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Fluorescence is transferred across the toad urinary bladder when fura-2/AM is added to the mucosal or serosal sides of the epithelium. It was now observed that: (1) Oxytocin (20 nM, serosal) increased fluorescence transfer from the mucosal to the serosal but not from the serosal to the mucosal baths. The ratio between the fluorescence intensities recorded with excitation wavelengths of 340 and 380 nm indicates that the calcium sensitive probe (free fura-2) was transferred to the serosal but not to the mucosal compartment by an oxytocin sensitive transport. (2) Preincubation with probenecid did not change fluorescence transfer in basal conditions but significantly reduced the oxytocin induced increase in free fura-2 transport. (3) Fluorescence accumulation inside the tissue was strongly reduced by oxytocin, but only when fura-2/AM was added to the mucosal side. (4) An osmotic gradient, in the presence of oxytocin, further increased the transfer of fluorescence at 380 nm but not at 340 nm. This indicated that the transfer of a calcium-insensitive fraction was being stimulated. (5) Preincubation with colchicine strongly inhibited fluorescence transfer across the tissue, at both 340 and 380 nm (the 340/380 ratio did not change). (6) Tissue accumulation was increased by colchicine. (7) Vanadate did not inhibit fura-2 transfer in the toad urinary bladder. We conclude that intracellularly-generated free fura-2 is only transported across the basolateral border, and that this transfer is stimulated by ADH. The calcium-insensitive fraction is transferred by a temperature-dependent process, sensitive to an osmotic gradient and colchicine.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.

Ca2(+)-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca2+ and Mg2+, and devoid of K+. The enzyme activity showed a high apparent affinity for Ca2+ (KCa:0.5 microM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl3 inhibition, and (3) not affected by 20 mM NaN3 or 0.1 mM ouabain. The addition of Mg2+ promoted the disappearance of Ca2(+)-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [gamma -32P]ATP resulted in the appearance of a 140 kDa band as revealed by SDS-gel electrophoresis. Labeling of this band with 32P was inhibited by 1 mM EGTA or 10 mM NH2OH, indicating that the isotope incorporation required the presence of Ca2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca2(+)-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca2+ in nerve terminals. Preincubation of synaptosomes in Ca2+ plus K+, a depolarizing procedure, induced a large and rapid decrease in the Ca2(+)-ATPase activity, possibly mediated via Ca2+ entry through voltage-gated Ca2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 microM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca2(+)-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.

(1) We have reexamined the effects of calmodulin and of the calmodulin antagonist, compound 48/80 on the interaction of ATP at its low-affinity site in the Ca2(+)-ATPase from human red cells. (2) At variance with our earlier proposal (Biochim. Biophys. Acta (1985) 816, 379-386) calmodulin increased the maximum effect of ATP without changing the apparent affinity for ATP at the low-affinity site. Accordingly, ATP increased the maximum activation by calmodulin without altering the apparent affinity of the Ca2(+)-ATPase for calmodulin. (3) Confirming our previous observation (Biochim. Biophys. Acta (1985) 816, 379-386) compound 48/80 lowered the apparent affinity of the Ca2(+)-ATPase for ATP at the low-affinity site. This has to be attributed to a direct effect of this compound on the enzyme rather than to its effect as calmodulin antagonist.

(1) The effects of treatments that mimic calmodulin in increasing the apparent affinity for Ca2+ were tested to see whether, like calmodulin, they also change the activation of the Ca2+-ATPase from human red cell membranes by ATP at the low-affinity site. (2) Short incubations with either trypsin or acidic phospholipids such as phosphatidylserine increased the apparent affinity for ATP at the low-affinity site. (3) Under conditions in which it increased the apparent affinity of the Ca2+-ATPase for Ca2+, EGTA failed to change the activation by ATP. (4) As in calmodulin-bound Ca2+-ATPase, compound 48/80 inhibited the activity of the enzyme in the presence of phosphatidylserine by lowering the apparent affinity for ATP at the low-affinity site, leaving the maximum velocity of the enzyme unaltered. (5) Compound 48/80 also inhibited the Ca2+-ATPase after partial proteolysis, but in this case it lowered the maximum activity, leaving the apparent affinity of the enzyme for ATP at the low-affinity site unaltered. (6) Inhibition of the Ca2+-ATPase by compound 48/80 in the absence of calmodulin suggests that the inhibitor can act directly on the enzyme.

Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg2+ and K+ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca2+ so that the activity becomes negligible above 30 microM Ca2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca2+. In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca2+ are needed and phosphatase activity is maximal in media without Ca2+. This is the first report of an activity sustained by the Ca2+-ATPase of red cell membranes in the absence of Ca2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca2+. These results strongly suggest that during activation by calmodulin, Ca2+ is needed only to form the calmodulin-Ca2+ complex which is the effective cofactor. Protection by ATP of the inhibitory effects of Ca2+ and the induction of phosphatase activity by ATP + Ca2+ suggests that activation of the phosphatase by Ca2+ in media with ATP requires the combination of the cation at sites in the ATPase. Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E1 and E2 towards E2. On the other hand, ATP + Ca2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E2 predominates over other conformers of the ATPase.

Compound 48/80, an anti-calmodulin agent, reduces the maximum effect of ATP and does not affect the apparent affinity for ATP of the high-affinity site of the Ca2+-ATPase from calmodulin-bound membranes of human red cells. In the same preparation, 48/80 reduces more than 50-times the apparent affinity for ATP of the low-affinity site with little change in the maximum effect of the nucleotide at this site of the Ca2+-ATPase. The effects of compound 48/80 are independent of the concentration of Ca2+ between 30 and 200 microM. The apparent affinity of the low-affinity site of the Ca2+-ATPase for ATP is almost 100-fold less in calmodulin-stripped membranes than in calmodulin-bound membranes. In calmodulin-stripped membranes, exogenous calmodulin increases the apparent affinity for ATP up to the control values. These results indicate that apart from increasing the apparent affinity of the transport site for Ca2+, calmodulin also increases the apparent affinity of the regulatory site of the Ca2+-ATPase for ATP. Since this effect is exerted within the physiological ranges of ATP concentrations, it may participate in the physiological regulation of Ca2+ pumping by calmodulin.

(1) Vanadate (pentavalent vanadium) inhibits with high affinity (K0.5 = 3 microM) the ATP-dependent Ca2+ efflux in reconstituted ghosts from human red cell. (2) To inhibit Ca2+ efflux vanadate has to have access to the inner surface of the cell membrane (3) The inhibitory effect of vanadate is potentiated by intracellular Mg2+ and by intracellular K+. (4) Ca2+ in the external medium antagonizes the inhibitory effect of vanadate.

The calmodulin antagonist compound 48/80 inhibits the phosphatase activity of the Ca2+-ATPase lowering its maximum velocity and leaving unaltered its apparent affinity for the substrate regardless on whether phosphatase activity is elicited by Ca2+ plus ATP or by calmodulin. Compound 48/80 has no effect on the Ki for ATP as inhibitor of the phosphatase. These results contrast sharply with the large increase that compound 48/80 induces in the apparent affinity of the regulatory site for the nucleotide of the Ca2+-ATPase and suggest that the active site for phosphatase activity is different from the regulatory site for ATP of the Ca2+-ATPase.

Ca2+-ATPase activity was measured in rat islet homogenates, in a medium of low ionic strength containing a low concentration of Ca2+ and Mg2+ and devoid of K+. The enzyme activity was highly sensitive to inhibition by compound 48/80 (a calmodulin inhibitor), stimulated by 120 nM calmodulin and slightly affected by 10 mM NaN3. The addition of Mg2+ to the assay medium promotes the disappearance of apparent Ca2+-ATPase activity. Ouabain (0.1 mM) did not modify this ATPase activity. The enzyme showed two kinetic components for Ca2+ as well as for ATP: one with high apparent affinity and low maximum velocity and the other with low apparent affinity and high maximum velocity. Incubation of islet homogenates in this assay medium with [gamma-32P]ATP in the presence of proteolytic inhibitors, results in the appearance of a single labelled band of 130 kDa, identified by gel electrophoresis. The incorporation of 32P into this band was similar in the presence of either 2.8 or 50 microM Ca2+ and susceptible to hydroxylamine attack. The results indicate that, under the conditions described above, the Ca2+-ATPase activity evidenced in the islet homogenates had characteristics resembling those of the enzyme which catalyzes the outward Ca2+ transport. On the other hand, the method could provide a useful tool to test the effect of different agents which affect insulin secretion upon the islet plasma membrane Ca2+-ATPase activity.

Plasma membrane Ca2+-ATPase activity was measured in rat islet homogenates. The enzyme was inhibited, in a dose-dependent manner, when the islets were preincubated for 5 min with different concentrations of glucose (2 to 16 mM). This inhibition disappeared almost entirely after 15 min incubation, regardless of the glucose concentration in the medium. Simultaneous measurement of insulin in the medium revealed a stimulatory effect of glucose upon insulin secretion. The Ca2+-ATPase activity was also inhibited when the islets were preincubated for 3 min with other stimulators of insulin secretion such as gliclazide (76 microM), tolbutamide (1.5 mM), glucagon (1.4 microM) + theophylline (10 mM) and ketoisocaproic acid (15 mM). Conversely, the activity of the enzyme was significantly enhanced when the islets were preincubated briefly with the insulin secretion blocker, somatostatin (1.4 microM). Neither glucose nor any of the other substances tested when added directly to the enzyme assay medium modified significantly the Ca2+-ATPase activity measured in the islet homogenates. These results would suggest that the activity of the islet plasma membrane is modulated by one or more of the intracellular metabolites produced when the islets are challenged by the insulin stimulator or blocking agents.