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The muscarinic receptor in the rabbit pancreas was characterized with the use of the labeled ligand (3H)-(-)-quinuclidinyl-benzylate ((3H)-(-)-QNB). Specific binding of (3H)-(-)-QNB to pancreatic acini was found to be reversible and of high affinity, with an equilibrium dissociation constant (KD) of 68 pmol/l and a receptor density (RT) of 170 fmol/mg protein. Agonist binding behaviour was investigated by displacement of (3H)-(-)-QNB binding by eight agonists like arecoline, arecaïdine-propargylester (APE) and carbachol, yielding only low affinity binding sites. The inhibition of (3H)-(-)-QNB binding by the selective antagonists pirenzepine, hexahydrosiladifenidol (HHSiD) and (11-(12-((diethyl - amino)- methyl)- 1- piperidinyl) acetyl)-5, 11-dihydro-6H-pyrido (2, 3-b) (1, 4) benzodiazepin-6-one) (AF-DX 116) confirmed the M3 nature of the rabbit pancreatic receptor.

Various claims, partially conflicting, have been made in recent years for abnormalities in cation pumps and fluxes in erythrocytes of patients with essential hypertension. In view of the obvious significance of such abnormalities for diagnostic purposes, and possibly for our understanding of the pathophysiology of essential hypertension, we have investigated these claims.We have determined the following parameters of erythrocytes from essential hypertensives and normotensives: 1. (Na++K+)-ATPase activity and the Km values for Na+, K+ and ATP; 2. ouabain-sensitive fluxes of Na+ and K+ in Na+-enriched cells after cold treatment and after treatment with p-chloromercuribenzenesulphonate; 3. furosemide-sensitive, ouabain-insensitive cotransport efflux of Na++K+.No significant differences were observed, except for a slight decrease in the ouabain-sensitive K+ influx after cold treatment in hypertensives. Hence, we conclude that determination of these parameters in erythrocytes does not seem to be useful for the diagnosis of essential hypertension.

1. The relative permeabilities for sodium, potassium and chloride in guinea pig mammary gland slices are determined by means of ion flux studies with radioisotopes.2. Assuming that there are no significant electrogenic potential components, we calculate permeability ratios PNa/PK = 0.97 and PCl/PK = 1.25.3. Substitution of these values in the Goldman equation yields membrane potentials of - 15 mV before and - 13 mV after ouabain treatment.4. This small change in membrane potential explains the absence of a significant change in chloride content upon ouabain application, which leads to large changes in intracellular sodium and potassium concentrations.

Summary Membrane fragments containing the H+K--ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na+K+-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J.4:3079–3085). Since H+K+-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K+. By separation of the H+ and K+ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K+ or at low concentrations of K+. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K+ transport during dephosphorylation is electrogenic, too, in contrast to the Na+K+-ATPase where the K+ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H+K+-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 µMfree Ca2+, accumulated 0.9–1.5 nmol of Ca2+/mg acinar protein in an energy-dependent pool. Uptake into this pool was not altered by pretreatment of acinar cells with the Ca2+mobilizing secretagogues carbamylcholine and cholecystokinin-octapeptide or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, indicating that the Ca2+pump of the internal Ca2+store was not affected by prolonged activation of the Ca2+messenger system. Inositol 1,4,5-trisphosphate (1,4,5-IP3) caused a rapid decrease in Ca2+content of the internal Ca2+store. The response was maximal within 30 s following addition of 1,4,5-IP3and no reuptake of Ca2+was observed over the next 60 s. Up to 55% of the amount of Ca2+stored in the energy-dependent pool was 1,4,5-IP3releasable with an EC50of 1.0 µM. Pretreatment of acinar cells with carbamylcholine or cholecystokinin-octapeptide significantly reduced the effectivity of 1,4,5-IP3to release Ca2+from the internal store. The dose-response curve for 1,4,5-IP3-induced release of actively stored Ca2+from carbamylcholine-treated acinar cells showed both a rightward shift (EC50value of 1.7 µM) and a decreased maximal response (maximal release value of 44%), which suggests that the affinity of 1,4,5-IP3for its receptor as well as the number of 1,4,5-IP3receptors or 1,4,5-IP3-operated Ca2+channels was reduced upon prolonged activation of the Ca2+messenger system. Moreover, analysis of the release values in a Hill plot suggested positive cooperativity for 1,4,5-IP3-induced Ca2+release from the internal store (nvalues of 1.3 and 1.7 for saline- and carbamylcholine-treated permeabilized acinar cells, respectively). Pretreatment of acinar cells with 12-O-tetradecanoylphorbol 13-acetate partly mimicked the inhibitory effect of carbamylcholine on 1,4,5-IP3-induced release of actively stored Ca2+in that the dose-response curve was shifted to the right but the maximal response was not affected, suggesting that the effects of carbamylcholine were at least in part mediated by protein kinase C. The results presented are in support of our previously published hypothesis (Willems, P. H. G. M., van Nooij, I. G. P., Haenen, H. E. M. G., and De Pont, J. J. H. H. M. (1987) Biochim. Biophys. Acta930,230–236) that activation of protein kinase C leads to negative feedback control on agonist-induced Ca2+mobilization at a level beyond receptor-mediated formation of 1,4,5-IPS and they suggest that protein kinase C directolry indirectly acts at the level of the 1,4,5-IPs-operated Ca2+channel of the internal Ca2+store.

(Na++ K+)ATPase contains two different subunits, a catalytic subunit (α) and a subunit with uncertain function (β). The enzyme binds ATP, ouabain and vanadate, and can be phos-phorylated by ATP as well as by inorganic phosphate. From the previously reported maximal binding and phosphorylation capacities of 3.5–4.3 nmol P per mg protein1–3(based on Lowry protein determination) and the earlier molecular weight value of ∼250,0001,2, a molar binding and phosphorylation capacity of 0.87–1.07 mol per mol enzyme was derived. As it is generally agreed that the enzyme molecule contains two α subunits1–6or even a multiple of two7,8, it has been suggested that the enzyme operates by means of a so-called ‘half-of-the-sites mechanism’, whereby only one of the two α subunits can be phosphorylated at any one time9,10. We now present evidence that every α subunit can be phosphorylated simultaneously, which rules out the operation of such a mechanism.