635th MEETING, ABERDEEN while in the absence of Na+, uptake was inhibited by 10 PMnitrobenzylthioinosine (NBMPR), an inhibitor of facilitated diffusion nucleoside transport. Thus the Na+-dependent and N a + -independent nucleoside transporter systems found in renal membrane vesicles were present in OK cells. The properties of Na+ -dependent nucleoside transport in OK cells were investigated further and are described in this report.Na +-dependent uridine uptake increased in activity from basal levels to maximum activity approximately 7 days after confluency. Uptake was also entirely dependent on the presence of Na+; replacement of Na+ by Li', K + , Rb' or choline + reduced uptake to levels not significantly different to control, defined as uptake in the presence of N-methyl+glucamine + .The uptake of ("Hlguanosine was saturable with respect to both guanosine and Na' and was apparently consistent with a one-site model. The apparent K, for guanosine uptake was 52 f 13 PM (mean f s.D., n = 3) and the V,,, was 47 f 15 pmol min-' mg protein-'. The K,, for Na' was 5 5 f 14 mM. Uridine uptake was also saturable with respect to Na' (K,, 63f5 mM), but, surprisingly, not with respect to uridine itself. This was explained by the inability of uridine to completely inhibit its own uptake (maximum inhibition was 40% with a concentration required to inhibit by 50n% (lC511) of 5 f 2 p~) .Similarly, adenosine only caused 40% inhibition of uridine uptake (lC5,1 value of 7.9 f 2.3 p~) .In contrast, the dose-response curves for inhibition of uridine uptake by inosine and guanosine were biphasic (lC51, values of 2.1 f 0.5 p~ and 5.0 k 0.7 mM for inosine and 14 f 5.7 and 590 f 270 p~ for guanosine). Thymidine did not cause any significant inhibition.Previous studies [7] have shown that bovine renal outer cortical brush-border-membrane vesicles possess two Na+dependent nucleoside transporters: one with a substrate specificity mainly for purine nucleosides and the other with a preference for pyrimidine nucleosides. Interestingly, adenosine and uridine are permeants for both systems. The inability of thymidine to inhibit uridine uptake in OK cells suggests that these cells possess only the purine nucleoside transporter. To investigate further the substrate specificity of the putative transporters, additive inhibition experiments of uridine uptake were carried out where the dose-response of one nucleoside (the secondary inhibitor) was analysed in the 1247 absence and presence of a fixed concentration ( 100 p~) of a second nucleoside (the primary inhibitor). In all experiments carried out with a variety of combinations of nucleosides, the primary inhibitor blocked the high-affinity component of the secondary inhibitor. This suggests that this component of inhibition for each nucleoside represents the same carrier with a substrate specificity similar to that of the Na+dependent purine nucleoside transport system in bovine renal brush-border vesicles.Following these observations, the saturability of uridine with respect to itself was analyse...

Oral administration of SaRI 59-801 (DL-alpha-[dimethylaminomethyl]-2-[3-ethyl-5-methyl-4-isoxazolyl]-1H- indole-3-methanol) has been reported to decrease blood glucose in several species and to elevate plasma insulin in rats and mice. In studies with isolated rat pancreatic islets incubated 1 h with 3 mM glucose, 0.05 mM 59-801 produced a significant increase in insulin secretion, and 0.3 mM produced maximum release. 59-801 (0.3 mM) stimulated insulin release 4-5-fold from islets incubated with 0, 3, or 5 mM glucose but had little effect on the high rates of release obtained at 10 or 20 mM glucose. Ten millimolar mannoheptulose, which inhibits phosphorylation of glucose and blocks glucose-stimulated insulin release, had little effect on the stimulation of insulin release by 0.3 mM 59-801 from islets incubated with 3 mM glucose. Stimulation of insulin release in the absence of glucose or in the presence of 3 mM glucose plus 10 mM mannoheptulose suggests that glucose metabolism is not required for the action of 59-801. The rate of conversion of 5 mM [5(-3)H]-glucose to 3H2O by islets, a measure of the rate of glycolysis, was not affected by 59-801. The potency, dependency on glucose concentration, lack of inhibition by mannoheptulose, and lack of effect on glycolysis of 59-801 were similar to that of tolbutamide. However, proinsulin synthesis by islets incubated with 5.55 mM glucose was not affected by 0.5 mM 59-801, but was inhibited 72% and 67% by 0.5 mM tolbutamide and 0.1 mM glibenclamide, respectively.

The mechanism of stimulation of insulin release from isolated rat islets by 0.3 mM SaRI 59-801 (DL-alpha-dimethylaminomethyl-2-[ 3-ethyl-5-methyl-4-isoxazoyl]-1H-indole-3-methanol) was investigated, considering cAMP concentration and Ca2+ uptake. Ten millimolar theophylline or 1 mM 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, each greatly increased the stimulation of insulin release by 59-801. Forskolin (0.1 mM), an activator of adenylate cyclase, or 1 mM dibutyryl cAMP also potentiated 59-801, suggesting that 59-801 does not elevate islet cAMP but is potentiated by other compounds that do. Measurement of cAMP in islets by radioimmunoassay confirmed that it was not significantly elevated by 59-801 but was increased sevenfold by forskolin or 1-methyl-3-isobutylxanthine. SaRI 59-801 was not effective in the absence of Ca2+ and presence of 1 mM EGTA. Agents that block entry of Ca2+ into beta-cells, verapamil, nifedipine, or CoCl2, inhibited the release of insulin in response to 59-801. Studies of 45Ca2+ uptake by isolated islets revealed an increased uptake in the presence of 59-801 and blockage of this effect by 50 microM verapamil. Thus, the stimulation of insulin secretion by 59-801 appears to involve a stimulation of Ca2+ uptake rather than an increase of cAMP concentration. The mechanism of stimulation of Ca2+ uptake by 59-801 requires further investigation.