Due to the high volume of blood contained in the venous circulation, a small increase in venous contraction can have a profound impact on systemic blood distribution and blood pressure. The endogenous vasoconstrictor peptide Endothelin-1 (ET-1) has been identified as a regulator of venous tone, since veins desensitize less than arteries to ET-1 and maintain contractility to ET-1 in hypertension. However, relatively little is known about the mechanisms regulating venous contraction. This project was designed to explore how increases in intracellular Ca2+ relate to venous contractility, and test the hypothesis that Ca2+ signaling induced by ET-1 differs between veins and arteries. Although contraction to ET-1 in rat aorta (RA) and rat vena cava (RVC) requires Ca2+, inhibition of voltage-gated calcium channels or nonselective cation channels did not significantly inhibit ET-1-induced contraction in either tissue. However, inhibition of the reverse-mode Na+/Ca2+ exchanger (NCX) by KB-R7943 (10 μM) significantly attenuated ET-1-induced contraction in RVC but not RA, suggesting that calcium influx by reverse-mode NCX is an important mechanism of Ca2+ influx during ET-1-induced contraction of RVC (chapter 3). We next investigated the mechanisms of intracellular Ca2+ stores release activated by ET-1 by examining the presence and function of ryanodine receptors (RyR) (chapter 4) and IP3 receptors (IP3R) (chapter 5) in RA and RVC. RA expressed mRNA for all 3 RyR subtypes, and the RyR activator caffeine (20 mM) caused a prolonged increase in intracellular Ca2+ associated with a rapid contraction. While RVC also expressed RyR mRNA, caffeine caused a small, transient increase in intracellular Ca2+ that was not associated with contraction. These data suggest that RyR, while present in both RA and RVC, are unable to release sufficient Ca2+ to cause contraction in RVC. RA and RVC express protein for all 3 IP3R subtypes, and the membrane-permeable IP3 analogue, Bt-IP3, caused contraction in both tissues. To measure ET-1-induced IP3R activation, Ca2+ wave characteristics were measured in RVC during exposure to ET-1 (100 nM). ET-1 increased Ca2+ wave frequency, occurrence and velocity in RVC, suggesting IP3-mediated Ca2+ release. However, ET-1-induced contraction was unchanged by the IP3R inhibitor 2-APB (100 μM), suggesting IP3-mediated Ca2+ release was not a significant source of Ca2+ during RVC contraction. To test if phospholipase-C (PLC) was activated by ET-1, isometric contraction was measured in RA and RVC rings exposed to vehicle, the PLC inhibitor U-73122 or its inactive analog U-73343 (1 μM). While U-73343 did not significantly inhibit contraction to ET-1, U-73122 significantly reduced maximum contraction to ET-1 in both tissues. These findings suggest that ET-1 activates PLC in RA and RVC, but DAG - and not IP3 - may regulate contraction to ET-1 in RVC.Taken together, these findings suggest that mechanisms of both extracellular Ca2+ influx and intracellular Ca2+ release are different in RVC than in RA, and that this may account for the differences in ET-1-induced contraction between RA and RVC.
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