INOSITOL 1,4,5-TRISPHOSPHATE AND CALCIUM REGULATE THE CALCIUM-CHANNELFUNCTION OF THE HEPATIC INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR

The regulation of the inositol 1,4,5-trisphosphate (IP3) receptor in l iver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with Ca-45(2+), and superfused at high flow rates to prov ide precise control over IP3 and Ca2+ concentrations ([Ca2+]) and to i solate Ca-45(2+) release from reuptake. Ca-45(2+) release was dependen t on both [Ca2+] and IP3. The initial rate of Ca-45(2+) release was a biphasic function of [Ca2+], increasing as [Ca2+] approached 3 mu M bu t decreasing at higher concentrations, suggesting that the hepatic IP3 receptor is regulated by [Ca2+] at two sites, a high affinity potenti ation site and a low affinity inhibitory site. The relationship betwee n initial rates and IP3 concentration was steep (Hill coefficient of 3 .4), suggesting that activation of the calcium channel requires bindin g of at least 3 IP3 molecules. IP3 concentrations above 10 mu M produc ed rapid decay of release rates, suggesting receptor inactivation. Sup erfusion with 10 mu M IP3 under conditions that minimize calcium relea se ([Ca2+] < 1 mM) inhibited Ca-45(2+) release in response to subseque nt stimulation (400 nM Ca2+). These data suggest sequential positive a nd negative regulation of the hepatic IP3 receptor by cytosolic calciu m and by IP3, which may underlie hepatocellular propagation of regener ative, oscillatory calcium signals.