This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-? constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C. [Neural Res 2003; 25: 268-270]
Keywords: Endothelin-1; protein kinase C; cerebral vasospasm
INTRODUCTION
The cellular mechanism responsible for the vasospasm following subarachnoid hemorrhage is not entirely known. The complexity of the mechanism is underscored by the recent demonstration in monkey middle cerebral artery that subarachnoid hemorrhage increased the expression of hundreds of genes, including numerous genes associated with signal transduction1. Since protein kinase C appears to play a major role in the spasm2, this study investigated whether subarachnoid hemorrhage functionally upregulated protein kinase C. This possibility was investigated based on the endothelin-1 dependence of the spastic rabbit basilar artery3 and testing whether the protein kinase C inhibitor, chelerythrine4, preferentially relaxed endothelin-1 constricted spastic vessels as compared to endothelin-1 constricted control vessels.
MATERIALS AND METHODS
Animal preparation
Procedures were approved by the Institutional Animal Care and Use Committee. Following i.m. injection of ketamine HCl OOmgkg^sup -1^) and xylazine (Bmgkg^sup -1^), New Zealand White male rabbits (weight 2.8-3.5 kg) were intubated and mechanically ventilated with room air supplemented with O2. The ear vein was then catheterized and sodium pentobarbital (~11 mg kg^sup -1^ h^sup -1^ and 5mgkg^sup -1^ as needed) and vecuronium administered as needed. Respiratory rate and tidal volume were adjusted to maintain the arterial pH at ~7.4. Under these conditions, pCO^sub 2^ was ~ 60 due to the anesthetics. Body temperature was maintained at 37[degrees]C and monitored via a needle probe inserted into the thigh muscle.
Basilar artery cranial window was prepared as previously described3. The cranial window was suffused (3 ml min^sup -1^) with Krebs Ringer bicarbonate solution (mM: NaCI 119, KCI 4.7, CaCI^sub 2^ 2.5, MgCI^sub 2^ 1.17, KH^sub 2^PO^sub 4^ 1.18, NaHCO^sub 3^ 25, EDTA 0.026, D-glucose 11). The solution was warmed and gassed with 12% O2/ 6% CO2/82%N^sub 2^ such that the temperature of the suffusate in the cranial window was 37[degrees]C with pH 7.4. The craniotomy was suffused with Krebs Ringer bicarbonate solution for 45 min prior to agent suffusion. Basilar artery diameter was recorded at the time of the plateau response to each agent. Each value of vessel diameter, measured in micrometers, was the mean of six consecutive measurements taken at approximately 120 sec intervals (Image-Pro 3.0, MediaCybernetics, Silver Springs, MD, USA).
Subarachnoid hemorrhage was performed essentially as previously described3. Rabbits were hemorrhaged on days 1 and 3, and in situ studies performed on day 7.
Protocol
Relaxations to cumulative chelerythrine concentrations were investigated in three groups:
Group 1 : Subarachnoid hemorrhage treated vessels.
Group 2: Subarachnoid hemorrhage treated vessels that, on day 7 after the initial hemorrhage, were first relaxed in situ with the endothelin converting enzyme inhibitor, phosphoramidon (100 [mu]M, 1 h), and were then constricted with 5 nM endothelin-1.
Group 3: Vessels unexposed to subarachnoid hemorrhage and constricted with 5 nM endothelin-1.
Calculations
Relaxation was expressed as a percentage of the constriction. Constriction was calculated as a percentage of baseline diameter. Baseline diameters were determined:
1. After addition of sodium nitroprusside in vessels exposed to subarachnoid hemorrhage and not treated with phosphoramidon (Group 1), as direct measurements of baseline diameter prior to subarachnoid hemorrhage could not be assessed.
2. After phosphoramidon treatment in subarachnoid hemorrhage exposed vessels (Group 2), thereby eliminated bias due to chelerythrine relaxation of any spasm remaining following phosphoramidon.
3. Prior to endothelin-1 challenge in vessels unexposed to subarachnoid hemorrhage (Group 3), thereby eliminating bias due to chelerythrine relaxation of basal tone. Magnitudes of relaxation in Group 1 were not compared to those in Groups 2 and 3.
Statistical methods
Statistical significance between chelerythrine induced relaxation in vessels from Groups 2 and 3 were determined with Student's unpaired f-test. Significance was accepted at the 0.05 level of probability. Values are expressed as mean + or - SEM; n represents the number of animals.
Materials
Reagent sources were American Peptide Company (Sunnyvale, CA, USA) for endothelin-1, Henry Schein (Port Washington, NY, USA) for ketamine and xylazine, Organon (West Orange, NJ, USA) for vecuronium bromide, Peptides International (Louisville, KY, USA) for phosphoramidon, Sigma Chemicals (St. Louis, MO, USA) for sodium nitroprusside, and Tocris Cookson (Ballwin, MO, USA) for chelerythrine.
RESULTS
Spastic vessels (Group 1) were relaxed by 0.1, 1, and 10 [mu]M chelerythrine by 6.6% + or -3.4%, 18.9% + or -8.1%, and 58.3% + or - 7.4%, respectively (n = 8). Thus, the spasm appears to involve protein kinase C activation, consistent with previous reports2. The magnitude of spasm due to subarachnoid hemorrhage was 32.6% + or - 4.3% (n = 8).
In 5 nM endothelin-1 constricted vessels unexposed to subarachnoid hemorrhage (Group 3), 0.1, 1, and 10 [mu]M chelerythrine induced 0%, 3.6% + or -2.2%, and 78.9% + or -2.1% relaxation, respectively (n = 5; Figure 7). Endothelin-1 (5 nM) constricted these unexposed vessels by 25.1% + or -3.0% (n = 5).
Chelerythrine also relaxed 5 nM endothelin-1 constricted vessels exposed to subarachnoid hemorrhage and treated with phosphoramidon (Group 2; Figure 7). Chelerythrine (0.1, 1, and 10 [mu]M) induced 5.8% + or - 2.3%, 17.8% + or -7.1%, and 57.1% + or -5.0% relaxation, respectively (n=7; Figure 1). Chelerythrine (10 [mu]M) relaxation was significantly less than in endothelin-1 constricted vessels unexposed to subarachnoid hemorrhage (Figure 1; p
DISCUSSION
The present study suggests that the protein kinase C constrictor pathway is not functionally upregulated in spastic vessels following subarachnoid hemorrhage. This suggestion is supported by the findings that relaxation to the protein kinase C inhibitor, chelerythrine, was not significantly increased in spastic vessels relaxed with phosphoramidon and reconstricted with endothelin-1, as compared to endothelin-1 constricted control vessels (unexposed to subarachnoid hemorrhage). Interestingly, KCI (25-35 mM) constricted control vessels were relaxed by chelerythrine, suggesting possible protein kinase C activation in KCI constriction of the rabbit basilar artery (unpublished observation).
These results, combined with our recent demonstration that the Rho-kinase constrictor pathway was also not functionally upregulated following subarachnoid hemorrhage5, suggest that the function of at least two major signal transduction pathways thought to underlie subarachnoid hemorrhage induced vasospasm, protein kinase C and Rho kinase2'6'7, remain unaltered in the rabbit basilar artery. It remains to be determined whether protein kinase C and Rho kinase are functionally upregulated in other models of subarachnoid hemorrhage, such as in the monkey middle cerebral artery, in which subarachnoid hemorrhage increased the expression of hundreds of genes, including numerous genes associated with signal transduction1.
ACKNOWLEDGEMENTS
Supported by grants from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs and the Department of Neurosurgery, University of Cincinnati, College of Medicine (Ohio), and the Mayfield Educational Research Fund (MERF).
REFERENCES
1 MacDonald RL, Zhang Z-D, Shigeki O, Kouro T. Dp-regulation of parathyroid hormone receptor in cerebral arteries after subarachnoid hemorrhage in monkeys. Neurosurgery 2002; 50: 1083-1093
2 Laher I, Zhang JH. Protein kinase C and cerebral vasospasm. J Cereb Blood Flow Metab 2001 ; 21: 887-906
3 Zuccarello M, Boccaletti R, Romano A, Rapoport RM. Endothelin B receptor antagonists attenuate subarachnoid hemorrhage-induced cerebral vasospasm. Stroke 1998; 29: 1924-1929
4 Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Com/nun 1990; 172: 993-999
5 Yoon SH, Sherman JD, Zuccarello M, Rapoport RM. Vasospasm following subarachnoid hemorrhage: Evidence against functional upregulation of the kinase constrictor pathway. Neuro! Res 2002; 24: 392-394
6 Miyagi Y, Carpenter RC, Meguro T, Parent AD, Zhang JH. Upregulation of rho A and rho kinase messenger RNAs in the basilar artery of a rat model of subarachnoid hemorrhage. J Neurosurg 2000; 93: 471-476
7 Sato M, Tani E, Fujikawa H, Kaibuchi K. Involvement of Rho-kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. OVc Res 2000; 87: 195-200
SeongHun Yoon*, Mario Zuccarello[dagger][double dagger] and Robert M. Rapoport*[sec]
* Department of Pharmacology and Cell Biophysics, [dagger] The Neuroscience Institute, Department of Neumsurgery, University of Cincinnati College of Medicine, Cincinnati, OH
[double dagger] Surgical Service, [sec] Research Service, Veterans Affairs Medical Center, Cincinnati, OH, USA
Correspondence and reprint requests to: Robert M. Rapoport, PhD, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, PO Box 670575, Cincinnati, OH 45267-0575, USA. [Robert.Rapoport@UC.EDU] Accepted for publication December 2002.
Copyright Forefront Publishing Group Apr 2003
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