Use of a newly developed technique to isolate rat pinealocytes and study the effects of adenosine agonists on melatonin production.

Recent studies have suggested a role for adenosine in the regulation of rat pineal melatonin synthesis. The data, however, are conflicting and therefore the aim of this study was to characterize adenosine receptors more fully in vitro by using a range of selective adenosine agonists and the adenosine antagonist 8-sulphophenyltheophylline (8-SPT). A simple method for the mechanical separation of rat pinealocytes was developed. Pinealocytes were briefly (15 min) incubated with drugs followed by a 4 hr drug-free incubation period after which melatonin concentrations in the incubation medium were measured by radioimmunoassay. The beta-adrenoceptor agonist isoprenaline gave a dose-related increase in melatonin production, demonstrating that this pinealocyte preparation technique is suitable to evaluate the effect of drugs on pineal melatonin synthesis. Our results show that adenosine, N6-(phenylisopropyl)adenosine (R-PIA) and 2-p-(2-carboxethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680) did not affect melatonin synthesis alone or in combination with isoprenaline. However 5'-N-ethylcarboxamidoadenosine (NECA) (100 microM) potentiated the stimulatory effect of isoprenaline (3 microM) on pineal melatonin production and this effect appeared to be antagonized by 8-SPT (50 microM). These results are consistent with activation by NECA of an A2b adenosine receptor.