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Alpha-adrenergic preservation of myocardial pH during ischemia is PKC isoform dependent
T F Rehring1, R S Friese, J C Cleveland
1Department of Surgery, University of Colorado Health Sciences Center, Denver 80262, USA.
Insights
Alpha1-adrenergic stimulation protects the heart from ischemic damage by reducing acidosis through a calcium-dependent protein kinase C (PKC) pathway. This preconditioning effect preserves cardiac function during ischemia.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Biochemistry
Background:
- Alpha1-adrenergic stimulation is known to precondition the heart against ischemia.
- This preconditioning effect involves protein kinase C (PKC) signaling.
- The specific PKC isoforms mediating this protection remain unclear.
Purpose of the Study:
- To investigate the role of calcium-dependent (cPKC) versus calcium-independent (nPKC) isoforms in alpha1-adrenergic mediated protection against ischemic acidosis.
- To determine the specific PKC pathway involved in alpha1-adrenergic preconditioning.
Main Methods:
- Isolated rat hearts were subjected to alpha1-adrenergic stimulation (phenylephrine) or PKC modulation before ischemia.
- Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy was used to measure intracellular pH (pHi).
- Immunohistochemistry was employed to assess PKC isoform translocation.
Main Results:
- Alpha1-adrenergic stimulation significantly attenuated ischemic acidosis.
- General PKC inhibition abolished this protective effect.
- Selective nPKC stimulation with SAG did not attenuate acidosis.
- Alpha1-adrenergic stimulation did not induce translocation of the PKC-epsilon isoform, unlike SAG.
Conclusions:
- Alpha1-adrenergic stimulation limits ischemic acidosis via a cPKC-dependent mechanism.
- The IP3 pathway, not DAG, is crucial for this protective effect.
- PKC-epsilon activation is not involved in alpha1-adrenergic mediated protection against ischemic acidosis.
Abstract:
alpha-adrenergic stimulation of patients with ischemic heart disease should intuitively impose a destructive stress. However, therapeutic alpha1-adrenergic receptor mediated cardioadaptation prior to myocardial ischemia protects ventricular mechanical function, promotes electrophysiologic stability, and preserves myocyte viability. Prior to an anticipated cardiac ischemic insult, alpha1-adrenergic preconditioning attenuates ischemic myocardial acidosis by a protein kinase C-(PKC) dependent mechanism. The alpha1-adrenoceptor can directly stimulate calcium-independent nPKC isoforms via diacylglycerol (DAG) or indirectly stimulate calcium-dependent cPKC isoforms through the release of intracellular calcium via inositol triphosphate, (IP3). We hypothesized that alpha1-adrenergic limitation of ischemic acidosis is mediated by the family of calcium-dependent PKC isoforms. [31P]NMR spectra were obtained in isolated, buffer perfused rat hearts treated with alpha1-adrenergic stimulation [phenylephrine (PE) 50 microM, 2 min]; PKC blockade [chelerythrine chloride, (Chel) 20 microM]; or stearoyl-arachidonoyl glycerol (SAG, a DAG analogue, 100 microM, 2 min) administered 10 min prior to ischemia. Control hearts were perfused under normoxic conditions for 20 min. All hearts were then subjected to global ischemia (20 min, 37.5 degrees C). Developed pressure (DP) and heart rate were recorded continuously. pHi was obtained from chemical shift of inorganic phosphate. Immunohistochemical staining was utilized to delineate the translocation and activation profiles of specific PKC profiles established with each stimulus. Pre-ischemic alpha1-adrenergic stimulation did attenuate the myocellular hydrogen ion accumulation during sustained normothermic ischemia (6.90 +/- 0.13 vs control 6.54 +/- 0.10; P < 0.05). General PKC inhibition abrogated this effect (end-ischemic pH 6.17 +/- 0.10; P < 0.05 vs control and PE). Ischemic acidosis was not attenuated following selective nPKC stimulation (SAG, 6.48 +/- 0.08; NS vs control). Myocellular immunohistochemical staining revealed translocation of the calcium-independent PKC-epsilon isoform in the calcium-dependent PKC (SAG) group, but not in response to alpha1-adrenergic stimulation. The results suggest that (1) alpha1-adrenoceptor stimulation limits ischemic acidosis, (2) alpha1-adrenergic stimulated attenuation of ischemic acidosis is PKC dependent, (3) direct nPKC stimulation with SAG does not limit ischemic acidosis, and (4) SAG stimulates nPKC-epsilon isoform activation where alpha1-adrenergic stimulation does not. We conclude that alpha1-adrenergic stimulation limits ischemic acidosis by a cPKC-dependent mechanism and that the mobilization of the IP3 arm by receptor stimuli suppresses PKC-epsilon thus permitting the limitation of ischemic acidosis.