfusion (103 +/- 18 to 207 +/- 11 pmol/mg protein), whereas release was not detected in normoxic myocardium. In agreement with this, neomycin (0.5 and 5 mmol/L) inhibited inositol phosphate release only under reperfusion conditions. Third, the reperfusion response, unlike the response in nonischemic tissue, required extracellular Ca2+. Longer periods of reperfusion resulted in a return to a pattern of inositol phosphate release that was not different from that seen in normoxic tissue. The rapid and transient release of inositol 1,4,5-trisphosphate at 2-minute postischemic reperfusion provides an explanation for the enhanced role of alpha 1-adrenergic receptors under these conditions and suggests an important role for this compound in initiating reperfusion-induced pathological events.

Inositol Phosphate Release and Metabolism in Rat Left Atria

The phosphatidylinositol (PtdIns) turnover pathway in intact heart tissue differs from that in most cell types in that products of the inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase pathway are not detected in 3H-labeling studies. In contrast, Ins(1,4,5)P3 kinase products are detected in isolated neonatal cardiomyocytes. To understand the basis for the observed properties of the cardiac pathway, a detailed study of inositol phosphate (InsP) release has been undertaken by using isolated adult rat left atria. Addition of norepinephrine to 3H-labeled atria caused a slow increase in 3H-labeled Ins(1,4,5)P3 and a more rapid increase in 3H-labeled Ins(1,4)P2, its immediate dephosphorylation product. The mass of Ins(1,4,5)P3 was high in unstimulated atria (13.5 +/- 1.1 pmol/mg tissue, mean +/- SEM, n = 4) and did not change with stimulation. Measurements of the specific activities of Ins(1,4,5)P3 and PtdIns(4,5)P2 provided an estimate of the turnover rate of Ins(1,4,5)P3 that was 20- to 40-fold lower than the rate of accumulation of 3H label in InsP1 and InsP2. In agreement with this, specific activities of InsP1 and InsP2 were higher than the specific activity of InsP3 in both control and stimulated atria. Neomycin (5 mmol/L) did not inhibit the accumulation of 3H-labeled InsP1 and InsP2 in left atria, even though it reduced the accumulation of 3H label in Ins(1,4,5)P3, providing evidence that InsP1 and InsP2 do not derive primarily from Ins(1,4,5)P3. Stimulation with norepinephrine for 20 minutes resulted in a parallel decrease in 3H-labeled Ins(1,4,5)P3 and in Ins(1,4,5)P3 mass, demonstrating that atria do not contain two different pools of Ins(1,4,5)P3.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Dietary Fat Supplementation on Inositol Phosphate Release and Metabolism in Rat Left Atria

Eight weeks dietary supplementation with oils enriched in saturated fats, n-6 polyunsaturated fats and n-3 polyunsaturated fats resulted in a reduced inositol phosphate response in isolated rat left atria. Reductions in both basal activity and norepinephrine-stimulated activity were observed. Diets supplemented with n-3 polyunsaturated fats produced a greater decrease in the norepinephrine-stimulated release than the other dietary groups. In addition, supplementation with n-6 polyunsaturated fats resulted in higher levels of the Ca(2+)-releasing compound inositol(1,4,5)trisphosphate while addition of n-3 fats eliminated accumulation of inositol(1, 4)bisphosphate in response to norepinephrine. The reduction in inositol phosphate accumulation observed in all fat-supplemented groups demonstrates the need for caution in choosing relevant control groups in such dietary studies. The specific effects of n-6 and n-3 polyunsaturated fats on individual inositol phosphate isomers demonstrates subtle effects on inositol phosphate metabolism, the significance of which requires further investigation.

THE NOREPINEPHRINE-STIMULATED INOSITOL PHOSPHATE RESPONSE IN HUMAN ATRIA

Inositol phosphate release and metabolism were studied in right atrial appendages obtained from 18 patients undergoing coronary artery bypass surgery and/or mitral valve replacement. [3H]Inositol-labeled human atria contained inositol(1,4. 5)trisphosphate, inositol(1,4)bisphosphate and the 1- (or 3) and 4-isomers of inositol monophosphate. Addition of norepinephrine (100 mumol/l) activated the release of inositol phosphates, as indicated by increased [3H]inositol label in all of these inositol phosphates. However, the phosphorylation product of inositol (1.4.5)trisphosphate, inositol-(1,3,4,5)tetrakisphosphate, and its metabolic products were not detected, either in control or stimulated atria. Similar inositol phosphate profiles were observed in rat right atria. Furthermore, both human and rat atria contained high concentrations of inositol(1,4,5)trisphosphate, which were not observed to increase with norepinephrine stimulation. The inositol phosphate responses to norepinephrine in rat and human cardiac tissue appear to be similar, except for the generally lower activity observed in human tissue. Thus, the rat provides a suitable model for the study of cardiac phosphatidylinositol turnover.

REPERFUSION FOLLOWING MYOCARDIAL ISCHAEMIA ENHANCES INOSITOL PHOSPHATE RELEASE IN THE ISOLATED PERFUSED RAT HEART

1. Global myocariial ischaemia (MI) for periods greater tan 5 min caused an inhibition of phosphatidylinositol specific phospholipase C (PtdIns‐PLC) activity.

Inositol 1,4,5-trisphosphate receptor function in neonatal cardiomyocytes

We have previously reported that the metabolism of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) is altered when rat neonatal ventricular cardiomyocytes are isolated and cultured. In the current study we show that the mass content of Ins(1,4,5)P3 is lower in the isolated cells than in the intact tissue. However, the properties of the Ins(1,4,5)P3 receptors were not different in the two preparations and the isolated cells remained insensitive to Ins(1,4,5)P3 in terms of 45Ca2+ release. Thus, despite the altered pattern of metabolism of Ins(1,4,5)P3 in isolated neonatal cells, the properties of the receptors were similar to those reported in other myocardial preparations.

Inositol-1,4,5-trisphosphate [ins(1,4,5)P3] and ins(1,4,5)P3 receptor concentrations in heart tissues.

1. The isolation and culture of neonatal cardiomyocytes causes changes in the metabolism of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) from primarily dephosphorylation in the intact tissue to a combination of phosphorylation and dephosphorylation in the cultured cells (Woodcock et al. 1992).

Lyophilization can generate artifacts in chromatographic profiles of inositol phosphates.

Lyophilized extracts of [3H]inositol-labelled rat heart or renal tubule preparations were found to contain unidentified 3H-labelled compounds in addition to the inositol phosphates. The appearance of these labelled substances was caused by the presence in the extracts of compounds which bound [3H]inositol when lyophilized together with it. These studies demonstrate a previously undescribed source of [3H]inositol-labelled compounds which can complicate chromatographic profiles of inositol phosphates. These problems can be overcome either by not lyophilizing the samples or by lyophilizing in the presence of 0.3 M urea, which prevents the association with [3H]inositol and does not interfere with the chromatography.

STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER IN ADULT RAT LEFT ATRIA DOES NOT INVOLVE RELEASE OF INOSITOL (1,4,5) TRI SPHOSPHATE

1. The turnover rate of inositol (1,4,5) tris phosphate (Ins(1,4,5)P3) in noradrenaline‐stimulated adult rat left atria was calculated from changes in specific activity and was found to equal 110 ct/min per mg tissue. In contrast, the isomers of inositol mono‐ and bis phosphates accumulated at a rate of 508 ct/min per mg.