Anti-diabetic biguanides inhibit hormone-induced intracellular Ca2+ concentration oscillations in rat hepatocytes.
Rat hepatocytes respond to glycogenolytic stimuli acting via phosphoinositide breakdown (e.g. alpha 1-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 microM, 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 microM phenformin. By contrast, when hepatocytes were loaded with fura-2/pentapotassium salt via a patch pipette, only the initial Mn(2+)-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 microM 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 micrograms/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.(ABSTRACT TRUNCATED AT 400 WORDS)
Ubl
J J
JJ
Pharmakologisches Institut, Universität Bern, Switzerland.
Chen
S
S
Stucki
J W
JW
D000818:Animals; D001645:Biguanides / Q000494:pharmacology*; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D017895:Manganese Compounds / Q000378:metabolism; D008687:Metformin / Q000494:pharmacology; D010629:Phenformin / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015290:Second Messenger Systems
D001645:Biguanides; D002712:Chlorides; D007328:Insulin; D017895:Manganese Compounds; D005576:Colforsin; D010656:Phenylephrine; D003994:Bucladesine; D015544:Inositol 1,4,5-Trisphosphate; D005934:Glucagon; D008687:Metformin; D010629:Phenformin; D000242:Cyclic AMP; C025340:manganese chloride; D002118:Calcium