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The role of extracellular calcium in hormone-induced glycogenolysis was examined in a rat liver perfusion system by manipulating the perfusate calcium concentration and by using calcium antagonistic drugs. When the perfusate contained 1 mM CaCl2, 5 microM phenylephrine, 20 nM vasopressin, and 10 nM angiotensin II caused a persistent increase in glucose output and phosphorylase activity as well as a transient increase in 45Ca efflux from 45Ca preloaded liver. Verapamil hydrochloride (20-100 microM) inhibited the activation of glucose output by these hormones in a dose-dependent manner. This inhibitory effect was also associated with the inhibition of hormone-induced activation of phosphorylase and 45Ca efflux. In the absence of CaCl2 in the perfusate, the glycogenolytic effect of phenylephrine and its inhibition by verapamil were obtained equally as in the presence of CaCl2. However, the effects of vasopressin and angiotensin II were markedly attenuated and were not inhibited any further by verapamil. The substitution of diltiazem hydrochloride for verapamil produced essentially identical results. Cyclic AMP concentrations in the tissue did not change under any of these test conditions. The results indicate that the glycogenolytic effect of alpha-adrenergic agonists depends on intracellular calcium but those of vasopressin and angiotensin II on extracellular calcium, and support the concept that calcium antagonistic drugs inhibit the glycogenolytic effects of calcium-dependent hormones at least by inhibiting the mobilization of calcium ion from cellular pools.

The glycogenolytic effect of catecholamine has been proposed to be mediated by calcium ions mobilized mainly from mitochondria. In this study, the contribution of mitochondria as a source of the mobilized calcium was evaluated by use of perfused rat livers. Phenylephrine-induced efflux of 45Ca from 45Ca preloaded liver was abrupt and transient, and almost ceased within 5 min. The perfusion with 5 microM phenylephrine for 5 min caused a significant decline in the content of 45Ca retained in the liver homogenate; control, 0.97 +/- 0.11 nmol of 45Ca/mg protein (mean +/- SEM) and phenylephrine, 0.41 +/- 0.08 nmol of 45Ca/mg protein (P less than 0.01), when calculated from the specific radioactivity of calcium in the perfusate used for loading perfusion. The contents of 45Ca in mitochondria isolated subsequently from the homogenate were 0.43 +/- 0.07 nmol/mg protein in controls and 0.22 +/- 0.03 nmol/mg protein (p less than 0.05) when phenylephrine was administered. Taking into consideration that mitochondrial protein is a fraction of homogenate protein, it is obvious that the decline in mitochondrial 45Ca represents only a small portion of the total reduction of radioactivity in the homogenate. In a series of experiments in which various perfusion periods were employed for 45Ca loading and for washing-out, it was found that phenylephrine induced 45Ca efflux also from a pool with a calcium turnover rate slower than that if mitochondria. These results suggest that alpha-adrenergic stimulation enhances release of calcium not only from mitochondria but also from other compartments with a slower turnover rate. The probability of plasma membrane is proposed as the other source of calcium released in response to alpha-adrenergic stimulation.

In an attempt to elucidate the role of Ca2+ flux in the initial events of hepatic glycogenolysis, extracellular Ca2+ concentration was manipulated in rat liver perfused with Ca2+ antagonistic drugs. After the liver had been perfused with a buffer containing verapamil and 1 mM CaCl2, either the addition of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid to the perfusate or the replacement of the perfusate with Ca2+-free buffer caused a rapid increase in glucose output as well as 45Ca2+ efflux. Substitution of diltiazem, but not 5-20 mM LaCl2, for verapamil also stimulated glucose output and 45Ca2+ efflux. However, when Ca+-free buffer was used throughout the experiment, any modes of verapamil or diltiazem perfusion were without significant effects on glucose output or Ca2+ efflux. The increases in glucose output and 45Ca2+ efflux were not affected by either 20 microM phentolamine or 300 microM ouabain, but they were inhibited significantly by 10-100 microM trifluoperazine. These results indicate that rapid decline in the extracellular Ca2+ concentration in verapamil- or diltiazem-perfused liver initiates the change in Ca2+ equilibrium on or across plasma membrane and activates glycogenolysis through a Ca2+-dependent mechanism.

The role of extracellular calcium in the glycogenolytic effects of calcium-dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II.