Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets.

We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state.

We tested the hypothesis that the adenylate cyclase system and Na+,K+-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+,K+-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+,K+-ATPase activity (44,62, and 43%, respectively rat kidney and in a purified porcine brain Na+,K+-ATPase preparation. The adenylate cyclase system and Na+,K+-ATPase may act reciprocally in pancreatic islets and in other tissues. In the B cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state. The sodium-and potassium-dependent adenosinetriphosphatase (EC 3.6.1.3) (Na',K'-ATPase) is responsible for the maintenance of the resting transmembrane potential in tissues, such as nerve and muscle (1, 2). It has been suggested that inhibition of this enzyme participates in the mechanical and ionic events that comprise, for example, myocardial contractility (3) and neurotransmitter release (4). Although stimulus-secretion coupling is similarly accompanied by ionic and intracellular contractile events (5), a role for inhibition of Na+,K+-ATPase has not been proven to modulate hormone secretion. Our laboratory has measured activity of this enzyme in pancreatic islets and has shown that most insulin secretagogues that we have tested inhibit the enzyme (6, 7); conversely, inhibitors of insulin secretion augment the activity of this enzyme in islets (6,8). In contrast, activation of the adenylate cyclase system is associated with p cell membrane depolarization (9) and insulin secretion ( Islets were visualized using a dissecting microscope and picked with a Pasteur pipette. The preparation of islets for the enzyme assay was as previously described (6) and is summarized below.

Islet Storage
Islets were harvested into a solution containing 0.25 M sucrose and 1 mM Tris-EDTA and frozen at -70 "C until homogenized for the enzyme assay. Islets were collected (250-350/rat) over a few days and pooled into each collecting tube until 1000 islets/tube were accumulated.

Membrane Preparation
Islets-One thousand islets were used for each experiment. Islets were thawed and homogenized in a 2-ml Ten Broeck tissue grinder with six or seven vigorous passes of the plunger. The homogenates were centrifuged at 35,000 x g for 35 min at 5 "C. The pellet was resuspended in 2.6 ml of cold 1 mM Tris-EDTA (pH 7.4) and divided into l.O-ml aliquots for subsequent assay. Each aliquot contained the membrane tissue of approximately 40 islets. The pellet material was used within 2-5 days after homogenization to prevent loss of enzyme activity.

A.
Kidney-Outer renal medulla was obtained and processed also as previously described (6

RESULTS
The dose response for inhibitory effects of theophylline upon islet Na+,K+-ATPase is shown in Fig. L4. Control activity of Na',K'-ATPase was 8.39 f 0.87 pmol of Pi/mg of protein/h. Over a dose range of 0.5-5 mM, theophylline evoked a dose-dependent inhibition of Na',K'-ATPase activity. Empirically, the approximate half-maximal inhibitory dose of theophylline was found to be 1 mM. Fig. 1B shows that control Mg'+-ATPase activity was 30.77 + 3.57 pmol of PJmg of protein/h, and this activity was not affected by theophylline. The effects of other substances that alter the activity of the adenylate cyclase system are shown in Fig. 2A 3), theophylline decreased both V,,,,, and apparent K,,, (ATP).
Similar to the effect of theophylline, BtzcAMP decreased V,.,; however, in contrast to theophylline, Bt+AMP increased apparent K,,, (ATP). Table I summarizes these kinetic findings with statistical analysis. Apparent K,,, was determined for individual experiments. The mean value for theophylline and Bt*cAMP was compared with that of control incubations using the unpaired t test. The changes in V,,,., and apparent K,, described above, were statistically significant. Table II summarizes effects of theophylline and Bt,cAMP upon renal and brain Na+,K+-ATPase, and M$+-ATPase activities. These compounds had effects upon Na',K'-ATPase which simulated those observed in the islet preparation. Theophylline and Bt*cAMP tended to enhance renal Mg*+-ATPase, but this was not significant.
This tendency was not seen in either islets or brain. Results shown as mean + SE. *, p < 0.001 uersas control. B, lack of effects of caffeine (1 mM) and B&CAMP (1 mM) on rat pancreatic islet Mg2'-ATPase. Results shown as mean + SE.

Both theophylline
and BtzcAMP inhibited islet Na+,K+-ATPase activity; however, neither acted by classical competitive or noncompetitive mechanisms. Both substances reduced the observed maximum velocity of the reaction. However, theophylline increased and B&CAMP decreased the measured affinity for ATP. Neither substance affected Mg2+-ATPase activity.
In the presence of glucose, theophylline, and Bt2cAMP both depolarize pancreatic B-cells and induce hormone release (9). Depolarization, accompanied by insulin secretion, is also produced by ouabain, the classic inhibitor of Na',K+-ATPase, and the electrical and secretory patterns can be quite similar to those with theophylline, depending on the concentration of ouabain and background glucose (16); thus, there may be an interplay between these substances upon the secretory mechanism, which these present studies begin to investigate.
These present experiments point out that there can be indirect inhibition of Na',K'-ATPase via CAMP production. Current ideas about glucose-induced insulin release suggest that a major depolarizing and secretogenic step is ATP blockage of K+ channels (18, 19). Using "jRb+ as a tracer which   to effects on K+(Rb+) permeability, but Na+,K+-ATPase activity was not measured ('20). We have described the inhibition of islet Na+,K+-ATPase by arginine and ouabain (6). We propose that the secretogenic properties of Adenylate Cyclase and Na+,K+-ATPase m Pancreatrc Islets cholmerglc agonists and argmme may be due to ouabam-like effects on the islet B-cell plasma membrane Glucose and leucme mhlblt K' efflux, and this 1s attributed to closure of ATP-sensitive K' channels (18-21) Slmdarly, tolbutamlde, the sulfonyl urea secretory stimulant, can close K' channels, while effects of an mhlbltor, dlazoxlde, have been ascribed to openmg of these channels (22) In contrast, the ammo acid secretagogues argmme and lysme increase K' efflux (23), but the authors theorize that the depolarlzmg and secretogemc effects of catlomc ammo acids are due to accumulation of these substances m B-cells Based on our observations, we suggest that Na+,K+-ATPase and adenylate cyclase act reciprocally during the process of secretion, smce theophyllme and BtzcAMP mhlblt Na+,K+-ATPase Reclproclty 1s further suggested by experiments m pancreatic islets showing that ouabam will enhance glucoseinduced CAMP when a CAMP phosphodlesterase mhlbltor 1s present (24) Reclproclty of these two enzyme systems has been reported m non-islet tissues, CAMP has been shown to mhlblt Na',K'-ATPase m rat brain (25) This 1s consistent with our present findings m porcine cerebral cortex and m rat renal particulate fractions Thus, It seems reasonable to consider modulation of Na',K'-ATPase by the adenylate cyclase system to be widespread m a variety of tissues Because adenylate cyclase actlvatlon 1s associated with msulm release, this enzyme system should utlhze ATP preferentially during secretion On the other hand, since, m a variety of tissues, Na',K'-ATPase 1s associated with cellular polanzatlon, this enzyme would utlhze ATP to mamtam the nonsecretory state Suppression of ATPases would be one mechanism to provide ATP at the plasma membrane for closure of ATPsensltlve K+ channels, adenylate cyclase actlvlty, phosphatldylmosltol turnover, and a variety of other cell functions Some secretagogues might stimulate secretion m a ouabamlike fashion These would enhance K' efflux Others, hke glucose, while suppressmg Na+,K+-ATPase (6) but also provldmg ATP during oxldatlon might evoke more intricate mteractlons with other islet structures, ion channels, and metabolic pathways 1 2 3 4 5 6 7 8