Studies on the role of cyclic guanosine 3':5'-monophosphate and extracellular Ca2+ in the regulation of glycogenolysis in rat liver cells.

R H Pointer,F R Butcher,J N Fain

doi:10.1016/s0021-9258(17)33489-0

Abstract

Catecholamines increased guanosine 3':5'-monophosphate (cyclic GMP) accumulation by isolated rat liver cells. The increases in cyclic GMP due to 1.5 muM epinephrine, isoproterenol, or phenylephrine were blocked by phenoxybenzamine but not by propranolol. The possibility that cyclic GMP is involved in the glycogenolytic action of catecholamines seems unlikely since cyclic GMP accumulation is also elevated by carbachol, insulin, A23187, and to a lesser extent by glucagon. Furthermore, carbachol had little effect on glycogenolysis while insulin actually inhibited hepatic glycogenolysis. The rise in cyclic GMP due to carbachol was abolished by atropine and that due to all agents was markedly reduced by the omission of extracellular calcium. However, the glycogenolytic action of glucagon and catecholamines was only slightly inhibited by the omission of calcium. The only agent which was unable to stimulate glycogenolysis in calcium-free buffer was the divalent cation ionophore A23187. There was a drop in ATP content of liver cells during incubation in calcium-free buffer which was accompanied by an inhibition of glucagon-activated adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The presence of calcium inhibited the rise in adenylate cyclase activity of lysed rat liver cells due to glucagon or isoproterenol but not that due to fluoride. These results suggest that the stimulation by catecholamines and glucagon of glycogenolysis is not mediated through cyclic GMP nor does it depend on the presence of extracellular calcium. Cyclic GMP accumulation was increased in liver cells by agents which either inhibit, have little affect, or accelerate glycogenolysis. The significance of elevations of cyclic GMP in rat liver cells remains to be established.

Highlights

Any simple interpretation which assumes that cyclic GMP is responsible for the (Y stimulation of glycogenolysis by epinephrine was complicated by the finding that other agents which either inhibited or stimulated glycogenolysis (A23187)
The rise in cyclic GMP due to insulin, carbachol, or A23187 was more rapid in onset than was that due to epinephrine with the maximal rise at 120 s being sustained at 5 min (Fig. 1)
On isolated rat Cyclic AMP’ at 2 min liver cells Cyclic GMPd at 2 min Inhibited No change No change Increased via a effect Increased

Summary

AND METHODS

$Predoctoral Fellow of the National Fellowship Fund. Present address, Department Medicine, Nashville,. Each experiment used cells obtained from the same rat but separate tubes were used for cyclic nucleotide and glycogen determinations. The succinylation reaction was carried out in 15.ml conical glass centrifuge tubes containing 200 ~1 of the sample or cyclic GMP standards dissolved in glass-distilled water. The total assay sample cyclic GMP or succinylated cyclic GMP standards, antigen, and antibody were dissolved in 50 rn~ sodium acetate, pH 6.2, and each added in 50.~1 aliquots to culture tubes (12 x 75 mm) in an ice bath. The liver ghosts (400 ~g of protein) were added to incubation tubes containing Tris buffers, 40 mu (pH = 8.0); M&l,, 5 mu; KCl, 30 rn~; ATP, 1 rn~; and an ATP-regenerating system consisting of 8 rn~ creatine phosphate and.

RESULTS

DISCUSSION

Summary