Ionic mechanism for contractile response to hyposmotic challenge in canine basilar arteries

A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca2+ and by the voltage-dependent Ca2+ channel (VDCC) blocker nicardipine, but it was unaffected by thapsigargin, which depletes intracellular Ca2+ stores. The contraction was also inhibited by Gd3+ and ruthenium red, cation channel blockers, and Cl− channel blockers DIDS and niflumic acid. The reduction of extracellular Cl− concentrations enhanced the hypotonically induced contraction. Patch-clamp analysis showed that a hyposmotic challenge activated outwardly rectifying whole cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl− concentration, indicating that the currents were carried by Cl−. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca2+. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca2+ influx, thereby activating Ca2+-activated Cl− channels. The subsequent membrane depolarization is likely to increase Ca2+ influx through VDCC and elicit contraction.

Nicotine Exposure, Mimicked Smoking, Directly and Indirectly Enhanced Protein Kinase C Activity in Isolated Canine Basilar Artery, Resulting in Enhancement of Arterial Contraction

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10−6 mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5′-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.