Regulation of Adrenoceptor Responses in Isolated Rat Hepatocytes

1. Both alpha1- and beta-adrenoceptors stimulate glycogenolysis in the liver. In the adult male rat, however, the a-response predominates. Under several conditions including primary culture of isolated hepatocytes, there is a switch from the predominantly alpha- to a beta-adrenergic response. The primary goal of this study was to arrest this time-dependent increase in beta-adrenergic responsiveness and thus, to elucidate the factors that regulate these changes. 2. Glycogenolysis was measured as an increase in glycogen phosphorylase a activity. Initial experiments, showed that cells were responsive to drugs and hormones such as noradrenaline, isoprenaline, glucagon and insulin. The cells were also responsive to Bt2cAMP and the ionophore, A23187, which act intracellularly. 3. Responses to phenylephrine (alpha-agonist) and isoprenaline (beta-agonist) was respectively inhibited by prazosin (alpha-antagonist) and propranolol (beta-antagonist). Bmax and KD for alpha1-adrenoceptors, deteimined by [3H]prazosin binding, were 0.13 +/- 0.03 nM and 319.8 +/- 18.5 fmol mg-1 protein respectively. Values for beta-adrenoceptors, determined by [3H]dihydroaIprenoloI binding, were 1.17 +/- 0.22 nM and 56.2 +/- 4.6 fmol mg-1 protein. 4. The results showed for the first time that the relative expression of the alpha- and beta-adrenergic response depended on the culture medium in which the hepatocytes were kept. In comparison to Krebs-Henseleit buffer, beta-responses were greatly enhanced in cells cultured in Williams' E. In Krebs-Henseleit, phenylephrine was ~7 fold more potent and ~2.5 fold more effective than isoprenaline. On the contrary, results obtained in cells kept in Williams' E, showed isoprenaline to be equally potent and almost as effective as phenylephrine. Also, the addition of amino acids to Krebs-Henseleit buffer increased the beta-response in the hepatocytes. This observation, therefore, suggests that the amino acid fraction of Williams' E was responsible for the increased beta-response. 5. To examine further the role of culture medium on adrenergic responses, individual amino acids were added to Krebs-Henseleit buffer. The effects of eight amino acids (glycine, L-arginine, L-cysteine, L-glutamic acid, L-lysine, L-methionine, L-proline, and L-valine) were investigated. Addition of L-proline (30 mg l-1) to Krebs' increased the beta-response in a manner similar to that in Williams' E. Furthermore, L-arginine, L-glutamic acid, and L-methionine caused an increase in the affinity but not the efficacy of isoprenaline. L-Glutamic acid, also, decreased the affinity of phenylephrine. 6. Consistent with previous workers, incubation of hepatocyte suspensions for up to 6 hours led to increased beta-adrenergic responsiveness. Increased beta-responsiveness was not necessarily accompanied by a complete switch from ?- to beta-response-in some instances, the beta-responses did not decrease with time. 7. Measurement of cyclic AMP accumulation showed no correlation between the generation of cyclic AMP and the activation of glycogen phosphorylase induced by isoprenaline in freshly isolated cells. These, data probably, suggest the involvement of a cAMP-independent mechanism in responses to the beta-agonist, isoprenaline. 8. Dimethyl sulfoxide (2%) and sodium butyrate (2 mM), two differentiating agents used successfully to maintain some liver-specific functions, failed to arrest the time-dependent increase in beta-response in hepatocyte suspensions. Furthermore, butyrate appeared to accelerate the process. However, both agents suppressed the emergence of the beta-response in short-term (24-48 h) monolayer cultures. This may suggest that the actions of DMSO and sodium butyrate require a much longer period to exert their effects, probably, through the synthesis of new, and not through the modification of preformed, protein(s). 9. Dexamethasone (0.1-1.0muM) failed to prevent the increased beta-adrenergic response in cultured hepatocytes. On the contrary, dexamethasone increased the beta-response, presumably, by upregulation of beta-adrenoceptors. The effect of dexamethasone was blocked by cycloheximide (2 muM). However, it must be pointed out that cycloheximide, also, inhibited the increased beta-adrenergic responses in control (untreated) cells. 10. The ornithine decarboxylase and arginase inhibitor, (+)-S-2-amino-5- iodoacetamidopentanoic acid (200 ?M), suppressed the time-dependent increase in adrenergic response in isolated hepatocyte suspensions. Also, putrescine (200 muM), a poly amine and a competitive inhibitor of poly amine biosynthesis, arrested the increased beta-adrenergic responsiveness