ATPase Pump Activity Research Articles

Increased functional Na(+)-K+ pump activity in the vasculature of fructose-fed hyperinsulinemic and hypertensive rats.

We hypothesized that hyperinsulinemia may alter insulin's ability to stimulate vascular Na+/K(+)-ATPase pump activity and modulate changes in vascular responsiveness associated with hypertension. We measured potassium-induced relaxation as an indicator of Na+/K(+)-ATPase pump activity in isolated femoral arteries from fructose-fed (FF) hyperinsulinemic, Sprague-Dawley rats. FF rats had higher mean arterial blood pressures than did normal diet-fed (NF) rats (FF, 125 +/- 2.2, n = 20, vs. NF, 113.5 +/- 2.5 mmHg, n = 20, p < 0.05) and were hyperinsulinemic (FF, 64 +/- 4 vs. NF, 37 +/- 2, microU/ml insulin, p < 0.01). FF rats were more sensitive to KCl in the Na+/K+ pump bioassay (FF, 0.86 +/- 0.07, n = 21 vs. NF, 1.18 +/- 0.08, n = 17, p < 0.05, expressed as ED50 in mmol/l KCl). Exogenous insulin (100 mU/ml) increased Na+/K+ pump sensitivity in FF rats as compared with a boiled insulin control (insulin 45 +/- 6%, n = 11, vs. control, 11 +/- 7%, n = 13, p < 0.01, expressed as percent increase in sensitivity, i.e., ED50). There were no significant differences in Na+/K+ pump sensitivity between insulin and control responses in the NF animals (insulin 29 +/- 6%, n = 11, vs. control 46 +/- 5%, n = 10, NS). Dose-response curves were obtained in tail and femoral arteries from the same animals to norepinephrine and acetylcholine, basally and after exogenous insulin. FF vessels had reduced sensitivity to norepinephrine as compared with the NF group. Insulin increased sensitivity to acetylcholine-induced relaxations and increased AII-induced contractions in FF-rat vessels. These data suggest that in the FF rat insulin's influence on the vascular Na+/K+ pump is enhanced and may modulate the changes in vascular responsiveness seen in this model.

Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.

Functional coupling of Na+,K+-ATPase pump activity to a basolateral membrane (BLM) K+ conductance is crucial for sustaining transport in the proximal tubule. Apical sodium entry stimulates pump activity, lowering cytosolic [ATP], which in turn disinhibits ATP-sensitive K+ (KATP) channels. Opening of these KATP channels mediates hyperpolarization of the BLM that facilitates Na+ reabsorption and K+ recycling required for continued Na+,K+-ATPase pump turnover. Despite its physiological importance, little is known about the regulation of this channel. The present study focuses on the regulation of the BLM KATP channel by second messengers and protein kinases using membrane patches from dissociated, polarized Ambystoma proximal tubule cells. The channel is regulated by protein kinases A and C, but in opposing directions. The channel is activated by forskolin in cell-attached (c/a) patches, and by PKA in inside-out (i/o) membrane patches. However, phosphorylation by PKA is not sufficient to prevent channel rundown. In contrast, the channel is inhibited by phorbol ester in c/a patches, and PKC decreases channel activity (nPo) in i/o patches. The channel is pH sensitive, and lowering cytosolic pH reduces nPo. Increasing intracellular [Ca2+] ([Ca2+]i) in c/a patches decreases nPo, and this effect is direct since [Ca2+]i inhibits nPo with a Ki of approximately 170 nM in i/o patches. Membrane stretch and hypotonic swelling do not significantly affect channel behavior, but the channel appears to be regulated by the actin cytoskeleton. Finally, the activity of this BLM KATP channel is coupled to transcellular transport. In c/a patches, maneuvers that inhibit turnover of the Na+,K+-ATPase pump reduce nPo, presumably due to a rise in intracellular [ATP], although the associated cell depolarization cannot be ruled out as the possible cause. Conversely, stimulation of transport (and thus pump turnover) leads to increases in nPo, presumably due to a fall in intracellular [ATP]. These results show that the inwardly rectifying KATP channel in the BLM of the proximal tubule is a key element in the feedback system that links cellular metabolism with transport activity. We conclude that coupling of this KATP channel to the activity of the Na+,K+-ATPase pump is a mechanism by which steady state NaCl reabsorption in the proximal tubule may be maintained.

Stimulation of Ca 2+-pumping ATPase activity in carrot plasma membrane by calmodulin

Activity of Ca 2+-pumping ATPase located at the plasma membrane of cultured carrot cells was appreciably stimulated when the enzyme was associated with calmodulin (CAM). Affinity of the ATPase toward the substrates was increased by the addition of CAM, and K m s of the enzyme for Ca 2+ were estimated to be 11.4 and 0.7 μM in the absence and presence of CAM, respectively, while the values for ATP decreased only slightly (914 and 670 μM). In contrast, relative V values of the enzyme were essentially not changed even after the addition of CAM. This ATPase was capable of utilizing several ATP analogues, such as GTP, ITP, UTP and CTP, instead of ATP, and Ca 2+-transport driven by these alternative substrates was also stimulated by CAM. The molar ratio of Ca 2+-transport and ITP hydrolysis of the enzyme reaction was estimated to be 2:1. These observations suggested that CAM-induced stimulation of Ca 2+- ATPase activity at the plasma membrane of cultured carrot cells is mainly due to the increase in the affinity of the enzyme toward Ca 2+ by association with the modulator protein, and the hydrolysis of 1 mol of ATP by the enzyme action results in the transport of 2 mol of Ca 2+ across the membrane. © 1997 Elsevier Science Ltd. All rights reserved

Influence of Bcl-2 overexpression on Na+/K(+)-ATPase pump activity: correlation with radiation-induced programmed cell death.

Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na+/ K(+)-ATPase pump activity, as measured by the uptake of Rubidium-86 (86Rb+), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses > or = 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 microM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na+/ K(+)-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). 86Rb+ uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of 86Rb+ uptake, suggesting that the effects of radiation on the Na+/K(+)-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na+/K(+)-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD.

Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: application to diagnosis of susceptibility to malignant hyperthermia.

A microassay is demonstrated for functional characterization of the Ca(2+)-release channel (CRC) of sarcoplasmic reticulum (SR) of skeletal muscle using swine with susceptibility to malignant hyperthermia (MH). Diluted muscle homogenates, indo-1 and ratiometric dual-emission spectrofluorometry are used to monitor Ca(2+)-lowering activity in real-time in the presence and absence of ryanodine at exposures that open and close the CRC. Reactions are initiated with 50 microM CaCl2 to raise ionized Ca2+ concentration near 1 microM and MgATP to activate the Ca(2+)-ATPase pump. Oxalate is included to precipitate Ca2+ within the SR. The assay requires less than 30 mg muscle, which may be cryopreserved, and is completed within 20 min of thawing the tissue. Maximum SR Ca(2+)-ATPase pumping and CRC activities, degree of CRC activation, and Ca(2+)-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca(2+)-ATPase pumping and CRC activities are normal, the CRC activity after addition of a bolus of Ca2+ is 50% greater in heterozygotes and 100% greater in homozygotes for the MH mutation. Hypersensitivity to CRC agonists, such as caffeine, and an associated hyposensitivity to CRC antagonists such as Mg2+ is also demonstrated. Genotypes for the MH mutation site can be discriminated from each other by determining Ca(2+)-lowering activities and the effect of ryanodine on them.

Effects of insulin and IGF-I on vascular smooth muscle glucose and cation metabolism.

Data is accumulating indicating that impaired insulin action predisposes to increased vascular smooth muscle (VSM) tone, the hallmark of hypertension associated with diabetes. During the last several years, it has been established that VSM is an insulin-sensitive tissue like skeletal muscle and adipocytes. Investigators have shown that insulin regulates VSM intracellular cation metabolism through attenuating effects on inward calcium (Ca2+) currents and by direct effects on VSM cells (VSMCs) Na+, K(+)-ATPase pump expression and activity and that insulin and IGF-I stimulate glucose uptake in VSMCs. Furthermore, recent data suggest that IGF-I, like insulin, attenuates cytosolic calcium [Ca2+]i transients and vasoconstrictive responses. IGF-I, like insulin, also stimulates the production of nitric oxide from both the endothelium and VSMCs. IGF-I and insulin are structurally related, share receptors, and have similar postreceptor actions. Unlike insulin, which must transverse the endothelium before acting on VSMCs in vivo, IGF-I is synthesized by VSMCs. Thus, it is likely that IGF-I has more relevance than insulin in regulating physiological parameters in VSMCs.

EFFECT OF CONTRACTION DURATION ON ENERGY COST AND FATIGUE IN CONTRACTING MUSCLE 836

The purpose of this study was to investigate the extent to which altering contraction duration (and presumably ion pump ATPase activity) can affect energy cost and fatigue in isolated contracting muscle. Canine gastrocnemius muscle (n=6) was isolated and 3 contractile periods (3 min of isometric, tetanic contractions) were conducted by each muscle in a balanced order design. Each of the 3 contractile periods had stimulation patterns that resulted in a 1:1 work-to-rest ratio, with the difference in the 3 contractile periods being in the contraction duration (short duration = 0.25 sec stimulation/0.25 sec rest; moderate duration = 0.50 sec stimulation/0.50 sec rest; and long duration = 1 sec stimulation/1 sec rest) with the total time of stimulation and number of stimulation pulses being the same over each 3 min contraction period. Muscle O2 uptake, the O2 cost of developed force, and the fall in developed force (fatigue) was significantly greater(p<0.05) with contractions of short duration (stimulation = 0.25 sec). Muscle [ATP] did not fall significantly from resting values during any contraction period and was not different among any of the 3 contraction periods. Lactate efflux from the working muscle and muscle [lactate] were significantly greater with contractions of short duration (stimulation = 0.25 sec). These results suggest that contraction duration, through the demand of cellular ion pump ATPases, can significantly affect the energy cost and fatigue (possibly related to increased glycolytic flux) of contracting muscle.

Decreased expression of cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase in congestive heart failure due to myocardial infarction.

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca(2+)-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca(2+)-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca(2+)-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.

Stimulation of sarcoplasmic reticulum Ca(2+)-ATPase by gingerol analogues.

We have reported previously that [8]-gingerol increased Ca(2+)-ATPase activity. Here we synthesized gingerol related compounds (AP-004, AP-005 and AP-015) and investigated the effects of gingerols ([6]-gingerol, [8]-gingerol and [10]-gingerol) and the synthesized compounds on the Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR). The Ca(2+)-ATPase activity and the Ca(2+)-pumping activity increased by these compounds in a concentration-dependent manner. It is probable that both the o-methoxyphenol and hydrocarbon chain in the molecule of gingerol analogues are necessary for the activation of the Ca(2+)-pumping ATPase activity of SR.

Thapsigargin‐ and cyclopiazonic acid‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Diminished brain synaptic plasma membrane ca 2+-atpase activity in spontaneously hypertensive rats: Association with reduced anesthetic requirements

We have recently reported that plasma membrane Ca 2+-ATPase ( PMCA) pumping activity in rat brain synaptic plasma membranes (SPM) was reduced by in vitro or prior in vivo exposure to inhalation anesthetics (IA). In addition, rats with streptozocin-induced diabetes were found to have diminished brain synaptic PMCA pumping and a decrease in the partial pressures of several IA required to prevent movement in response to stimulation, defined as the minimum effective dose or MED. Diminished PMCA activity in erythrocytes of spontaneously hypertensive rats (SHR) has been noted. Because PMCA is ubiquitous, it seemed possible that PMCA pumping might be decreased in the brain of SHR and perhaps associated with decreased IA requirement. Eighteen SHR and 18 control, normotensive Wistar-Kyoto rats (WKY) were studied. PMCA activity was assessed by measurement of Ca 2+ uptake into synaptic plasma membrane vesicles prepared from cerebrum and diencephalon-mesencephalon (D-M) in WKY and SHR. Ca 2+ pumping was significantly less in SHR than in WKY, 85% of control in the cerebrum and 90% in the D-M (p < 0.01). The MEDs for halothane, isoflurane and desflurane were also lower in SHR than in WKY, 91%, 90% and 89%, respectively, of control (p < 0.05). Thus, an animal model of primary hypertension (SHR) manifested diminished brain synaptic PMCA activity and reduced MED for several volatile anesthetics. These findings provide further evidence for a role for PMCA in anesthetic action.

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca(2+)-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca2+ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.

Halothane, Isoflurane, Xenon, and Nitrous Oxide Inhibit Calcium ATPase Pump Activity in Rat Brain Synaptic Plasma Membranes

Perturbation of neuronal calcium homeostasis may alter neurotransmission in the brain, a phenomenon postulated to characterize the anesthetic state. Because of the central role of plasma membrane Ca(2+)-ATPase (PMCA) in maintaining Ca2+ homeostasis, the authors examined the effect of several inhalational anesthetics on PMCA function in synaptic plasma membranes (SPM) prepared from rat brain. Ca(2+)-ATPase pumping activity was assessed by measurement of ATP-dependent uptake of Ca2+ by SPM vesicles. ATPase hydrolytic activity was assessed by spectrophotometric measurement of inorganic phosphate (Pi) released from ATP. For studies of anesthetic effects on PMCA activity, Ca2+ uptake or Pi release was measured in SPM exposed to halothane, isoflurane, xenon, and nitrous oxide at partial pressures ranging from 0 to 1.6 MAC equivalents. Halothane and isoflurane exposures were carried out under a gassing hood. For xenon and nitrous oxide exposures, samples were incubated in a pressure chamber at total pressures sufficient to provide anesthetizing partial pressures for each agent. Dose-related inhibition of Ca(2+)-ATPase pumping activity was observed in SPM exposed to increasing concentrations of halothane and isoflurane, confirmed by ANOVA and multiple comparison testing (P < 0.05). Concentrations of halothane and isoflurane equivalent to one minimum effective dose (MED) depressed PMCA pumping approximately 30%. Xenon and nitrous oxide also inhibited Ca2+ uptake by SPM vesicles. At partial pressures of these two gases equivalent to 1.3 MAC, PMCA was inhibited approximately 20%. Hydrolysis of ATP by SPM fractions was also inhibited in a dose-related fashion. An additive effect occurred when 1 vol% of halothane was added to xenon or nitrous oxide at partial pressures equivalent to 0-1.6 MAC for the latter two agents. Plasma membranes Ca(2+)-ATPase is significantly inhibited, in a dose-related manner, by clinically relevant partial pressures of halothane, isoflurane, xenon, and nitrous oxide. Furthermore, these anesthetics inhibit PMCA activity in accordance with their known potencies, and an additive effect was observed. How inhalational anesthetics inhibit the PMCA pump is not known at this time. It is noteworthy that the only shared characteristic of this group of agents of widely different structure is anesthetic action. The relevance of this dual commonality, anesthetic action and PMCA inhibition, to actual production of the anesthetic state remains to be determined.

Recognition of markers of response to potassium-canrenoate in essential hypertension

Potassium canrenoate (K-Can) prevents hypertension in Milan hypertensive strain (MHS) but not in spontaneously hypertensive rats (SHR). Essential hypertensive patients (HT) may have differential sensitivity to diuretics, since a subgroup of HT insensitive to hydrochlorothiazide (HCTZ) but sensitive to K-Can has previously been found. The aims of this study were: 1) to seek markers of response in essential hypertensive patients selectively sensitive to K-Can; and 2) to test whether selective sensitivity to furosemide may also be demonstrated. After 2 weeks of placebo (P) 50 uncomplicated, mild to moderate HT (46 ± 9 yrs, mean ± SD) received K-Can (50 mg/day) for 4 weeks. After 2 more weeks of P, patients received HCTZ (25 mg) and furosemide (25 mg) for 4 weeks each in a single blind crossover design, with 2 weeks P between each treatment. Dosages were doubled after 2 weeks if diastolic blood pressure (DBP) was >90 mmHg. Responders (R) were those HT whose DBP was ⩽90 mmHg and/or at least 10 mmHg lower than before treatment. Systolic blood pressure (SBP)/DBP was measured every 2 weeks with plasma renin activity (PRA), red blood cell Na +-K +-Cl − cotransport (COT) and Na +-K + ATPase pump activity measured at the end of the first P period, and serum electrolytes at the end of each period. Four HT dropped out because of low compliance, 6 because of reversible side effects, and 1 because blood pressure was not back to pre-treatment value after the second placebo period. Seven HT were “selective R” to K-Can since DBP did not change in response to furosemide and HCTZ. Five HT did not respond to any drug. PRA and COT were respectively significantly lower and higher in HT “selective R” to K-Can. We thus concluded that, in our subgroup of HT selectively sensitive to K-Can, hypertension may be initiated and maintained by a particular mechanism.

Altered sarcolemmal calcium channel density and Ca 2+-pump ATPase activity in tachycardia heart failure

Whether sarcolemmal (SL) calcium handling is altered in endstage heart failure produced by chronic rapid pacing is not known. To investigate this we paced 7 rabbits at a rate of 400 beats/min for 35 ± 11 days. 6 animals served as non-paced controls. Purified left ventricular SL membranes were then prepared and tested for [ 3H]-nitrendipine binding and (Ca 2++Mg 2+)-dependent ATPase (Ca 2+-pump) activity. Results show a 50% reduction in calcium channel antagonist binding sites with B max values reduced from 450 ± 40 to 230 ± 8 fmoles/mg protein in response to chronic rapid pacing ( P < 0.01). This change was accompanied by a modest decrease in K d from 0.29 ± 0.09 to 0.22 ± 0.03 nM (not significant). V max values for the SL Ca 2+-pump ATPase were decreased from 387 to 164 nmoles/mg protein/min ( P < 0.01) with K Ca 2+ values reduced from 0.91 to 0.28 μM Ca 2+ ( P < 0.05) in response to tachycardia induced failure as compared to controls. ATPase activity in both groups was very sensitive to 25 μM calmidazolium and 5 μM vanadate. Results from this study indicate that both a reduction in SL calcium channel density and decrease in SL Ca 2+-pump ATPase activity are evident in tachycardia heart failure. We conclude that sarcolemmal calcium handling is altered in heart failure induced by chronic rapid pacing and that such changes may contribute to systolic dysfunction associated with this model of heart failure.

Oxygen free radicals and calcium homeostasis in the heart.

Many experiments have been done to clarify the effects of oxygen free radicals on Ca2+ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analayze the results. It is, however, evident that Ca2+ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca2+ binding is increased, Ca2+ influx through Ca2+ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca2+ can enter the myocytes is Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-K+ ATPase and Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-H+ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na+ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na(+)-Ca2+ exchange in Ca2+ influx process from extracellular space. Another question is 'which way does Na(+)-Ca2+ exchange work under oxidative stress? Net influx or efflux of Ca2+?' Membrane permeability for Ca2+ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca(2+)-pump ATPase activity is depressed by oxygen free radicals, Ca2+ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca2+ release from sarcoplasmic reticulum and inhibit Ca2+ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca2+ handling systems could cause the Ca2+ overload due to oxygen free radicals.

Oxygen free radicals and calcium homeostasis in the heart.

Many experiments have been done to clarify the effects of oxygen free radicals on Ca2+ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca2+ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Through ATP-independent Ca2+ binding is increased, Ca2+ influx through Ca2+ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca2+ can enter the myocytes is Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-K+ ATPase and Na(+)-H(+) exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na(+) level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na(+)-Ca2+ exchange in Ca2+ influx process from extracellular space. Another question is 'which way does Na(+)-Ca2+ exchange work under oxidative stress? Net influx or efflux of Ca2+?' Membrane permeability for Ca2+ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca(2+)-pump ATPase activity is depressed by oxygen free radicals, Ca2+ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca2+ release from sarcoplasmic reticulum and inhibit Ca2+ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca2+ handling systems could cause the Ca2+ overload due to oxygen free radicals.

Decreased Calcium Pump Activity in Duodenal Epithelial Cells from Spontaneously Hypertensive Rats

Recently, hypertension research has focused on altered cytosolic free Ca2+, [Ca2+]i, in cells of spontaneously hypertensive rats (SHR). In this work, a mechanism(s) responsible for regulating [Ca2+]i was studied with duodenal epithelial cells isolated from SHR and their control, normotensive Wistar-Kyoto rats (WKY). The equal specific activity of sucrase, an enzyme characteristic of microvilli, in both mucosal scrapings and isolated cells from SHR and WKY suggested that mucosal density of enterocytes was not largely different between the two strains. [Ca2+]i of the isolated cells was estimated with fura-2. Upon incubation in the presence of CaCl2, [Ca2+]i increased to a larger extent in SHR than in WKY cells. Ca2+ efflux based on measurements of [Ca2+]i was decreased in SHR cells. Correspondingly, it was found that 45Ca efflux at 10 sec was lower for SHR cells than for WKY cells. Specific activities of Ca(2+)-stimulated and Ca2+/calmodulin-stimulated ATPases in basolateral plasma membrane preparations were reduced in SHR cells. These results indicated that Ca2+ efflux was decreased by reduction in Ca(2+)-pumping ATPase activity in SHR enterocytes.

In Vitro Aging of Bovine and Human Retinal Pigment Epithelium: Number and Activity of the Na/K ATPase Pump

We have previously observed that the density of Na/K ATPase pumps is lower in RPE cells in the posterior pole of both bovine and human eyes. The posterior pole in human eyes includes the macula, a region which is predisposed to aging pathology. In this study we examined the effect of age on the sodium pump using cultures of bovine (bRPE) and human (hRPE) RPE cells that were aged in vitro by repeated passage. The cultures were assayed for cell number, protein, pump density (specific binding of [3H]ouabain) and pump activity (specific uptake of 86Rb) at confluency at each passage. In culture, bRPE had more pumps per cell (3·2 × 106) than hRPE (1·2 × 106), but the bRPE pumps were less active so the pumping capacity per cell was nearly equal. Bovine RPE aged more rapidly in vitro (survived fewer passages) than hRPE. With aging, RPE cells from both species showed declines in cell number at confluency. Pump number and pump activity per cell remained constant. Because cell number declined, the pumping capacity per unit area of confluent epithelium was diminished with culture aging. RPE cell number is known to decline with age in situ, especially in the macula. If Na/K ATPase pump number and activity per RPE cell remain constant with aging in vivo as shown here in vitro, the effective pumping capacity of the RPE per unit area of 'monolayer' would decline in aged eyes. Diminished pumping capacity with aging could affect the ability of the RPE to transport ions and other metabolites especially in the aging macula where pump density is lower even in normal young eyes.