We examined the effects of reduced Na ؉ /K ؉ -ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na ؉ /K ؉ -ATPase, augmented 16.7 mmol/l glucose-induced insulin release in the early period but suppressed it after a delay of 20 -30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na ؉ /K ؉ -ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca 2؉ -depleted or Na ؉ -depleted conditions. That mitochondrial membrane hyperpolarization and O 2 consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, ␣-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion. efficacy in stimulation-secretion coupling, which also is dependent on the accelerated glucose metabolism that correlates with increments in the ATP/ADP ratio. Regulation of the ATP level in pancreatic ␤-cells, therefore, plays a crucial role in insulin secretion. Na ϩ /K ϩ -ATPase is involved in maintaining Na ϩ and K ϩ gradients across the ␤-cell plasma membrane and is thought to consume a large amount of ATP in the maintenance of homeostasis (2,3). Accordingly, the role of Na ϩ / K ϩ -ATPase in the regulation of the intracellular ATP levels in ␤-cells is of interest. We have reported that the ATP content of an islet unexpectedly decreases in the presence of glucose when Na ϩ /K ϩ -ATPase activity is diminished by ouabain, despite reduced consumption of ATP by the enzyme, whereas decrease of ATP content is not observed using thapsigargin, an inhibitor of another ATP consumer, the Ca 2ϩ -ATPase in endoplasmic reticulum (4,5). Inhibition by ouabain of glucose oxidation (6) and glucose utilization (7) has been reported previously in rat islets. However, the effect of ouabain on glucose-induced insulin release is complex. Ouabain has a stimulatory effect on glucose-induced insulin secretion in the early phase (8 -10), probably owing to increased Ca 2ϩ influx (10 -12). Such increased Ca 2ϩ influx by ouabain should be due to depolarization, since the electrogenic effect of Na ϩ /K ϩ -ATPase is to hyperpolarize the membrane potential (2). On the other hand, ouabain decreases glucoseinduced insulin release in th...

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Islet transplantation corrects chronic hyperglycemia by augmentation of insulin supply from the graft tissue, but the role of endogenous beta-cells after transplantation is not clear. In the present study, we examined endogenous beta-cell function after glucose homeostasis had been reestablished by islet graft in streptozotocin (STZ)-induced diabetic rats. Fed plasma glucose levels in diabetic rats transplanted with a large number of islets (2500 islets) into the left kidney capsule soon became lower (139.8 +/- 8.2 mg/dl) and close to the level in controls (129.7 +/- 11.3 mg/dl), and IPGTT exhibited a pattern of plasma glucose response almost identical to control. The insulin and DNA contents, islet area, and the distribution of beta-cells that were markedly deteriorated in islets of STZ rats were significantly restored in transplanted rats. The insulin release in response to glucose or alpha-ketoisocaproate was less in STZ rats, while in islets of transplanted rats the secretion recovered to levels similar to controls. On the other hand, arginine-induced insulin release was conversely hyperresponsive in STZ rats, but in transplanted rats, the response was decreased similar to controls. Thus, as the plasma glucose level normalizes, residual beta-cells show a recovery of function that cannot be accounted for by the increase in mass alone.

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.

Inner ear fluids were studied biochemically in guinea pigs from 1 to 16 weeks after producing endolymphatic hydrops by obstructing the endolymphatic duct. Fluid collected from beneath the footplate showed changes of 50% of the animals indicating distension of the saccule. There was an increase in potassium concentration and decrease in sodium concentration of the collected fluid, indicating a traumatic mixing of endolymph and perilymph produced by rupture of the saccule during collection of the fluid. The smallest changes occurred at one week, and the greatest changes were found four months after endolymphatic sac obstruction. Similar findings were observed in studying inner ear fluids obtained from patients with Ménière's disease. Cochlear endolymph showed biochemical alterations after endolymphatic duct blockage. There was a two- to three-fold increase in sodium concentration and a decrease in potassium concentration with the total ionic concentration remaining approximately the same. Some guinea pigs showed similar changes in vestibular endolymph. This study indicates that in the guinea pig, endolymph obtained in the distended endolymph compartment has a slightly different sodium-potassium ratio as compared to the normal ear. In patients with Ménière's disease there may also be an elevation of endolymph sodium concentration. The significance of this change and hearing loss observed in experimental endolymphatic hydrops and Ménière's disease is open to speculation at the present time.