Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during "metabolic" preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U · ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Objective: The aim was to discriminate among several hypotheses of preconditioning of isolated rabbit cardiomyocytes and to determine if ischaemic preincubation would evoke a protective response. Methods: Isolated myocytes were subjected to 5 min of preincubation, in the presence or absence of glucose, and incubated in the presence of 1 mM iodoacetic acid during the final sustained ischaemic period. In a second series, the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA), ingenol 3, 20-dibenzoate, and thymeleatoxin were added during preincubation. In a third series, preincubation periods were substituted by brief ischaemic pelleting of cells. Final prolonged ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury was determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 5 min glucose-free preincubation. Addition of iodoacetic acid into the final ischaemic pellet increased the rates of rigor contracture and injury, but did not abolish the protective response. Direct protein kinase C activation with PMA, a non-selective phorbol ester, and ingenol, an ε, δ-PKC isozyme selective activator, protected cells, but thymeleatoxin, an α,β,γ-PKC isozyme selective activator, did not. A 10 min ischaemic preincubation preconditioned, but the protection was not enhanced when ischaemia was extended to 30 min, or when PMA was included during the initial ischaemic preincubation. Adenosine partially inhibited the response. Conclusions: (1) Preconditioning of isolated myocytes is not dependent on glycolysis or glucose transport. (2) Preconditioning appears dependent on activation of the ε-PKC isoformn. (3) Ischaemia is capable of preconditioning isolated myocytes in vitro, and initiation of this effect is modified by simultaneous additional of adenosine but not by direct protein kinase C activation with PMA. Induction of protection by PMA and ingenol shows that protection requires protein kinase C activation, but direct potassium channel activation by regulatory G proteins is not critical.Cardiovascular Research 1994;28:1700-1706.

The role of protein kinase C (PKC) in mediating contractile responses in the rat vas deferens was studied. Phorbol-12,13-di-acetate (PDA) in the presence of 20 mM K+ elicited a concentration-dependent response with an EC50 of 190 nM. The non-PKC activator 4α-phorbol (2 μM) was unable to elicit contraction in 20 mM K+ buffer. Incubation of rat vas deferens with the PKC inhibitor iso-H7 (30 μM) attenuated the response to norepinephrine (NE) ane neurokinin A, with maximal effects depressed to 42 and 39% of control, respectively. Responses to 60 mM K+ and 2 μM PDA (20 mM K+) was also significantly inhibited by iso-H7. In the presence of 2 μM PDA and 20 mM K+, the NE concentration-effect curve was shifted 3,6-fold to the right of the control curve in a parallel manner. 4α-Phorbol (20 mM K+) at the same concentration did not produce this effect. These results suggest a significant role for PKC in the contractile response of the rat vas deferens.

Cardiomyocytes isolated from rabbit hearts were preconditioned in vitro by 10 min of ischemia or treatment with 100 μM adenosine. Protection was assessed as average integrated mortality following osmotic swelling and determination of viability by trypan blue exclusion over 60-180 min ischemia. Repetitive submaximal stimulations with 1 μM adenosine amplified the protective response. Treatment with adenosine only at the onset of prolonged ischemia afforded a dose-dependent protection. The PKC inhibitor calphostin C (500 nM) blocked preconditioning and, when added during ischemic incubation of non-preconditioned cells, significantly increased injury. The memory of adenosine-induced preconditioning decayed over a 60 min post-incubation period. Light activation of calphostin C initially added to preconditioned ischemic cells in the dark indicated that a 10 min period of PKC activity at the onset of ischemia affords full protection. The reversible PKC inhibitors chelerythrine (5 μM) or staurosporine (100 nM) added only to bracket induction of ischemia, reduced but did not abolish protection. Protection was abolished when either drug was present during induction and a subsequent 30 min post-incubation period. Staurosporine included during initiation and post-incubation but washed out in the final 5 min of post-incubation allowed significant protection to occur. It is concluded that a single adenosine receptor-stimulation induces protection as it preconditions, and PKC activity appears to be required for both induction and protection. Memory may reside in post-receptor amplification of an initial protective response.

This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning. Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions. Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells, the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta. Preconditioning with adenosine (100 μM) or 10 min ischemia had no significant effect on these percentages. Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation. PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon. The distribution of PKC zeta between fractions was not affected by any drug, The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the final ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37°C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOsm media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 μm) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 μm) added with pinacidil and calphostin C (200nm) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 μm) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 μm) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil’s protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3, dipropylxanthine (DPCPX) (10 μm) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

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