Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells

Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca(2+)-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca(2+)-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca(2+)-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca(2+) loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase V(max) values much higher than those ever measured by PMCA-mediated Ca(2+) extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.     

Ovariectomy worsens secondary hyperparathyroidism in mature rats during low-Ca diet

Estrogen deficiency impairs intestinal Ca absorption and induces bone loss, but its effects on the vitamin D-endocrine system are unclear. In the present study, calciotropic hormones levels, renal vitamin D metabolism, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-dependent intestinal calcium absorption, and bone properties in 3-mo-old sham-operated (sham) or ovariectomized (OVX) rats fed either a normal-Ca (NCD; 0.6% Ca, 0.65% P) or a low-Ca (LCD; 0.1% Ca, 0.65% P) diet for 2 wk were determined. LCD increased serum 1,25(OH)2D3 levels in both sham and OVX rats. Serum parathyroid hormone [PTH(1-84)] levels were highest in OVX rats fed LCD. Renal 25-hydroxyvitamin D1alpha-hydroxylase (1-OHase) protein expression was induced in both sham and OVX rats during LCD, while renal 1-OHase mRNA expression was highest in OVX rats fed LCD. Renal vitamin D receptor (VDR) and mRNA expressions in rats were induced by ovariectomy in rats fed NCD but suppressed by ovariectomy in rats fed LCD. The induction of intestinal calcium transporter-1 and calbindin-D9k mRNA expressions by LCD were not altered by ovariectomy. As expected, bone Ca content, cancellous bone mineral density, and bone strength index in proximal metaphysis of rat tibia were reduced by both ovariectomy and LCD (P<0.05) as analyzed by two-way ANOVA. Taken together, the data demonstrate that ovariectomy alters the responses of circulating PTH levels, renal 1-OHase mRNA expression, and renal VDR expression to LCD. These results suggest that estrogen is necessary for the full adaptive response to LCD mediated by both PTH and 1,25(OH)2D3.     

Detrimental effect of eicosapentaenoic acid supplementation on bone following ovariectomy in rats

Although several studies have reported a positive effect of n-3 essential fatty acids (EFAs) on bone density post-ovariectomy, the role of specific EFAs has yet to be fully elucidated. In this study, ovariectomised (OVX) rats were supplemented with 0.1 g (LOW) or 1.0 g (HIGH) of eicosapentaenoic acid (EPA)/kg body weight for 9 weeks. Bone mineral density (BMD), 25-hydroxyvitamin D(3) and plasma fatty acid profile were compared to those of OVX and sham animals fed a non-supplemented diet. BMD decreased significantly in all OVX (P<0.001) but not sham rats. There was no difference in BMD between the LOW group and OVX controls. BMD was significantly lower in the HIGH group compared to OVX and sham controls. 25-hydroxyvitamin D(3) levels were significantly higher in both the LOW and HIGH groups compared to OVX controls (P=0.0006 and 0.02, respectively). In conclusion, high-dose EPA supplementation exacerbated the effects of ovariectomy on BMD.     

Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats

We investigated the effect of dietary calcium:phosphorus (Ca:P) ratio on bone mineralization and intestinal Ca absorption in ovariectomized (OVX) rat models of osteoporosis and sham-operated rats. Thirty 12-wk-old female Wistar rats were divided into three groups of OVX rats and three groups of sham rats. Thirty days after the adaptation period, OVX rats and sham rats were fed a diet formulated Ca:P, 1:0.5, 1:1 or 1:2 (each diet containing 0.5% Ca), respectively for 42 d. In both sham and OVX rats, serum osteocalcin, a marker of bone turnover, was increased by decreasing Ca:P ratio (1:2). In contrast, rats fed the Ca:P = 1:0.5 diet (dietary P restriction) suppressed the increased serum parathyroid hormone, osteocalcin and urinary deoxypyridinoline, and increased Ca absorption in both sham and OVX rats compared to the Ca:P = 1:1 and 1:2 diets. Especially, in OVX rats, the decreased bone mineral density of the fifth lumbar was also suppressed when rats were fed the Ca:P = 1:0.5 diet. These results indicated that the elevation of dietary Ca:P ratio may inhibit bone loss and increase intestinal Ca absorption in OVX rats.     

Lack of response of (Ca2+ + Mg2+) ATPase to atrial natriuretic peptide in basolateral membranes from kidney cortex of chronic diabetic rats

Incubation of basolateral membranes obtained from control rat kidney cortex in the presence of atrial natriuretic peptide (ANP) increased (Ca2+ + Mg2+) ATPase activity in a dose-dependent manner. Such response was absent in membranes obtained from animals made diabetic by streptozotocin injection (65 mg/kg, iv). The differential responses in the ATPase activity were not due to changes in the affinity for Ca2+ and insulin treatment in the diabetic animals completely reversed the situation. Our data suggest that ANP may mediate its cellular effects in part by changes in cellular Ca2+ homeostasis in kidney cortex and the lack of response of (Ca2+ + Mg2+) ATPase to ANP in chronic diabetes may contribute to the development of intracellular Ca2+ overload and nephropathy.     

Effect of estrogen on calcium-handling proteins, beta-adrenergic receptors, and function in rat heart

Regulation of cellular Ca(2+) cycling is central to myocardial contractile function. Loss of Ca(2+) regulation is associated with cardiac dysfunction and pathology. Estrogen has been shown to modify contractile function and to confer cardioprotection. Therefore, we investigated the effect of estrogen on expression of rat heart myocardial Ca(2+)-handling proteins and beta-adrenergic receptor (beta(1)-AR) and examined functional correlates. Female rats were sham-operated (SHAM) or ovariectomized. Two weeks after ovariectomy rats were injected (i.p.) daily with estradiol benozoate (OVX+EB) or sesame oil (OVX) for 2 weeks. Protein abundance was measured by immunoblotting and mRNA was quantified by real-time RT-PCR. OVX significantly decreased estrogen and progesterone levels and EB replacement returned both estrogen and progesterone to physiological levels. OVX induced a 75% reduction of uterine weight and a gain in body weight. Replacement restored weights to SHAM level. OVX increased and estrogen-replacement normalized abundance of beta(1)-AR and L-type Ca(2+) channel (Cav1.2) protein. OVX decreased sodium-Ca(2+) exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. Sarcoplasmic reticular ATPase (SERCA), phospholamban (PLB), and ryanodine receptor (RyR) abundance was not altered by hormone status. Levels of mRNA encoding for beta(1)-AR, Cav1.2, and NCX were not influenced by OVX or estrogen replacement. OVX had no effect on SERCA and PLB mRNA level but estrogen replacement elicited a significant increase compared to OVX and SHAM. Estrogen-dependent changes in Ca(2+)-handling proteins and beta(1)-AR are theoretically consistent reduced myocellular Ca(2+) load. However, hormone-dependent alterations in protein were not associated with changes in contractile function.     

Regulation of duodenal insulin-like growth factor I and active calcium transport by ovariectomy in female rats.

There is a significant body of data that supports the concept that reproductive hormones in females have effects on duodenal calcium transport that are not mediated via altered circulating concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Previously, we have shown parallel alterations in duodenal Ca transport and longitudinal bone growth rate in sexually maturing female rats in response to ovariectomy and estradiol (E) treatment of ovariectomized (OVX) rats (OVX+E) without any change in circulating levels of 1,25(OH)2D or parathyroid hormone. Results are presented here from experiments designed to: (i) further explore the relationship between 1,25(OH)2D and ovarian status in the regulation of duodenal calcium transport, and (ii) determine whether OVX and E replacement alter circulating and duodenal levels of insulin-like growth factor I (IGF-I) that might be related to effects on Ca transport. Growth hormone, which has been shown to affect intestinal Ca absorption and vitamin D metabolism, is thought to act indirectly by stimulating IGF-I. Six-week-old female rats were OVX, given estradiol implants (OVX+E), and fed a diet containing either 0.5% or 0.1% Ca for 3 weeks. In both diet groups, the OVX animals exhibited a higher level of Ca transport, as measured by the everted gut sac method, than either the intact controls or the OVX+E group; there was no difference in calcium transport between the different diet groups. Although there was no difference in circulating levels of 1,25(OH)2D among the intact, OVX, and OVX+E groups fed either diet, animals fed the 0.1% Ca diet had higher circulating levels of 1,25(OH)2D than those fed the 0.5% Ca diet. There was no difference in duodenal levels of calbindin9K among intact, OVX, and OVX+E animals in either diet group, although the animals fed the 0.1% Ca diet had higher levels of calbindin9K than the animals fed the 0.5% Ca diet. In animals fed the 0.5% Ca diet, OVX resulted in elevated serum and duodenal levels of IGF-1, as compared with intact and OVX+E animals on the same diet. In animals fed the 0.1% Ca diet, there was no elevation of IGF-I in the OVX group relative to intact and OVX+E animals. These results lend additional support to the concept that alterations in duodenal active calcium transport that occur with alterations in ovarian hormones are not mediated by changes in serum levels of 1,25(OH)2D, but may be related to some factor related to growth, possibly IGF-I.     

Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.     

Hemodynamic effects of chronic urotensin II administration in animals with and without aorto-caval fistula

Urotensin II (UTII) is a potent vasoactive peptide. Recent studies have demonstrated increased expression of both UTII and its receptor (UTR) expression in end-stage congestive heart failure (CHF), but it is unclear whether UTII and UTR are late stage markers of decompensation, or earlier adaptive responses. The purpose of this study was to measure the effects of chronic UTII administration in normal and volume overloaded animals. Chronic 4 weeks administration of UTII produced decreases in hemodynamic function in animals not subjected to volume overload while returning function to control levels in animals with overload. Expression levels of calcium regulatory proteins phospholamban (PLN), sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), and Na(+)/Ca(2+) exchanger (NCX) were measured to determine if administration of UTII resulted in aberrant Ca(2+) handling. Changes in protein expression revealed that UTII influenced Ca(2+) handling proteins in normal animals although these changes are not seen in the volume overload.     

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.     

Characteristics of sarcoplasmic reticulum from normal and denervated rat skeletal muscle.

Denervation of rat skeletal muscle produces after 14 days a decrease in Ca2+ uptake of a heterogeneous population of sarcoplasmic-reticulum vesicles, when measured in the presence of oxalate. The Mg2+-dependent ATPase (Ca2+-independent) activity increased after the same period and the Ca2+ + Mg2+-dependent ATPase activity decreased. Concomitant with these changes, there was an increase in vesicle size and calcium content. The observations are discussed in terms of changes in altered membrane structure, manifested in the shift of the equilibrium of the ATPase from an enzyme involved in calcium transport to a phosphoenzyme giving rise to an increase in the Mg2+-dependent ATPase activity.     

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.     

Changes in skeletal muscle SR Ca2+ pump in congestive heart failure due to myocardial infarction are prevented by angiotensin II blockade

In order to understand the mechanisms of exercise intolerance and muscle fatigue, which are commonly observed in congestive heart failure, we studied sarcoplasmic reticulum (SR) Ca(2+)-transport in the hind-leg skeletal muscle of rats subjected to myocardial infarction (MI). Sham-operated animals were used for comparison. On one hand, the maximal velocities (Vmax) for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities in skeletal muscle of rats at 8 weeks of MI were higher than those of controls. On the other hand, the Vmax values for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities were decreased significantly at 16 weeks of MI when compared with controls. These alterations in Ca(2+)-transport activities were not associated with any change in the affinity (1/Ka) of the SR Ca(2+)-pump for Ca2+. Furthermore, the stimulation of SR Ca(2+)-stimulated ATPase activity by cyclic AMP-dependent protein kinase was not altered at 8 or 16 weeks of MI when compared with the respective control values. Treatment of 3-week infarcted animals with angiotensin-converting enzyme (ACE) inhibitors such as captopril, imidapril, and enalapril or an angiotensin receptor (AT1R) antagonist, losartan, for a period of 13 weeks not only attenuated changes in left ventricular function but also prevented defects in SR Ca(2+)-pump in skeletal muscle. These results indicate that the skeletal muscle SR Ca(2+)-transport is altered in a biphasic manner in heart failure due to MI. It is suggested that the initial increase in SR Ca(2+)-pump activity in skeletal muscle may be compensatory whereas the depression at late stages of MI may play a role in exercise intolerance and muscle fatigue in congestive heart failure. Furthermore, the improvements in the skeletal muscle SR Ca(2+)-transport by ACE inhibitors may be due to the decreased activity of renin-angiotensin system in congestive heart failure.     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.     

Identification of Ca(2+)-activated Mg(2+)-dependent ATP-hydrolase activity of membrane microsomal fraction of loach embryo (Misgurnus fossilis L.)

The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.     

Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca(2+)+Mg(2+)-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2mug/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca(2+)+Mg(2+)-stimulated ATPase by increasing the Ca(2+)-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca(2+)+Mg(2+)-dependent ATPase in addition to its effect on the Ca(2+)-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca(2+)+Mg(2+)-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca(2+) concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5mum-cyclic AMP inhibited Ca(2+)+Mg(2+)-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase. Secondly, trifluoperazine (20mum) antagonized the stimulation of Ca(2+)+Mg(2+)-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase by PKI.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.