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(Circulation Research. 1995;76:261-268.)
© 1995 American Heart Association, Inc.

Articles

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

Karen E. Anderson, Anthony M. Dart, Elizabeth A. Woodcock

From the Cellular Biochemistry Laboratory (K.E.A., E.A.W.) and the Alfred Baker Medical Unit (A.M.D.), Baker Medical Research Institute, Prahran, Australia.

Correspondence to Dr Elizabeth Woodcock, Baker Medical Research Institute, Commercial Rd, Prahran 3181, Australia.

	Abstract

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Abstract 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 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 ₁-adrenergic receptors under these conditions and suggests an important role for this compound in initiating reperfusion-induced pathological events.

Key Words: inositol 1,4,5-trisphosphate • rat hearts • myocardial ischemia • reperfusion

	Introduction

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Extrinsic control of myocardial function involves primarily the autonomic nervous system, the stimulatory sympathetic arm of which is mediated largely by ß-adrenergic receptors and related to changes in cAMP. Heart tissue also contains ₁-adrenergic receptors, which can initiate inotropic reponses and release of atrial natriuretic factor, but they do not appear to contribute substantially to sympathetic responses under physiological conditions. However, ₁-adrenergic receptors appear to play a more important role under pathological conditions, such as myocardial ischemia and reperfusion, where extensive norepinephrine release has been documented.¹ ₁-Adrenergic receptor stimulation is capable of inducing ventricular arrhythmias during both ischemia and reperfusion,² a response not observed in nonischemic tissue. Myocardial ischemia has been reported to double ₁-adrenergic receptor density in the rat myocardium,³ adult rat myocytes,⁴ and other myocardial preparations and to increase sensitivity to norepinephrine stimulation,^{3 4} both of which may contribute to arrhythmogenesis.^{5 6} Reperfusion of ischemically damaged myocardium is associated with large Ca²⁺ accumulations, which are inhibited by ₁-adrenergic receptor blockade.⁷ Furthermore, ₁-adrenergic receptor blockade has been shown to be antiarrhythmic during both early ischemia and postischemic reperfusion,^{8 9} but not under nonischemic conditions. These and other studies suggest a link between ₁-adrenergic receptor stimulation and arrhythmias produced by myocardial ischemia and reperfusion.

In heart, as in other tissues, ₁-adrenergic receptors are coupled to the phosphatidylinositol (PtdIns) turnover pathway, and it is likely that components of this pathway are responsible for some of the effects of ₁-adrenergic receptor stimulation in the myocardium. The PtdIns turnover pathway is a complex signal transduction system that mediates a diverse range of neurotransmitter- and hormone-induced responses in a wide variety of cells.¹⁰ In most cell types, the pathway involves the receptor-mediated hydrolysis of membrane phospholipid PtdIns(4,5)P₂ by a specific phospholipase C (PtdIns-PLC) to generate two well-described second messengers sn-1,2-diacylglyerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. DAG activates various isoforms of protein kinase C (PKC) within the plasma membrane, and Ins(1,4,5)P₃ releases Ca²⁺ from specific intracellular stores. These effects, separately or in concert, control a wide range of cellular responses, including contraction, secretion, and mitogenesis.¹¹ Ins(1,4,5)P₃ is metabolized rapidly within the cell, both by dephosphorylation to inositol 1,4-bisphosphate [Ins(1,4)P₂] and inositol 4-monophosphate [Ins(4)P₁] and by phosphorylation to Ins(1,3,4,5)P_4. The latter compound is further metabolized to produce a wide range of inositol phosphate (InsP) isomers.¹²

The PtdIns pathway in the heart is activated by ₁-adrenergic and muscarinic receptor stimulation,¹³ endothelin,¹⁴ and stretch.¹⁵ However, a number of characteristics of the heart PtdIns pathway differ from those in other cells. First, adult heart tissue contains predominately Ca²⁺-independent isoforms of PKC.¹⁶ Second, the heart is relatively insensitive to Ins(1,4,5)P₃ in terms of Ca²⁺ release,^{17 18 19} with the addition of high concentrations of Ins(1,4,5)P₃ causing a slow leakage of Ca²⁺¹⁷ rather than the rapid release seen in most cells.¹¹ In addition, the Ca²⁺ released by Ins(1,4,5)P₃ does not enhance Ca²⁺-induced Ca²⁺ release,¹⁹ the mechanism of excitation-contraction coupling, but enhances Ca²⁺ oscillations, and such oscillations can be associated with arrhythmogenesis.^{19 20} Third, we have previously reported an apparent absence of labeled phosphorylation products of Ins(1,4,5)P₃, indicating that metabolism occurs primarily by dephosphorylation, such that only low levels of Ins(1,3,4,5)P₄ and its metabolic products are observed.²¹ In the accompanying article in this issue of Circulation Research,²² these observations have been expanded, and a model of cardiac PtdIns turnover has been proposed whereby Ins(1,4)P₂ rather than Ins(1,4,5)P₃ is the major InsP released in myocardial tissue under ₁-adrenergic receptor stimulation. Properties of the pathway in atria and ventricle were similar, as demonstrated by metabolites generated, specific activities, and effects of inhibitors.

The unusual properties of the cardiac PtdIns turnover pathway may relate in some way to the relatively minor role of myocardial ₁-adrenergic receptors under physiological conditions. Furthermore, the enhanced activity of ₁-adrenergic receptors under conditions of myocardial ischemia and reperfusion might relate to some functional change in this signal transduction pathway. On the basis of these considerations, the effect of myocardial ischemia and reperfusion on the release and metabolism of InsPs has been examined by using isolated perfused rat hearts.

	Materials and Methods

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[³H]Inositol Labeling of Isolated Perfused Rat Hearts
Adult male Sprague-Dawley rats (250 to 300 g) were used. Where indicated, rats were treated with reserpine (5 mg/kg IP, 18 hours) to deplete endogenous norepinephrine. Rats were heparinized (1 U/g body wt IP) 30 minutes before killing by decapitation. Hearts were rapidly excised and immediately arrested in ice-cold saline. Under ice-cold solution, hearts were cannulated via the ascending aorta as described by Langendorff and perfused retrogradely at a rate of 5 mL/min with HEPES-buffered Krebs' solution (37°C) equilibrated with 5% CO₂/95%O₂. Initial perfusion was in a nonrecirculating manner to remove blood. Hearts were allowed to gradually warm up, and when free of blood, they were transferred to a 37°C water-jacketed organ bath containing carbogenated Krebs' medium. Perfusion was continued in a recirculating manner. After a 15-minute equilibration period, hearts were labeled with [³H]inositol (2 µCi/mL) for 2 hours. Krebs' medium containing [³H]inositol was then removed and replaced with medium containing propranolol (1 µmol/L) and LiCl (50 mmol/L) to block ß-adrenergic receptors and to inhibit InsP metabolism, respectively.¹² In initial experiments, 10 mmol/L LiCl did not fully inhibit InsP₁ breakdown; therefore, 50 mmol/L LiCl was used in subsequent studies. NaCl concentrations in the medium were lowered accordingly. Hearts were perfused with medium containing propranolol and LiCl for 10 minutes. Antagonists, when administered, were included in this 10-minute period. Normothermic global ischemia was produced by terminating perfusion for various periods up to 30 minutes. Reperfusion was achieved by resuming perfusion at 5 mL/min. At the indicated time points, ventricles were rapidly frozen in liquid N₂ after excision at the atrioventricular junction. Frozen ventricles were weighed, and InsPs was extracted as described below.

Extraction and Quantification of InsPs
InsPs were extracted from frozen ventricles as described for atria in the accompanying article,²² except that the extraction volume was 3.5 mL and the initial trichloroacetic acid (TCA) pellets were reextracted with 1.5 mL TCA. The final aqueous phase was collected and treated with proteinase K (50 µg/mL, 2 hours, 50°C) to prevent deterioration of the high-performance liquid chromatography (HPLC) column. Samples were then passed through a 1-mL Dowex-50 column (4% cross-linked; mesh size, 4 to 400) and eluted with 1 mL water.²³ Urea (final concentration, 0.05 mol/L) was added,²⁴ and samples were lyophilized before HPLC analysis. InsP responses measured in whole ventricular tissue were derived from cardiomyocytes, because activators specific for stimulation of other cell types present in the myocardium were ineffective.²⁵

Other Assays
Norepinephrine concentrations in the coronary effluent and heart perfusate at various times of ischemia and reperfusion were determined by HPLC analysis, as described previously.^{26 27}

Protein concentration was determined in aliquots of TCA pellet after InsP extraction by a modified Lowry method²⁸ with bovine serum albumin used as a standard.

Statistics
Values presented are mean±SEM (n=4) unless otherwise stated. Statistical analysis of results involved either Student's unpaired t test for single comparisons or one-way ANOVA for multiple comparisons. Significance by either method was determined at P<.05.

Materials
[³H]Inositol, [³H]Ins(1,4,5)P₃, assay kit for measurement of Ins(1,4,5)P₃ mass, and unlabeled Ins(1,4,5)P₃ were obtained from the Radiochemical Centre Amersham. All ³H-labeled InsPs were checked for purity by HPLC before use. All other chemicals were analytical reagent grade, and reagents were dissolved in Milli Q water.

	Results

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Norepinephrine Overflow During Ischemia and Reperfusion
It is well established that endogenous norepinephrine accumulates in myocardial extracellular space under ischemic conditions.¹ Norepinephrine was measured in the heart perfusate to establish levels achieved under the conditions used for ischemia and reperfusion in the present experiments. The accumulation of norepinephrine in the extracellular space over the 20-minute ischemic period was estimated by measuring the initial washout of norepinephrine with reperfusion and assuming that extracellular space was 20% (vol/wt) of heart wet weight²⁹ and that the initial washout reflected norepinephrine accumulated in extracellular space.³⁰ This provided a value of 0.8±0.3 µmol/L, which agreed well with previous reports.^{30 31} Norepinephrine was rapidly released into the perfusate with reperfusion, elevating perfusate levels from 1.0±0.3 before ischemia to 15.4±8 nmol/L after 2-minute reperfusion. These levels dropped significantly after 20-minute reperfusion to a concentration not different from values before ischemia (1.1±0.5 nmol/L, P>.3).

Effects of Ischemia on Accumulation of ³H-Labeled InsPs
TCA extracts of [³H]inositol-labeled hearts contained compounds identified as Ins(1/3)P₁, Ins(4)P₁, Ins(1,4)P₂, and Ins(1,4,5)P₃. As reported previously,²¹ [³H]Ins(1,3,4,5)P₄ and its metabolic products were not evident (Fig 1). Thirty-minute perfusion with oxygenated medium, in the presence of LiCl, resulted in increased accumulation of ³H-labeled Ins(1/3)P₁, Ins(4)P₁, and Ins(1,4)P₂ isomers. Total accumulation of ³H-labeled InsPs increased from 144±7 to 477±57 cpm/mg protein (P<.001), indicating slow turnover of the pathway in unstimulated myocardium under normoxic conditions. In contrast, myocardial ischemia caused increases in Ins(1/3)P₁ and Ins(4)P₁ accumulation and concurrent decreases in Ins(1,4)P₂ and Ins(1,4,5)P₃ (Fig 1, lower panel). Such changes were observed 10 minutes after the initiation of ischemia and increased thereafter (Fig 2). The decrease in ³H-labeled Ins(1,4)P₂ preceded that in Ins(1,4,5)P₃. This is consistent with most of the Ins(1,4)P₂ not being derived from Ins(1,4,5)P₃, as proposed in the accompanying article.²²

View larger version (24K): [in this window] [in a new window]   Figure 1. Anion-exchange high-performance liquid chromatographic profiles of 3H-labeled inositol phosphates (InsPs) in isolated perfused rat hearts before and after 30-minute global myocardial ischemia. Hearts were labeled with [3H]inositol and subsequently made ischemic by terminating perfusion. InsPs were extracted as described in "Materials and Methods." Shown are representative experiments. Average values from four experiments are shown in Fig 2.

View larger version (13K): [in this window] [in a new window]   Figure 2. Graphs showing the effect of global myocardial ischemia on inositol phosphates (InsPs) in isolated perfused rat hearts. Shown are the time courses of accumulation of 3H-labeled InsPs ( or ) and Ins(1,4,5)P3 mass () with global myocardial ischemia or 30-minute normoxic perfusion ( or ). Analyses of labeled InsPs were performed as described in Fig 1. Values shown are mean±SEM of four separate determinations. Statistical analyses were performed by one-way ANOVA (*P<.05 vs nonischemic zero-time controls). Closed symbols represent Ins(4)P1, Ins(1,4)P2, and Ins(1,4,5)P3. Open squares and circles represent Ins(1/3)P1.

There was no change in total [³H]InsP content over the 30-minute ischemic period (138±8 cpm/mg protein at 30-minute ischemia, P>.10) relative to hearts before ischemia. The finding that total [³H]InsP accumulation was unchanged with myocardial ischemia supports the concept that the primary effect of ischemia is a cessation of release of InsPs, most likely reflecting a functional inhibition of PtdIns-PLC, either directly or indirectly. InsPs initially present at the onset of ischemia are degraded to InsP₁, with further breakdown being inhibited by 50 mmol/L LiCl.

Ins(1,4,5)P₃ mass levels were measured in experiments parallel with those described above. Nonischemic myocardium contained a high concentration of Ins(1,4,5)P₃ (Table 1 and Fig 2). As shown in Fig 2, the decrease in mass of Ins(1,4,5)P₃ closely paralleled the decrease in ³H-labeled Ins(1,4,5)P₃, indicating that Ins(1,4,5)P₃ in ventricle, as in left atria,²² is of uniform-specific activity.

View this table: [in this window] [in a new window]   Table 1. Changes in Mass of Inositol 1,4,5-Trisphosphate in Isolated Perfused Rat Hearts After Norepinephrine Stimulation, Ischemia, and Postischemic Reperfusion in the Presence and Absence of Neomycin

The effect of ischemia on InsP distribution was unaffected by depletion of endogenous norepinephrine stores by reserpinization, showing it to be independent of norepinephrine release.

Effects of Postischemic Reperfusion on Release of ³H-Labeled InsPs
Reperfusion following 20-minute global myocardial ischemia resulted in a rapid release of [³H]InsPs between 1 and 2 minutes of reperfusion, followed by a slow, quantitatively smaller, secondary accumulation, which continued up to the 20-minute reperfusion period studied (Fig 3). Two-minute postischemic reperfusion, the time point that gave maximal InsP release, was used in subsequent studies.

View larger version (15K): [in this window] [in a new window]   Figure 3. Graphs showing 3H-labeled inositol phosphates (InsPs) in isolated perfused rat hearts subjected to global ischemia () and subsequent reperfusion following 20-minute ischemia (). Analyses were performed as described in Fig 1. [3H]Inositol-labeled hearts were subjected to myocardial ischemia or 20-minute ischemia followed by reperfusion as described in "Materials and Methods." Values shown are mean±SEM of four separate determinations. Statistical analyses were performed by one-way ANOVA (*P<.05 vs 20-minute ischemia).

Two-minute reperfusion following 5-minute ischemia did not significantly alter the profile of InsPs from that observed before reperfusion. Total [³H]InsP accumulation increased with 2-minute reperfusion following 10-minute ischemia, from 156±7 to 232±18 cpm/mg protein (P<.01), but did not cause detectable increases in Ins(1,4,5)P₃ in either mass or ³H-labeled studies. As mentioned above, 2-minute reperfusion following 20-minute myocardial ischemia resulted in rapid increases in InsP release, with total ³H-labeled InsPs increasing from 144±5 to 466±37 cpm/mg protein (P<.001), with Ins(1,4,5)P₃ rising from 19±3 to 52±4 cpm/mg protein (P<.0025). The InsP response after 2-minute reperfusion following 30-minute ischemia was similar to that observed after 20-minute ischemia but quantitatively smaller. Total ³H-labeled InsPs increased from 138±8 to 284±30 cpm/mg protein (P<.005), whereas Ins(1,4,5)P₃ increased from 10±1 to 31±4 cpm/mg protein (P<.025). Thus, the InsP response with 2-minute reperfusion was quantitatively greatest after 20-minute ischemia. Subsequent reperfusion studies were performed after this ischemic period. The transient nature of the early reperfusion response most likely explains why it was not observed in previous studies.³²

Two-Minute Postischemic Reperfusion
³H-Labeled InsPs
Two-minute reperfusion following 20-minute myocardial ischemia was characterized by a large release of ³H-labeled InsPs (Fig 3 and Table 2). When similar experiments were performed in hearts from reserpinized animals, InsP profiles after 2-minute reperfusion were not different from those at 20-minute ischemia (Table 2). Addition of a maximally effective concentration of norepinephrine (100 µmol/L) to the perfusate during reperfusion of norepinephrine-depleted hearts restored the reperfusion-induced InsP response (Table 2). The response observed with exogenous norepinephrine during reperfusion in hearts from reserpinized animals was not different from that of 2-minute reperfusion in untreated animals. Therefore, the norepinephrine released under these conditions of ischemia and reperfusion was sufficient to maximally activate InsP release. Perfusion with the ₁-adrenergic receptor antagonist prazosin (10 µmol/L) throughout the ischemia/reperfusion protocol inhibited the rise in InsPs over 2-minute postischemic reperfusion (Table 2). Thus, the 2-minute reperfusion response depended on the release of endogenous norepinephrine and was mediated via ₁-adrenergic receptors.

View this table: [in this window] [in a new window]   Table 2. Accumulation of Inositol Phosphate Isomers in Isolated Perfused Rat Hearts Stimulated With Norepinephrine or Subjected to Ischemia or Ischemia Followed by Reperfusion

Perfusion of nonischemic hearts with norepinephrine (100 µmol/L) caused release of InsPs, but the increase was smaller than observed during 2-minute reperfusion. The total increase in InsP accumulation with 2-minute reperfusion was 466±37 cpm/mg protein, compared with 345±29 cpm/mg protein after 2-minute norepinephrine stimulation of nonischemic myocardium (P<.05). This is in contrast to the response to added norepinephrine under reperfusion conditions, which was quantitatively similar to that observed with 2-minute reperfusion (see above). This shows that the heart does not differentiate between endogenous release of norepinephrine and norepinephrine in the perfusate. Rather, the heart responds to norepinephrine differently under reperfusion conditions. The profile of InsP accumulation was qualitatively different between 2-minute norepinephrine stimulation of nonischemic myocardium and 2-minute postischemic reperfusion. Norepinephrine stimulation of nonischemic hearts resulted in increased accumulation of ³H-labeled Ins(1,4)P₂ and Ins(4)P₁. Rises in ³H-labeled Ins(1,4)P₂ and Ins(4)P₁ were seen with 2-minute reperfusion. However, in addition, increased accumulation of Ins(1/3)P₁ and the second-messenger Ins(1,4,5)P₃ was also observed (Table 2).

Mass of Ins(1,4,5)P₃
In parallel with ³H-labeled studies, 2-minute reperfusion following 20-minute ischemia produced a rapid increase of Ins(1,4,5)P₃ mass. In contrast, 2-minute stimulation of nonischemic hearts with norepinephrine (100 µmol/L) did not increase the mass of Ins(1,4,5)P₃ (Table 1).

Effect of Neomycin
The aminoglycoside antibiotic neomycin has been well characterized as an inhibitor of Ins(1,4,5)P₃ release.^{33 34} The effects of neomycin on the 2-minute reperfusion–induced InsP response were investigated. Neomycin (0.5 and 5 mmol/L), added 10 minutes before the initiation of ischemia and maintained throughout the procedure, inhibited the release of InsPs during the 2-minute reperfusion period. ³H label in all InsP isomers was decreased similarly (Fig 4). Lower concentrations of neomycin (0.05 mmol/L) did not significantly inhibit InsP release. Neomycin (5 mmol/L) inhibited the rise in mass in Ins(1,4,5)P₃ with 2-minute reperfusion, similar to the inhibition of the rise in counts (Table 2). Neomycin at 5 mmol/L had no inhibitory effect on the accumulation of any ³H-labeled InsP with 2-minute norepinephrine stimulation of nonischemic myocardium.

View larger version (21K): [in this window] [in a new window]   Figure 4. Bar graphs showing the effect of neomycin on the reperfusion-induced inositol phosphate (InsPs) response. Hearts were subjected to 20-minute myocardial ischemia followed by 2-minute reperfusion in the absence or presence of neomycin. Neomycin (hatched bars, 0.05 mmol/L; crosshatched bars, 0.5 mmol/L; and vertically striped bars, 5 mmol/L) was added to the perfusate 10 minutes before ischemia and maintained throughout the ischemic and reperfusion procedure. Values shown are percentages of the accumulation in the absence of neomycin (open bar). Values shown are mean±SEM of four separate determinations. Analyses were performed by one-way ANOVA (*P<.05).

Ca²⁺ Dependence
Perfusion with Ca²⁺-free Krebs' medium significantly inhibited the 2-minute reperfusion–induced release of ³H-labeled InsPs and abolished the rise in Ins(1,4,5)P₃ (Fig 5). In contrast, the InsP accumulation with 2-minute norepinephrine stimulation of nonischemic hearts was not significantly inhibited in Ca²⁺-free medium (Fig 5). Thus, the 2-minute reperfusion response differs from the response in normoxic tissue in its requirement for extracellular Ca²⁺.

View larger version (22K): [in this window] [in a new window]   Figure 5. Bar graphs showing the effect of Ca2+ on the inositol phosphate (InsP) response to 2-minute reperfusion. A, 3H-labeled hearts were subjected to 20-minute myocardial ischemia followed by 2-minute reperfusion in oxygenated Krebs' medium either Ca2+ free (hatched bars) or containing 2 mmol/L CaCl (open bars). B, The effect of Ca2+-free medium on the response to 2-minute norepinephrine stimulation of normoxic hearts is shown for comparison. Analyses were performed as shown in Fig 1. Values shown are mean±SEM of four separate determinations (*P<.001).

InsP Release Between 5- and 20-Minute Postischemic Reperfusion
The initial transient 2-minute reperfusion response was followed by a smaller secondary accumulation, which continued to the 20-minute reperfusion period studied (Fig 4). This secondary phase, from 5- to 20-minute reperfusion, exhibited significant increases in accumulation of ³H-labeled Ins(4)P₁ and Ins(1,4)P₂ but no further increase in Ins(1,4,5)P₃. Ins(1,4,5)P₃ mass also did not increase (Table 1). This pattern of response is similar to that observed with 20-minute perfusion of nonischemic myocardium in the absence of added agonist. In contrast to the 2-minute reperfusion response, InsP accumulation after 20-minute reperfusion was unaffected either by reserpinization or by prazosin. Thus, this secondary accumulation, unlike the 2-minute response, does not depend on endogenous norepinephrine or ₁-adrenergic receptors. Addition of exogenous norepinephrine during 20-minute reperfusion of hearts from reserpinized rats resulted in total InsP accumulation greater than with 20-minute reperfusion alone (856±76 cpm/mg protein compared with 372±15 cpm/mg protein, P<.005). Similar responses to norepinephrine were obtained by using hearts from nonreserpinized animals.

Neomycin (5 mmol/L) had no significant inhibitory effect on the accumulation of any of the ³H-labeled InsPs between 5- and 20-minute postischemic reperfusion. Increases in InsPs observed with addition of exogenous norepinephrine during 20-minute reperfusion were also insensitive to 5 mmol/L neomycin. Furthermore, the accumulation of InsPs between 5- and 20-minute reperfusion was not dependent on extracellular Ca²⁺ (data not shown). Thus, the release of InsPs between 5- and 20-minute postischemic reperfusion is different from the immediate release observed at 2 minutes and is indistinguishable from responses in normoxic tissue by any criteria investigated.

	Discussion

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The data presented here show that myocardial ischemia and subsequent reperfusion have complex effects on InsP release and metabolism in the isolated perfused rat heart. Release of InsPs was inhibited under ischemic conditions, but in contrast, postischemic reperfusion caused a rapid transient release of Ins(1,4,5)P₃. This reperfusion response was dependent on release of endogenous norepinephrine and was mediated via ₁-adrenergic receptors but differed from the ₁-adrenergic receptor–mediated norepinephrine response of normoxic myocardium.

Ventricular tissue slowly accumulates InsPs under normoxic conditions when perfused in the presence of LiCl to inhibit dephosphorylation of InsP₁, indicating some basal activity of PtdIns-PLC. Stimulation with norepinephrine increases InsP accumulation, but the pattern of release of the InsPs and their subsequent metabolism appears to be similar under both basal and stimulated conditions. Accumulation of ³H-labeled InsPs was restricted to Ins(1,4)P₂ and the isomers of InsP₁. No change in Ins(1,4,5)P₃ was detected at any time point investigated either in control or norepinephrine-stimulated ventricles.

In contrast to normoxic myocardium, no net increase in InsPs was observed under ischemic conditions. Instead, ischemia for periods >5 minutes caused a redistribution of ³H-labeled InsPs, while overall content remained unchanged. Decreases in Ins(1,4)P₂ and Ins(1,4,5)P₃ were observed, together with concurrent increases in their metabolic products Ins(1/3)P₁ and Ins(4)P₁. These findings are most readily explained by an inhibition of InsP production under ischemic conditions, with InsPs present in the myocardium before ischemia being metabolized normally. Inhibition of PtdIns-PLC under ischemic conditions has been suggested in two previous studies that reported progressive decreases in DAG content over a similar timescale.^{32 35} InsP metabolism appears to be similar under ischemic and normoxic conditions, because the same isomers of InsP₂ and InsP₁ were generated under both conditions. It is of interest that the decrease in [³H]Ins(1,4)P₂ preceded that of Ins(1,4,5)P₃. If the decreases in these products are due to the inhibition of InsP release, then this finding provides evidence that in the myocardium, Ins(1,4)P₂ does not derive from Ins(1,4,5)P₃ as it does in other tissues. The observed decrease in [³H]Ins(1,4,5)P₃ was paralleled by a decrease in Ins(1,4,5)P₃ mass, indicating that [³H]Ins(1,4,5)P₃ in ventricle is of uniform-specific acitvity. Thus, even though the content of Ins(1,4,5)P₃ in ventricle is high, it exists in only one functional compartment.

Our data show an inhibition of InsP release after 10-minute myocardial ischemia. Ischemia of 10 minutes or longer is associated with the development of ischemic damage, as indicated by decreased pH_i, depletion of intracellular substrates, loss of ATP stores, norepinephrine release, and accumulation of potentially toxic metabolites (for review, see Reference 36³⁶ ). Any one or a combination of these factors might be involved in the observed inhibition of PtdIns-PLC. Prior reserpinization of rats did not alter the effect of ischemia on InsP profiles, eliminating norepinephrine as a causative factor, and as outlined below, decreased ATP levels alone also are unlikely to be responsible. Whatever the mechanism, the observed cessation of InsP release means that any stimulation of ₁-adrenergic receptors is unlikely to activate PtdIns turnover under ischemic conditions, despite the increased release of norepinephrine and the reported increases in receptor density^{5 9 37} and sensitivity.³ Therefore, any observed effects of ₁-adrenergic receptor stimulation under ischemic conditions must be independent of InsPs.

In contrast to the suppression of InsP release observed during ischemia, reperfusion with oxygenated medium, following 20-minute myocardial ischemia, produced an activation of InsP release. This response was transient, with maximal release occurring at 2 minutes, followed by a decline toward basal levels and a smaller secondary rise in InsP accumulation that continued up to the 20-minute reperfusion point studied. The characteristics of the secondary accumulation were indistinguishable from that of normoxic tissue. The 2-minute reperfusion–induced InsP response was greatest after 20-minute ischemia. Ischemic periods of 10 or 30 minutes produced smaller responses. This indicates a relation between the extent of ischemia and the subsequent reperfusion-induced InsP response.

The initial 2-minute reperfusion response following 20-minute ischemia was dependent on endogenous norepinephrine and was mediated via ₁-adrenergic receptors. However, despite this, the reperfusion-induced InsP release was different from the norepinephrine-stimulated responses in nonischemic tissue in a number of ways. First, the observed response with 2-minute reperfusion was quantitatively greater, in terms of total [³H]InsPs released, than 2-minute stimulation with maximal concentrations of exogenous norepinephrine in nonischemic tissue. Second, the profiles of InsP release differed qualitatively between the two responses, with reperfusion causing an increase in Ins(1,4,5)P₃, measured in both mass assays and ³H-labeled studies. Studies with neomycin, which inhibits Ins(1,4,5)P₃ release, confirmed the release of Ins(1,4,5)P₃ with reperfusion and demonstrated that this, rather than Ins(1,4)P₂ (as proposed for normoxic myocardium),²² was the source of most of the accumulated InsPs. Increased Ins(1,4,5)P₃ accumulation was not observed in normoxic ventricles in either ³H-labeling studies or mass measurements, and neomycin was ineffective in inhibiting accumulation of ³H-labeled InsPs. Third, the 2-minute reperfusion response was decreased in Ca²⁺-free medium, whereas no effect of perfusate Ca²⁺ was observed during norepinephrine stimulation under normoxic conditions. All of these findings support the contention that the 2-minute reperfusion response is mechanistically different from responses observed in healthy tissue. This mechanistic difference does not reflect a different response to exogenous and endogenous norepinephrine, because addition of norepinephrine to the perfusate fully restored the reperfusion InsP response in hearts from reserpinized rats. Norepinephrine release occurs throughout the ischemic period, and synaptic concentrations are unlikely to rise substantially further between 1 and 2 minutes after the initiation of reperfusion, the time period over which maximal InsP release is observed. Thus, it is unlikely that the transient nature of the InsP response to reperfusion reflects the levels of ambient norepinephrine. Rather, the data suggest that the heart responds unusually to norepinephrine only for a short period of time, changing the nature of the PtdIns pathway such that Ins(1,4,5)P₃ is released.

The question remains as to the factor or factors that initiate the immediate InsP response under reperfusion conditions. Restoration of levels of ATP alone is unlikely to be important, because these require long periods (from hours to days) to return to preischemic levels.^{38 39} ₁-Adrenergic receptor density has been reported to increase with myocardial ischemia and to remain elevated at 2-minute reperfusion but to return to basal levels by 15-minute reperfusion.⁵ This increased ₁-adrenergic receptor density may contribute to the increased InsP release at this time. However, an increase in receptors alone cannot explain the observed change in the nature of the PtdIns turnover pathway. A more likely explanation for the switch in the nature of the PtdIns turnover pathway could be the rapid reversal of intracellular acidosis, or Ca²⁺ overload, either separately or in concert. However, these factors alone are not sufficient to initiate the response, because norepinephrine, either exogenous or endogenous, also was required.

Longer periods of reperfusion (5 to 20 minutes), following the initial transient reperfusion-induced InsP release, resulted in smaller InsP accumulation. This secondary accumulation of InsPs, in contrast to the initial transient response, was independent of endogenous norepinephrine and ₁-adrenergic receptor stimulation, in agreement with previous studies.³² This was not due to a loss of norepinephrine responsiveness, because 20-minute reperfusion in the presence of 100 µmol/L norepinephrine stimulated InsP accumulation further. Norepinephrine concentrations at 20-minute reperfusion were significantly lower than after 2-minute reperfusion. The difference in InsP accumulations at 2- and 20-minute reperfusion is not, however, simply a result of norepinephrine levels. Qualitatively different InsP accumulation profiles were observed at 2- and 20-minute reperfusion, with no further increase in Ins(1,4,5)P₃ detectable between 5 and 20 minutes after the initiation of reperfusion. Furthermore, this secondary InsP accumulation was qualitatively similar to that observed in nonischemic heart in terms of InsP isomers accumulated, resistance to neomycin, and independence of extracellular Ca²⁺. Thus, after the initial transient enhancement of PtdIns-PLC hydrolysis of PtdIns(4,5)P₂ and the release of Ins(1,4,5)P₃ with early reperfusion, a return to the pathway observed in nonischemic ventricle is observed.

Taken together, data presented here indicate that myocardial ischemia causes an cessation of InsP release. This suggests that the products of the PtdIns turnover pathway are unlikely to be involved in myocardial damage during the ischemic period. Previous reports have demonstrated enhanced PKC activity under conditions of global ischemia.⁴⁰ Our studies demonstrate that this is unlikely to be due to release of DAG from inositol phospholipids. This agrees with the observation that the PKC activation was independent of norepinephrine. Reperfusion following 20-minute ischemia produced a rapid and transient increase in InsP accumulation between 1 and 2 minutes after initiation of reperfusion, the time at which reperfusion-induced arrhythmias develop. This was followed by a return to basal activity by 5 minutes. Two-minute reperfusion resulted in an enhanced PtdIns-PLC activity, giving a rapid rise in Ins(1,4,5)P₃. Such a rapid increase in Ins(1,4,5)P₃, coinciding with the time at which reperfusion arrhythmias develop, suggests a possible causative role for Ins(1,4,5)P₃ in reperfusion arrhythmias. Such arrhythmias could conceivably be initiated via Ca²⁺ oscillations caused by the rapid release of Ins(1,4,5)P₃.¹⁹ These findings provide an explanation for earlier observations that the heart is unusually sensitive to ₁-adrenergic receptor stimulation under reperfusion conditions and also suggest a reason for the unusual pattern of InsP release observed in heart.

	Acknowledgments

 
This study was supported by a grant-in-aid from the National Heart Foundation of Australia and by the Australian National Health and Medical Research Council. K. Anderson is the recipient of a Dora Lush Biomedical Post-Graduate Research Scholarship. We would like to thank Andrea Turner (Human Endocrinology Laboratory, Baker Medical Research Institute) for performing the norepinephrine determinations.

Received May 13, 1994; accepted October 6, 1994.

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