R. Mouton

Increased myocardial inositol trisphosphate levels during α1-adrenergic stimulation and reperfusion of ischaemic rat heart

Although stimulated [3H] inositol phosphate turnover has been demonstrated in isolated, perfused [3H] inositol prelabelled rat hearts, there is still no information regarding Ins (1,4,5)P3 levels in intact cardiac muscle. Using a D-myo-Ins(1,4,5)P3 assay system, Ins(1,4,5)P3 levels were determined in isolated perfused rats hearts during ischaemia, reperfusion and alpha 1-adrenergic stimulation via noradrenaline (3 x 10(-5) M). Control hearts contained +/- 674 pmols Ins(1,4,5)P3/g dry heart weight. Myocardial Ins(1,4,5)P3 levels were significantly decreased (+/- 389 pmols/g dry heart weight) after exposure to 20 mins of normothermic ischaemic cardiac arrest (NICA). Reperfusion produced a marked increase in Ins(1,4,5,)P3 levels (+/- 1,115 pmols/g dry heart weight) after only 30 s. Noradrenaline caused a 3-4 fold increase in tissue Ins(1,4,5)P3 levels within 30 s. After 20 mins stimulation with noradrenaline, the Ins(1,4,5)P3 levels were still significantly elevated. The rise in tissue Ins(1,4,5)P3 levels during reperfusion as well as during noradrenaline administration was counteracted by neomycin (0.5 x 10(-3) M), an inhibitor of phosphoinositidase specific phospholipase C. In both events neomycin restored the Ins(1,4,5)P3 levels to control values. For correlation of tissue Ins(1,4,5)P3 levels with mechanical events, noradrenaline (3 x 10(-5) M), in the presence of 10 mM LiCl, 10(-7) M propranolol and 10(-7) M atropine, was administered to isolated perfused rat hearts and the mechanical performance recorded over a period of 20 mins. Noradrenaline caused a significant increase in peak systolic pressure and work performance which was maintained for at least 10 mins, suggesting that the positive inotropic effects of noradrenaline may be provoked by Ins(1,4,5)P3. Furthermore, the finding that 20 min NICA followed by 30 s reperfusion causes an immediate significant increase in Ins(1,4,5)P3 content suggests a role for the phosphatidylinositol pathway in the intracellular Ca2+ overloading, characteristic of ischaemia-reperfusion.

Myocardial membrane cholesterol: Effects of ischaemia

Evidence has recently been presented that myocardial ischaemia is associated with a significant increased mitochondrial cholesterol content, suggesting a redistribution of cholesterol within the ischaemic cell (Rouslin et al. 1980, 1982). The aim of this study was therefore to determine the effects of different periods of ischaemia and reperfusion on the cholesterol content of myocardial mitochondria, sarcoplasmic reticulum and sarcolemma. Using the isolated perfused rat heart as experimental model, it was demonstrated that increasing periods of ischaemia (15-60 min) caused a progressive loss of cholesterol from the tissue as well as from the sarcolemma and sarcoplasmic reticulum, concomitant with a significant increase in mitochondrial cholesterol content. These compositional changes were associated with a marked increase in sarcolemmal and mitochondrial microviscosity, while that of the sarcoplasmic reticulum was reduced. To gain more insight into the mechanisms controlling intracellular cholesterol distribution, control and ischaemic hearts were perfused with either exogenous cholesterol or its precursor [U-14C]acetate as an indicator of endogenous cholesterol synthesis. Perfusion with exogenous cholesterol resulted in significant increases in the membrane cholesterol content of control hearts. However, hypoxic, low flow perfusion prevented cholesterol enrichment of the sarcolemmal and sarcoplasmic reticulum membranes, while the cholesterol content of the mitochondria was increased from 99.48 +/- 12.75 to 127.61 +/- 1.84 nmols/mg protein, indicating specific incorporation into this membrane system. Incorporation of [U-14C]acetate into cholesterol in the sarcoplasmic reticulum was increased by 120% in ischaemic conditions. However, a marked redistribution of newly synthesized cholesterol was observed within the ischaemic cell: under control conditions most of the labelled cholesterol was transferred to the sarcolemma and least to the mitochondria, while this distribution pattern was reversed in ischaemia. In view of the fact that exchange of cholesterol between membranes is affected by both phospholipid polar head-group composition and acyl chain length and saturation, it is suggested that prior ischaemia-induced membrane compositional changes might lead to intracellular cholesterol redistribution. Finally, to determine whether cholesterol loss affects sarcolemmal permeability, hearts enriched in sarcolemmal cholesterol were subjected to 15 or 30 min global ischaemia followed by reperfusion and the rate of enzyme release determined. However, enzyme release was similar in treated and untreated hearts, indicating that sarcolemmal cholesterol loss probably does not affect its permeability.

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [3H]inositol phospholipids and [3H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP3) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [3H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [3H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2), as well as in the cytosolic [3H]inositol phosphates, [3H]InsP2 and [3H]InsP3. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [3H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P2. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P2, remained unchanged. [3H]Insp, [3H]InsP2 and [3H]InsP3 also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca2+ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca2+ overload, characteristic of postischaemic reperfusion.

The effect of ischaemia-reperfusion on [3H]inositol phosphates and Ins(1,4,5)P3 levels in cardiac atria and ventricles - A comparative study

In this study incorporation of [3H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P3 levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [3H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2–3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[3H]Ins(1,4,5)P3 assay system, the Ins(1,4,5)P3 levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles.Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P3 levels as well as incorporation of [3H]inositol into ventricular InSP2 and InSP3. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P3 levels and also [3H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P3 levels as well as the incorporation of [3H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.

Signal transduction in myocardial ischaemia and reperfusion

Recent studies in the non-ischaemic myocardium indicated that drugs stimulating cAMP formation inhibit α1-mediated inositol phosphate generation, while α1-adrenergic stimulation lowered tissue CAMP levels, implicating cross-talk between α1,- and β-adrenergic signalling pathways in normal physiological conditions. Massive amounts of endogenous catecholamines, predominantly noradrenaline, are released during myocardial ischaemia and reperfusion, causing stimulation of both α1- and β-adrenergic receptors which, in turn, may contribute to intracellular Ca2+ overload and subsequent cell damage. Since no information is available regarding cross-talk in pathophysiological conditions, the aim of this study was to evaluate the interactions between α1- and β-adrenergic signalling pathways during different periods of ischaemia and reperfusion.Isolated rat hearts were perfused retrogradely for 30 min before being subjected to (i) 5–25 min global ischaemia and (ii) 1–5 min of reperfusion after 20 min global ischaemia. Drugs (prazosin, 10−7 M; propranolol, 10−6 M; phenylephrine 3 × 10−5 M; isoproterenol 10−9 M) were added 10 min before the onset of ischaemia and were present during reperfusion.Increasing periods of ischaemia caused an immediate rise and progressive lowering in tissue cAMP and Ins(1,4,5)P3 levels respectively. In contrast, reperfusion caused an elevation in Ins(1,4,5)P3 levels and reduced cAMP. Prazosin elevated cAMP levels during both ischaemia and reperfusion, while propranolol had no effects on tissue Ins(1,4,5)P3−. The activity of the α1-adrenergic signal transduction pathway appears to have an inhibitory effect on the activity of the β-adrenergic system during ischaemia and reperfusion.

Defective stretch-induced release of atrial natriuretic peptide from aging hypertensive rat heart : possible role of phosphatidylinositol pathway

Because the phosphatidylinositol pathway may be part of the signaling system associated with stretch-induced release of atrial natriuretic peptide (ANP), we tested the hypothesis that formation of the intermediate inositol-1,4,5-trisphosphate (IP3) is impaired when ANP release is decreased in response to atrial stretch in hearts from aging genetically hypertensive (GH) rats. Immunoreactive ANP release into the coronary effluent and IP3 levels were studied in cardiac tissues of isolated perfused hearts from normotensive control (WAG) or GH rats aged 4,11, or 16 months. Left atria were repeatedly distended and released with a latex balloon. ANP was measured in coronary effluent, and IP3 was measured in cardiac tissues. In all age groups, stretch and relief of stretch evoked considerably less ANP release in spontaneously beating hearts from GH than from WAG rats. Hearts from GH rats aged 16 months released no ANP, but electrical pacing restored some stretch-induced ANP secretion. With repeated stretch and release of stretch of the left atrium for 2 min, IP3 levels increased in left atrial tissue in WAG but not in GH hearts of all age groups. IP3 levels in (unstretched) left ventricles were much lower than in left atria and were unaltered by atrial stretch. In aging GH rats, the capacity to release ANP on atrial stretch is largely lost, in association with complete suppression of stimulus-induced increase in IP3 levels. These data support a role for IP3 in stretch-mediated atrial ANP secretion and suggest a progressive uncoupling of this signaling pathway in aging hypertensive rats.

Massive atrial natriuretic peptide (ANP) release in ischemia reperfusion

Recent studies reported the stimulatory effects of Ca 2+ channel and al receptor agonists [1,2] on ANP release by atrial tissue. Therefore the increased cytosolic Ca z÷ [3] and release of endogenous catecholamines [4] occurring during ischemia-reperfusion may be associated with ANP release. Hypoxia has previously been shown to be a potent stimulus for ANP release by the isolated perfused heart [5,6], as well as in conscious animals [7] and humans [8]. In view of the marked differences in coronary flow rates in ischemia and hypoxia, and the role of coronary hemodynamics in ANP secretion [9], ANP release during reperfusion was monitored in this study after exposure of the isolated rat heart to either 15 or 25 minutes normothermic ischemic arrest (representing reversible and irreversible ischemic injury, respectively [10]). ANP released during ischemia alone was evaluated by determining the amounts released into the recirculating buffer of hearts subjected to 35 minutes normothermic cardioplegic arrest (a condition causing overt ischemic damage). To eliminate the possible contribution of a high K+-cardioplegic solution to ANP release, myocardial ANP release was also monitored during exposure to 90 minutes of hypothermic arrest, a procedure that has been shown to offer complete protection against ischemic damage [11]. Male Wistar rats (weight +200 g), fed ad libitum until experimentation were used in all experiments. The hearts were perfused retrogradely for 20 minutes, during which time the pulmonary vein was cannulated. Atrial stretch was then induced by perfusion in the working mode for 10 minutes (preload 15 cm H20; afterload 100 cm H20). The perfusion conditions and buffer, as well as induction of total global ischemia, have been described previously [10]. The hearts were reperfused in the retrograde mode for 10 minutes, followed by 5 minutes perfusion in the working mode. Normothermic (37°C) as well as hypothermic (20°C) cardioplegic arrests were induced by using 20 ml Plasmaiyte B (Sabax, South Africa) to which CaC12 (1.25 mM) and KC1 (25 mM) were added (final composition in mM: Na ÷ 130; K ÷ , 29; C1109; bicarbonate 28; Ca 2+ 1.25). The cardioplegic solution was gassed with 95% 02/5% CO2 and administered for 1 minute at 10-minute intervals at a pressure of 40 mmHg during the arrest period. Reperfusion of these hearts was done as described above. At least six hearts were studied in each group. Samples for measurement of perfusate ANP were collected over a period of 1 minute at the following time intervals during the control perfusion period: 10 and 20 minutes retrograde perfusion, 5 and 10 minutes working heart perfusion. During reperfusion after normothermic or hypothermic arrest, samples were collected at 1, 2, and 5 minutes of retrograde perfusion and at 1, 2, and 5 minutes of working heart perfusion. In the case of the cardioplegic hearts, the recirculating cardioplegic fluid was collected at the end of the cardioplegic arrest. Samples were kept frozen at -70°C until assayed. At the end of the experimental period, the hearts were blotted dry, dried in an oven for 24 hours, and weighed. ANP release was expressed as fentomol/g dry weight/min. Perfusate ANP was analyzed using Amershams ~ANP [12~I] radioreceptor assay system. This system utilizes a high specific activity (3 [125I] iodotyrasy128) aANP tracer, together with a specific and sensitive ANP binding receptor preparation from bovine adrenal gland. Crossreactivity with human and rat a-ANP is 100% and 73%, respectively. The assay involves a 90-minute room temperature incubation and measures aANP in the range 1-128 fentomol/tube. The ED50 and the intraassay coefficient of variation averaged 25-29 fmol and 6%, respectively. Nonspecific binding was 4%. The standard curve and unknowns were evaluated by a RIA-calc computer program. In view of the fact that similar values were obtained in the amounts of ANP released during control perfusion, the values of the four series of experiments were pooled (Table 1). Atrial stretch, induced by switching from retrograde to working heart perfusion, caused an 88-100% increase (p < 0.001) in ANP release. The severity of ischemic damage markedly affected the pattern and amounts of ANP released: (a) Reperfusion in the retrograde mode after induction of 15 minutes ischemia (reversible damage) caused a massive ANP release within 2 minutes (62059 -+ 14037 and 49466 + 8604 fmoles/g/min). Although the release tended to decline after 5 minutes of retrograde reperfusion, the amounts of ANP released were still significantly higher (25384 fmoles/g/min) than that observed during atrial stretch in the control period. ANP release during reperfusion in the working mode after 15 minutes of ischemia was similar to that occurring during the corresponding control period, indicating that the washout of the ischemia-induced ANP release was probably completed. (b) Irreversible damage (25 minutes ischemia) was associated with an initial lesser degree of ANP release, which might be due to lower coronary flow rates during retrograde perfusion (mean coronary flow rates during first 2 minutes of retrograde perfusion after 15 and 25 minutes of ischemia were 16 -+ 1 and 11 -+ 1 ml/min, respectively). However, ANP release remained high throughout the reperfusion period and was not further affected by atrial stretch. (c) The magnitude of ANP released during ischemia is reflected by the massive amounts of ANP discharged into the cardioplegic fluid. Although high concentrations of KCI have been shown to induce ANP release in rat hypothalamus [12], it was unable to do so in Langendorff-

EFFECTS OF ISCHAEMIA, REPERFUSION AND ALPHA 1-ADRENERGIC RECEPTOR STIMULATION ON THE INOSITOLTRISPHOSPHATE RECEPTOR POPULATION IN RAT HEART ATRIA AND VENTRICLES

Binding sites specific for inositol 1,4,5-trisphosphate (InsP3) have been demonstrated in sarcoplasmic reticulum vesicles isolated from heart muscle. Scatchard analysis of a binding isotherm indicated a high as well as a low affinity binding site [1]. In this study a comparison was made between InsP3 binding to crude microsomal membranes prepared from rat heart atria and ventricles respectively. Results obtained showed a four-fold higher incidence of binding to atrial membranes. Furthermore, the receptor populations of the atria and ventricles behaved differently during conditions causing fluctuations in tissue InsP3 levels, viz. ischaemia, reperfusion and α1-adrenergic stimulation. Reperfusion, as well as phenylephrine stimulation, caused an increase in InsP3 levels associated with down-regulation of the ventricular InsP3 receptor population while binding to atrial binding sites was elevated. In the ventricular population this down-regulation was the result of a reduction in Bmax alone with no changes in the Kd values of the high- or the low-affinity binding sites. The reason(s) for the differential response of the atrial and ventricular InsP3 receptor populations to changes in InsP3 levels, remains to be established.