Abstract-Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences.Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca 2ϩ -selective and K ϩ -selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K ϩ channel (K v ) currents by Ϸ30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca 2ϩ channels or large conductance Ca 2ϩ -activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of K v channel currents and oxyhb-induced constriction of isolated cerebral arteries. The K v channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in K v channel activity could represent either channel block, or a decrease in K v channel density on the plasma membrane. To explore whether oxyhb altered trafficking of K v channels to the plasma membrane, we used an antibody generated against an extracellular epitope of K v 1.5 channels. In the presence of oxyhb, staining of K v 1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with K v 1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of K v currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

No abstract

Abstract-Cerebral aneurysm rupture and subarachnoid hemorrhage (SAH) inflict disability and death on thousands of individuals each year. In addition to vasospasm in large diameter arteries, enhanced constriction of resistance arteries within the cerebral vasculature may contribute to decreased cerebral blood flow and the development of delayed neurological deficits after SAH. In this study, we provide novel evidence that SAH leads to enhanced Ca 2ϩ entry in myocytes of small diameter cerebral arteries through the emergence of R-type voltage-dependent Ca 2ϩ channels (VDCCs) encoded by the gene Ca V 2.3. Using in vitro diameter measurements and patch clamp electrophysiology, we have found that L-type VDCC antagonists abolish cerebral artery constriction and block VDCC currents in cerebral artery myocytes from healthy animals. However, 5 days after the intracisternal injection of blood into rabbits to mimic SAH, cerebral artery constriction and VDCC currents were enhanced and partially resistant to L-type VDCC blockers. Further, SNX-482, a blocker of R-type Ca 2ϩ channels, reduced constriction and membrane currents in cerebral arteries from SAH animals, but was without effect on cerebral arteries of healthy animals. Consistent with our biophysical and functional data, cerebral arteries from healthy animals were found to express only L-type VDCCs (Ca V 1.2), whereas after SAH, cerebral arteries were found to express both Ca V 1.2 and Ca V 2.3. We propose that R-type VDCCs may contribute to enhanced cerebral artery constriction after SAH and may represent a novel therapeutic target in the treatment of neurological deficits after SAH.