Biomedical subjects

M V Cohen

Publications and source records attributed to M V Cohen.

At least 37 records · Page 2Linked to original sources

PubMed2000

SB 203580, an inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts.

There is debate concerning the involvement of p38 mitogen-activated protein kinase (MAPK) in ischemic preconditioning (PC). At the center of the controversy are data obtained after administration of SB 203580, a specific inhibitor of p38 MAPK. Whereas several studies have reported that SB 203580 abolishes the cardioprotective effect of PC, others claim that this compound is actually cardioprotective against ischemia. Many of these latter observations have been made in isolated myocardial cells. Accordingly the present study was designed to test the effect of SB 203580 in a model of preconditioning in intact rabbit hearts in which infarct size was the end-point. Isolated hearts experienced 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size was measured with triphenyltetrazolium chloride. In control hearts infarction was 30.2 +/- 3.3% of the risk zone. PC with 5 min of global ischemia and 10 min of reperfusion before the 30-min period of ischemia significantly reduced infarct size to 10.2 +/- 2.4% (P < 0.05 vs. control). SB 203580 (2 microM) added to the perfusate for 20 min starting 5 min before the index ischemia totally blocked the protection from PC (27.4 +/- 3.3% infarction). SB 203580 alone had no effect on infarct size (28.6 +/- 4.6% infarction). These results reveal that SB 203580 does not affect infarct size on its own, but selectively blocks preconditioning's anti-infarct effect in the intact rabbit heart.

Animals↗

PubMed2000

Do mitochondrial K(ATP) channels serve as triggers rather than end-effectors of ischemic preconditioning's protection?

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Adenosine Triphosphate↗

PubMed2000

Ischemic preconditioning: from basic mechanisms to clinical applications.

When the heart is subjected to a transient nonlethal period of ischemia, it quickly adapts itself to become resistant to infarction from a subsequent ischemic insult. This adaptation is called preconditioning. This cardioprotection has been shown to be mediated by stimulation of receptors linked to protein kinase C (PKC) (adenosine, bradykinin, opioids, etc.), and these receptors protect by activating PKC. PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in the preconditioned heart. Recent studies imply that p38 mitogen-activated protein kinase carries the signal from PKC to the mitochondrial K(ATP) channels, causing them to open and thus protect the heart. The cardioprotection of preconditioning occurs in all species tested to date, and possibly also humans. It is expected that as the mechanism of preconditioning is more thoroughly understood, pharmacological preconditioning will become practical for clinical use.

Animals↗

PubMed2000

Adenosine A1 agonist at reperfusion trial (AART): results of a three-center, blinded, randomized, controlled experimental infarct study.

Adenosine A1 receptor agonists given prior to myocardial ischemia limit ischemic injury in several species. However, the ability of adenosine receptor agonists to limit infarct size when given at reperfusion has proved controversial. We designed a three-center experimental study using a blinded, randomized treatment protocol to test the hypothesis that adenosine A1 receptor activation during early reperfusion can attenuate lethal reperfusion injury, thereby reducing infarct size. Sixty anesthetized rabbits (20 in each laboratory) underwent 30 minutes coronary artery occlusion followed by 120 minutes reperfusion. The selective adenosine A1 receptor agonist GR79236 (10.5 microg/kg, a dose shown to limit infarction in this model when given before ischemia) or vehicle were administered IV 10 minutes before reperfusion. Infarct size was assessed by tetrazolium staining and, after the randomization code was revealed, data from the three laboratories were pooled for statistical analysis. Infarct size was not modified by administration of GR79236. In the vehicle-treated group, the infarct-to-risk ratio was 28.9 +/- 2.7% (n = 24) compared with 31.9 +/- 2.6% (n = 26) in the GR79236-treated group (not significant). Risk zone volume was similar in the two groups (1.06 +/- 0.05 cm3 vs 1.00 +/- 0.05 cm3, respectively). A modest reduction in rate-pressure product was noted following the administration of GR79236, but this effect was transient. The same dose of GR79236 was found to limit infarct size when given prior to coronary artery occlusion. We conclude that A1 receptor activation does not modify lethal reperfusion injury in myocardium.

Adenosine↗

PubMed2000

Ischemic preconditioning: from adenosine receptor to KATP channel.

Ischemic preconditioning is a phenomenon whereby exposure of the myocardium to a brief episode of ischemia and reperfusion markedly reduces tissue necrosis induced by a subsequent prolonged ischemia. It is hoped that elucidation of the mechanism for preconditioning will yield therapeutic strategies capable of reducing myocardial infarction. In the rabbit, the brief period of preconditioning ischemia and reperfusion releases adenosine, bradykinin, opioids, and oxygen radicals. The combined effect of the release of these substances on G proteins and the cell's phospholipases induces the translocation and activation of the epsilon isozyme of protein kinase C. Protein kinase C appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in preconditioned hearts. Current evidence indicates that this cascade contains at least one tyrosine kinase and ultimately leads to the activation of p38 mitogen-activated protein kinase. p38 Mitogen-activated protein kinase phosphorylates mitogen-activated protein kinase-activated protein kinase 2. Mitogen-activated protein kinase-activated protein kinase 2 phosphorylates HSP27, a 27-kDa heat shock protein that controls actin filament polymerization, and, therefore, affects the integrity of the cytoskeleton. Finally, mitochondrial adenosine 5'-triphosphate-sensitive K+ channels open, and the latter may be the final mediator of protection for ischemic preconditioning. The protective pathway has many built-in redundancies, perhaps creating a safety factor. These redundancies may also explain some of the species-related differences seen in ischemic preconditioning in which one redundant pathway may predominate over another.

ATP-Binding Cassette Transporters↗

PubMed1999

Second window of protection against infarction in conscious rabbits: real or artifactual.

To date, most studies of the second window of protection against infarction (SWOP) have evaluated infarct size by staining with triphenyltetrazolium chloride (TTC) soon after reperfusion. However, early TTC staining has been found to be an unreliable indicator of the ultimate infarct size following some interventions. Therefore, we tested whether SWOP could induce a sustained limitation of infarct size. Instrumented, conscious rabbits underwent 30 min of coronary occlusion. Infarct size was determined by either TTC staining after 3 h of reperfusion or conventional histology after 72 h of reperfusion. In the TTC study, 43.5+/-3.1% of the risk zone infarcted in the control group. Four cycles of 5 min ischemia/10 min reperfusion 24 h prior to 30 min ischemia significantly reduced infarct size measured by TTC to 32.5+/-2.3% (P<0.05 v control). In the histological study 57.8+/-3.6% of the risk zone infarcted in the control group. However, ischemic preconditioning 24 h prior to the 30 min ischemia did not protect the heart (59.3+/-4.4% infarction). Thus the infarct-limiting effect of SWOP evaluated with early TTC staining could not be demonstrated when infarction was assessed by histology after 3 days of reperfusion. These data suggest that SWOP may not have a sustained anti-infarct effect, but rather may simply delay the progression to infarction.

Animals↗

PubMed1999

Protein kinase C-epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes.

The role of protein kinase C (PKC) in the protection of ischemic preconditioning (PC) is still controversial, partly because of the multiple isozymes of PKC and the inability to directly measure PKC activity in vivo. In this study we have used novel peptide inhibitors which correspond to part of the amino acid sequence from the isozyme-specific RACK-binding site on the PKC molecule. The peptides prevent binding of a specific activated PKC isozyme to its RACK, thus halting isozyme translocation and function. The inhibitor peptides are cross-linked to the membrane-translocating antennapedia homeodomain peptide that allows their entry into cells. The effect of inhibitors of PKC-beta, -delta, -epsilon and -eta were evaluated. Rabbit adult ventricular myocytes were obtained by enzymatic dissociation. Ischemia was simulated by centrifuging the myocytes into an oxygen-free pellet for 180 min. PC was induced by 10 min of pelleting followed by resuspension in oxygenated medium for 15 min. During simulated ischemia cells undergo a predictable increase in osmotic fragility as judged by determination of the number of stained cells following their incubation in hypotonic (85 mOsm) trypan blue. The percentage of cells experiencing membrane rupture, and thus cell staining, was considered to be an index of ischemic injury. PC significantly delayed the progression of osmotic fragility during simulated ischemia (P<0.01). The protection of PC was abolished by the peptide inhibitor of PKC-epsilon but not by the peptide inhibitors selective for PKC-beta, PKC-delta, or PKC-eta; each was applied at 100 n N. Protection could also be induced by the PKC activator oleoylacetyl glycerol, and that protection was aborted by the inhibitor selective for PKC-epsilon, but not by the inhibitor for PKC-delta. None of the above peptide treatments affected the osmotic fragility in non-PC cells during simulated ischemia. Our studies further support PKC as a critical part of the signal transduction pathway in PC and indicate that PKC-epsilon alone is responsible for the early phase of PC's protection in rabbit cardiomyocytes.

Animals↗

PubMed1999

Failure of N-2-mercaptopropionyl glycine to reduce myocardial infarction after 3 days of reperfusion in rabbits.

Recent studies have reported that prolonged infusion of N-2-mercaptopropionyl glycine (MPG), a diffusible antioxidant, could limit infarct size in dogs. However, there are no comparable studies testing this agent in other species. We examined the efficacy of MPG in a rabbit model of infarction. Rabbit hearts were subjected to a 30-min coronary artery occlusion. Infarct size expressed as a percentage of risk zone was determined by either triphenyltetrazolium chloride (TTC) staining after 3h of reperfusion (study 1) or by histology after 72 h of reperfusion (study 2). In study 1, 37 +/- 2.6% of the risk zone infarcted in the control group. Intravenous MPG at a rate of 100 mg/kg/h starting 15 min after the onset of ischemia and continuing until 1 h after reperfusion had no effect on infarct size (35.4 +/- 3.4% infarction). However, infusion of MPG until the end of reperfusion significantly reduced infarct size as measured with TTC to 17.2 +/- 2.5% (p < 0.01 vs. control group). In study 2, 48.6 +/- 4.0% of the risk zone infarcted in the control group. In the treatment group MPG was started as above and was continued for 4 h of reperfusion followed by an intramuscular injection at the termination of the intravenous infusion. No protection was seen after 72 h of reperfusion (43.8 +/- 2.1% infarction). These findings reveal that MPG at a dose and schedule that appeared to protect the dog heart could not effect sustained protection in the rabbit heart. TTC staining revealed that MPG appeared to have preserved viability for up to 3 h of reperfusion suggesting that failure may have been due to early withdrawal of the drug. Alternatively, early TTC staining may yield spurious results under conditions in which protection is dependent upon antioxidant or free radical scavenger treatment as has previously been suggested. It is concluded that MPG as administered in the previous canine studies does not limit infarct size in all species, thus raising a concern about MPG's potential efficacy in man.

Animals↗

PubMed1999

Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart.

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (p.c.) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic p.c. and if the mechanism of protection was similar to that of p.c. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 +/- 2.3%. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 +/- 2.1% (P < 0.05). The tyrosine kinase inhibitor genistein (50 microM) given around the insulin infusion blocked protection (28.9 +/- 2.8%). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 +/- 2.4%; P < 0.05). Inhibition of either PKC by polymyxin B (50 microM) or KATP channels by 5-hydroxydecanoate (100 microM) also failed to prevent protection by insulin (17.5 +/- 3.2% and 17.6 +/- 3.0%, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (PI3K, 28.3 +/- 2.2%). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 +/- 3.4%). P.c. reduced infarction to 12.8 +/- 2.0% (P < 0.05) and still offered significant protection in the presence of wortmannin (22.1 +/- 2.4%; P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of p.c. and involves activation of PI3K but not PKC or KATP channels.

Animals↗

PubMed1999

The warm-up phenomenon and ischaemic preconditioning: an uncertain relationship.

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Animals↗

PubMed1999

Signal transduction in ischemic preconditioning: the role of kinases and mitochondrial K(ATP) channels.

Animals↗

PubMed1999

Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton.

Both mitochondrial ATP-sensitive K+ (KATP) channels and the actin cytoskeleton have been proposed to be end-effectors in ischemic preconditioning (PC). For evaluation of the participation of these proposed end effectors, rabbits underwent 30 min of regional ischemia and 3 h of reperfusion. PC by 5-min ischemia + 10-min reperfusion reduced infarct size by 60%. Diazoxide, a mitochondrial KATP-channel opener, administered before ischemia was protective. Protection was lost when diazoxide was given after onset of ischemia. Anisomycin, a p38/JNK activator, reduced infarct size, but protection from both diazoxide and anisomycin was abolished by 5-hydroxydecanoate (5-HD), an inhibitor of mitochondrial KATP channels. Isolated adult rabbit cardiomyocytes were subjected to simulated ischemia by centrifuging the cells into an oxygen-free pellet for 3 h. PC was induced by prior pelleting for 10 min followed by resuspension for 15 min. Osmotic fragility was assessed by adding cells to hypotonic (85 mosmol) Trypan blue. PC delayed the progressive increase in fragility seen in non-PC cells. Incubation with diazoxide or pinacidil was as protective as PC. Anisomycin reduced osmotic fragility, and this was reversed by 5-HD. Interestingly, protection by PC, diazoxide, and pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data support a role for both mitochondrial KATP channels and cytoskeletal actin in protection by PC.

Actins↗

PubMed1999

Smaller infarct after preconditioning does not predict extent of early functional improvement of reperfused heart.

We evaluated the ability of ischemic preconditioning to restore function to salvaged myocardium in rabbits. Although ischemic preconditioning reduces infarct size, few investigators studying recovery of function after coronary occlusions lasting >/=30 min have reported any mechanical benefit in preconditioned hearts. However, because myocardial function was seldom evaluated beyond 5 h after reperfusion stunning may have masked the benefit. Accordingly, rabbits were chronically instrumented with a pneumatic occluder around a branch of the left coronary artery, a pair of 1-mm ultrasonic crystals in the myocardial territory destined to become ischemic, and electrocardiogram (ECG) leads. One week after surgery the ECG and segment length tracing were recorded at rest, during 30-min occlusion and 1 h of reflow, and again at 24, 48, and 72 h. In ischemically preconditioned rabbits, 5-min coronary occlusion and 10-min reperfusion preceded the long occlusion. The beginning and end of systole were determined by recording the first and second heart sounds with a hand-held precordial microphone. Postmortem infarct size was measured with triphenyltetrazolium chloride. During the 30-min coronary occlusion all segments became nearly akinetic or bulged during systole. After 60 min of reflow there was little return of function in either group. Between 24 and 72 h there was minimal recovery in the control group (segment shortening equals 13.3 +/- 4.1% of baseline), whereas function was much better in preconditioned hearts (44.2 +/- 7.4% of baseline, P < 0.02). Infarct size as a percentage of risk zone was much smaller in preconditioned hearts (10.2 +/- 1.4 vs. 29.7 +/- 1.8%, P < 0.001). Thus there is a gradual recovery of systolic function of reperfused myocardium after a coronary occlusion. Although early mechanical recovery is significantly better after preconditioning, it is much less than would be predicted by the reduction of infarct size.

Animals↗

PubMed1999

S-T segment voltage during sequential coronary occlusions is an unreliable marker of preconditioning.

During coronary angioplasty, a stair-step decrease in peak S-T segment elevation from the first to the second coronary occlusion has been assumed to indicate a preconditioning (PC) effect. This association was evaluated with myocardial electrograms in rabbits, which revealed that two sequential 5-min coronary occlusions resulted in a marked decrease in the area under the S-T segment voltage-time curve (P < 0.05) with no change during a third occlusion. Pretreatment with either 5-hydroxydecanoate, a mitochondrial ATP-sensitive potassium (K(ATP)) channel blocker, or anisomycin, an activator of stress-activated protein kinases, had no effect on the stair-step decline in the S-T segment voltage between the first two occlusions. HMR-1883, a potent closer of sarcolemmal K(ATP) channels, abolished changes in S-T segment elevation after brief coronary occlusions but had no effect on the infarct-sparing property of the two preconditioning 5-min occlusions. Interestingly, HMR-1883 blocked myocardial protection from diazoxide, raising doubt that the latter opens only mitochondrial channels. Therefore, myocardial protection and S-T segment changes during ischemia are dissociated. These data suggest that it is the mitochondrial K(ATP) channel that protects the myocardium, and it is the sarcolemmal channel that is responsible for changes in S-T elevation. Therefore, it cannot always be inferred that changes in S-T segment elevation reflect the state of myocardial protection.

Adenosine Triphosphate↗

PubMed1999

Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.

Animals↗

PubMed1998

Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia.

BACKGROUND: The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. METHODS AND RESULTS: Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. CONCLUSIONS: Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Alkenes↗

PubMed1998

Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning's anti-infarct effect in the rabbit heart.

The present study tested the hypothesis that one or more tyrosine kinase(s) are downstream of protein kinase C (PKC) in the signal transduction pathway responsible for the cardioprotective effect of ischemic preconditioning (PC). Isolated rabbit hearts were subjected to 30 min of regional ischemia followed by 2 h of reperfusion. Infarct size was measured by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarction in control hearts was 32.9+/-1.8%. Ischemic PC with 5-min ischemia/10-min reperfusion reduced infarct size to 11.5+/-1.5% (P<0.05). Infusion of the tyrosine kinase inhibitors, genistein (50 microM) or lavendustin A (0.5 microM), alone did not affect the level of infarction. When infused around the 5-min PC ischemia genistein failed to block protection (13.7+/-1.0%). However, when present at the onset of the 30-min ischemia both genistein and lavendustin A completely aborted protection (31.4+/-2.0 and 28.1+/-1.5%, respectively). Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 0.05 nmol) was as protective is ischemic PC (14.9+/-3.0%; P<0. 05). Similar to PC, PMA-induced protection was completely prevented by both genistein and lavendustin A. Conversely, anisomycin (50 ng/ml), an activator of MAP kinase kinases (dual tyrosine and threonine kinases), was very protective (7.5+/-1.6%; P<0.05) and this protection was still present when PKC was inhibited by 5 microM chelerythrine (12.1+/-1.6%; P<0.05). In conclusion, activation of a tyrosine kinase during the long ischemia appears to be required for cardioprotection in the rabbit heart. Furthermore, the ability of tyrosine kinase inhibitors to block PMA-induced protection in conjunction with the failure of PKC inhibition to prevent anisomycin-induced protection suggests that the tyrosine kinase is downstream of PKC and that the tyrosine kinase may be a MAP kinase kinase.

Alkaloids↗

PubMed1998

The PKC activator PMA preconditions rabbit heart in the presence of adenosine receptor blockade: is 5'-nucleotidase important?

While there is good evidence that both protein kinase C (PKC) and adenosine are involved in ischemic preconditioning, their sequence in the intracellular signaling cascade is in dispute. One hypothesis proposes that PKC activation causes release of adenosine which then protects the heart, while the other proposes that adenosine stimulates PKC which in turn causes protection. Accordingly, we studied the effects of specified sequences of pharmacologic triggers and blockers on the infarct-sparing effect of a preconditioning protocol. The combination of the adenosine receptor agonist R(-)N6-(2-phenylisopropyl) adenosine (PIA) and the PKC blocker chelerythrine would be protective only if the first hypothesis were correct. On the other hand, the combination of the adenosine receptor blocker 8-(p-sulfophenyl) theophylline (SPT) and a PKC activator would be protective only if the second hypothesis were correct. Isolated, Krebs-perfused rabbit hearts experienced 30 min of regional ischemia and 2 h of reperfusion. Infarct size was quantitated by triphenyltetrazolium chloride staining. In untreated control hearts, 30.0 +/- 2.7% of the risk zone infarcted. Fifty nmol/l PIA for 20 min starting 10 min prior to ischemia resulted in only 8.4 +/- 1.9% infarction (P<0.01), while the combination of PIA and 5 micromol/l chelerythrine resulted in large infarcts of 27.8 +/- 3.2%. This attenuation of the protective effect continued to be observed even when the PIA infusion was continued to the end of the reperfusion period. Conversely, 0.2 nmol of the PKC activator phorbol 12-myristate 13-acetate (PMA) infused during the 10-min interval prior to ischemia protected the hearts (6.5 +/- 1.3% infarction, P<0.01 v control). And protection persisted when PMA-treated hearts were also exposed to 100 microM SPT for 35 min starting 5 min prior to ischemia (9.5 +/- 1.9% infarction, P<0.01 v control). When PKC activation by the PKC-coupled agonist phenylephrine was continued to the end of ischemia and adenosine blockade was extended throughout the reperfusion period by prolonged infusion of SPT, protection was unaffected. The administration of either SPT or chelerythrine alone did not confer any protection (32.5 +/- 3.3 and 34.0 +/- 3.2% infarction, respectively). Thus, because the combination of PKC activation and adenosine receptor blockade was protective while that of adenosine receptor agonist and PKC blockade was not, adenosine receptors must be upstream of PKC in preconditioning.

Adenosine↗