Abstract
In isolated rat hepatocytes, ATP and ADP (10(-6) M) rapidly mobilize intracellular Ca2+ and increase the concentration of free cytosolic Ca2+ ([Ca2+]i) within 1-2 s. The increase in [Ca2+]i is maximal (2.5- to 3-fold) by about 10 s and is dose-dependent, with ATP and ADP being half-maximally effective at 8 X 10(-7) and 3 X 10(-7) M, respectively. At submaximal concentrations, the rise in [Ca2+]i is transient due to hydrolysis of the agonist. The increase in [Ca2+]i in response to ATP or ADP can be potentiated by low concentrations of glucagon (10(-9) M). In addition, the [Ca2+]i rise can be antagonized in a time- and dose-dependent manner by the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. Adenosine, at concentrations as high as 10(-4) M, does not alter [Ca2+]i. AMP is ineffective at 10(-5) M, but at 10(-4) M it increases [Ca2+]i approximately 1.5-fold after a 30-s lag and at a slow rate. Conversely, high concentrations (10(-4) M) of adenosine and AMP increases cell cAMP about 2- to 3-fold. ATP and ADP, at concentrations (10(-6) M) which near-maximally increase [Ca2+]i, do not affect hepatocyte cAMP. ATP and ADP increase the cellular level of myoinositol 1,4,5-trisphosphate (IP3), the putative second messenger for Ca2+ mobilization. The increase in IP3 is dose-dependent and precedes or is coincident with the [Ca2+]i rise. There is an approximate 20% increase in IP3 with concentrations of ATP or ADP which near-maximally induce other physiological responses. It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+ and activate phosphorylase in hepatocytes due to generation of IP3. These effects may involve P2-purinergic receptors. In contrast adenosine and AMP interact with P1 (A2)-purinergic receptors to increase cAMP.
Highlights
From the Laboratories for the Studies of Metabolic Disorders, Howard Hughes Medical Institute and Departmntof Physiology, Vanderbilt University School of Medicine, Nashville,Tennessee37232
At concentrations as high as M, does not alter [Ca2+Ii.AMP is ineffective at lo-‘ M, but at investigated in the liver, only a few studies have been concerned with the hepatic responses to adenosine/AMP and ADP/ATP which are mediated by PI- and Pa-purinergic receptors, respectively
Stock solutions of ATP (100 mM), ADP (100 mM), AMP (10 mM), and adenosine (10 mM) were prepared in water, titrated to pH6.8, and the concentration verified by the absorbance a t 259 nm
Summary
It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+and activate phosphorylase in hepatocytes due to generation of IPS To examine the hydrolysis of ATP orADP by isolated hepatocytes, 1-ml aliquots of cell suspension were removed into liquid nitrogen at various times after the addition of tritiated ATP or ADP (1 pCi/ml abolishes its ability to raise [Ca"']: (data not shown). This potency series suggests that ATP andADP act through the P2-purinergicreceptor [13, 14]. At a concentration (1 p ~ w)hich near-maximally raises [Ca2+Ii,neither ATP nor ADP is able to significantly
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