ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic beta-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 microM and as high as 500 microM, with maximal activation between 10 and 100 microM ADP in mouse beta-cell membrane patches. At concentrations greater than 500 microM, ADP inhibited K(ATP) channels while 10 mM virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mM ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of beta-cell membrane potential, increasing ADP in the 10 and 100 microM range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize beta-cells. The results suggest that (i) K(ATP)-channel activity is not well predicted by the "ATP/ADP ratio," and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10-100 microM range as suggested by biochemical studies.

I Potential changes in isolated superior cervical ganglia of the rat produced by muscarinic-receptor agonists were recorded by an extracellular 'air-gap' method. Depolarization was accompanied by facilitation of submaximal ganglionic transmission. 6 Muscarine (1 to 100 gM) initially reduced, then increased, the rate of 86Rb+-efflux from isolated ganglia at both 6 and 120 mM [K ]. These effects were reduced by 1 AM hyoscine.7 No consistent change in the amounts of cyclic 3',5'-guanosine monophosphate in isolated ganglia accompanying muscarinic depolarization could be detected.8 Mean against ED5o values (pM) for contracting the rat isolated ileum were: oxotremorine 0.012, methylfurmethide 0.29, (±)muscarine 0.48, pilocarpine 7.8 and AHR-602 9.9. Mean antagonist K, values (nM) were: hyoscine 0.17, atropine 0.34 and lachesine 0.27. 9 It is concluded that ganglionic muscarinic receptors are quite similar to ileal receptors in terms of agonist ED50 and antagonist K, values, and that the major difference between them lies in the greater 'efficacy' of certain agonists (pilocarpine, AHR-602 and McN-A-343) on the ganglion.

Extracellular inorganic phosphate (ePi) is a key regulator of cementoblast behavior, both in vivo and in vitro, and results in a marked increase in osteopontin expression in vitro. To examine the molecular mechanisms involved in ePi induction of osteopontin gene expression, we transfected a series of osteopontin promoter-luciferase constructs into OCCM-30 cementoblasts. Our results demonstrate that ePi can directly induce osteopontin gene transcription. The region responsive to ePi signaling was localized to a 53-bp region of the promoter between −1454 and −1401 that contains a glucocorticoid response element (GRE). Mutation of the GRE abolished the ePi response, suggesting that glucocorticoid receptor (GR) signaling is required for ePi-mediated transcription. In addition, treatment of cells with the GR antagonist RU-486 (Mifepristone) prevented promoter activation by ePi. The results presented support a model demonstrating that inorganic phosphate regulates OPN gene transcription in cementoblasts through a pathway that requires a functional GR.