ANTIDIABETIC BIGUANIDES INHIBIT HORMONE-INDUCED INTRACELLULAR CA2+ CONCENTRATION OSCILLATIONS IN RAT HEPATOCYTES

Rat hepatocytes respond to glycogenolytic stimuli acting via phosphoinositide breakdown (e.g. a,-adrenergic agonists, vasopressin) by oscillations of the free intracellular Ca2+ concentration ([Ca2+](i)). We have investigated the action of metformin and phenformin, two anti-diabetic drugs of the biguanide type, on phenylephrine-induced [Ca2+](i) oscillations. Metformin and phenformin lowered the frequency of the [Ca2+](i) oscillations in a concentration-dependent manner with an IC50 of 0.1 mM and 1 mu M, respectively. Simultaneous addition of the biguanides and insulin resulted in a further reduction of the frequency. By contrast, agents which increase the cellular cyclic AMP (cAMP) concentration (glucagon, forskolin, N,2'-O-dibutyryl-cAMP) reversed this inhibition. Furthermore, we investigated whether biguanides influenced the agonist-induced Ca2+ influx across the plasma membrane. When hepatocytes were loaded with the acetoxymethyl ester of fura-2 (fura-2/AM), addition of Mn2+ led to a quench of cellular fura-2, measured at the isosbestic excitation wavelength of 360 nm, until a new steady state was reached. Surprisingly, however, this addition of Mn2+ caused a marked increase of the fluorescence ratio simultaneously measured at 340 and 380 nm during the approach of the 360 nm signal to a new steady state. This observation can be understood on the basis of a compartmentalization of fura-2/AM into intracellular stores sensing the [Ca2+] therein. Subsequent application of phenylephrine resulted in a further decline of the fura-2 signal at 360 nm and a concomitant decrease of the fluorescence ratio. This second phase of the Mn2+ quench and the decrease of the fluorescence ratio could be diminished by addition of either 3 mM metformin or 30 mu M phenformin. By contrast, when hepatocytes were loaded with fura-2/pentapotassium salt via a patch pipette, only the initial Mn2+-induced quench, measured at 360 nm, but no change of the fluorescence ratio, could be observed. The subsequent addition of phenylephrine and biguanides during the on-going quench caused no further changes, except for a fading oscillatory response. After loading hepatocytes with fluo-3 acetoxymethyl ester, the cells were permeabilized with 5 mu M digitonin. Addition of inositol-1,4,5-trisphosphate (IP3) caused a rapid decrease of the remaining cellular fluorescence which could be effectively inhibited by 20 mu g/ml heparin, indicating a release of Ca2+ from intracellular compartments mediated by IP3. This IP3-induced release of Ca2+ from intracellular stores could be diminished by prior addition of metformin and phenformin. Therefore we concluded that the biguanides exerted their inhibitory effect on phenylephrine-induced [Ca2+](i) oscillations by a direct negative interference with the IP3-sensitive channel of intracellular Ca2+ stores.