Cellular Signalling Vol. 2. No. 3. pp. 235-243. 1990. 0898--6568/90 $3.00 + .00 Printed in Great Britain. © 1990 Pergamon Press plc MODULATION OF GLUCAGON ACTIONS BY PHORBOL MYRISTATE ACETATE IN ISOLATED HEPATOCYTES. EFFECT OF HYPOTHYROIDISM J. ADOLFO GARCiA-S,~INZ,* t MARINA MACiAS-SILVA,* S. M. TERESA HERNANDEZ-SOTOMAYOR,* MA. EUGENIA TORRES-M,~RQUEZ,*~ DEV TRIVEDI§ and VICTOR J. HRUBY§ *Instituto de Fisiologia Celular, Universidad Nacional Aut6noma de M6xico; ~Departamento de Bioquimica, Instituto Nacional de Cardiologia, M6xico; and §Department of Chemistry, University of Arizona, Tucson, Arizona, U.S.A. (Received 20 October 1989; and accepted 8 November 1989) Abstract--Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumu- lation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2÷] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action. Key words." Glucagon, protein kinase C, phorbol esters, ureagenesis, cyclic AMP, calcium. INTRODUCTION PHORBOL 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, inhibits glucagon-stimulated adenylate cyclase activity in liver membranes and the accumulation of cyclic AMP in hepatocytes [I-5]. Such dimin- ution of glucagon-stimulated cyclic AMP accumulation is accompanied by a slight shift to the right in the dose-response curve to gluca- gon for some metabolic actions, such as phos- phorylase activation [3] or ureagenesis [2] in rat *Author to whom correspondence should be addressed at: lnstituto de Fisiologia Celular, UNAM, Ap. Postal 70- 248, 04510 M~xico D.F., Mexico. Ahhreviations." Butz-cAMP--dibutyryl cyclic adenosine 3', 5' monophosphate: MIX--methyl isobuthyl xanthine; PMA--phorbol 12-myristate 13-acetate; THG--(I-Nct- trinitrophenyl histidine, 12-homoarginine)-glucagon; KRB--Krebs-Ringer bicarbonate buffer. hepatocytes from euthyroid animals. Interest- ingly, using hepatocytes from hypothyroid ani- mals, we observed that PMA markedly inhibits the stimulation of ureagenesis by glucagon [4, 5]. These data prompted us to investigate (com- paratively) the effect of this tumour promoter on glucagon actions, using hepatocytes from euthyroid and hypothyroid rats, in order to gain an insight into the reason(s) for such dif- ference in sensitivity to PMA. 235 MATERIALS AND METHODS Phorbol 12-myristate 13-acetate, 6-N-propyl-2- thiouracil, rotenone, methyl-isobutyl-xanthine (MIX), L-glutamine, L-ornithine, urease, glucose oxi- dase, peroxidase, dibutyryl cyclic AMP (But2-cAMP) and Quin 2/AM were obtained from the Sigma Chemical Co. THG, ([l-N~-trinitrophenyl-histidine, 12-homoarginine]-glucagon), an analogue of gluca- gon, was synthesized and purified as described [6].