Differential effects of proinsulin C-peptide fragments on Na +, K +-ATPase activity of renal tubule segments

Summary Proinsulin C-peptide has been shown to stimulate the activity of Na ⁺ K ⁺ ATPase of rat renal tubule segments. Thirty-six peptides and amino acids, corresponding to parts of the intact rat C-peptide and suitable controls were screened for capacity to stimulate Na ⁺, K ⁺-ATPase in an attempt to determine potential active sites in the C-peptide molecule. The carboxy-terminal tetra and penta peptides were found to elicit 92–103 % of the intact molecule's activity, and the remaining segment, des-(27–31) C-peptide, did not possess stimulatory activity. Peptides from the middle C-peptide segment, however, centering around a GGPEAG sequence, stimulated Na ⁺, K ⁺-ATPase activity (36–80 % of the intact molecule's effect) but this effect was not balanced by corresponding inactivity of other parts. Furthermore, it was paralleled by activity of a non-native dipeptide d-form. It is concluded that the latter effect and that of the middle segment may represent complex interactions other than the apparently specific effects of the C-terminal segment. [Diabetologia (1998) 41: 287–291] Received: 31 July 1997 and in revised form: 30 October 1997     

Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.

A panel of nine mouse monoclonal antibodies has been prepared against purified preparations of rat kidney Na+,K+-ATPase (EC 3.6.1.3). Selection for specific antibody was based upon the ability of crude hybridoma fluids to inhibit Na+-ATPase activity (using luciferase-linked ATPase assays) and upon antibody binding to both the purified kidney membrane enzyme and to glutaraldehyde-fixed hepatocytes by using standard enzyme-linked immunoadsorbent assays. After immunoaffinity purification, two of the antibodies (both of the IgG1 subclass) fully inhibit kidney and liver membrane Na+,K+-ATPase activity with Ki (apparent) values of 30 nM ("9-A5") and 600 nM ("9-B1"). Immunoblots demonstrate directly that three different 125I-labeled antibodies (6-4, 9-A5, and 9-B1) bind predominantly to a 94,000 Mr protein that comigrates in NaDodSO4/polyacrylamide gels with the fluorescein isothiocyanate-labeled alpha subunit of the Na+,K+-ATPase. Indirect immunofluorescence studies with these antibodies on paraformaldehyde-fixed liver slices reveal staining patterns congruent with bile canalicular membrane domains. These results together suggest that the antibodies exert inhibitory effects by recognizing alpha subunits of both liver and kidney Na+ pumps in their native conformations.     

C-peptide, Na+,K(+)-ATPase, and diabetes

Na+,K(+)-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na+,K(+)-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na+,K(+)-ATPase activity was strongly related to blood C-peptide levels in non-insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene. A polymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na+,K(+)-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na+,K(+)-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na+,K(+)-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na+,K(+)-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na+,K(+)-ATPase activity. This impairment in Na+,K(+)-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetes-induced decrease in Na+,K(+)-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na+,K(+)-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na+,K(+)-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na+,K(+)-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

Erythrocyte Na+, K+-ATPase activity in patients with congestive heart failure.

In order to determine if the Na+, K+-ATPase activity in erythrocyte membranes is altered in congestive heart failure, and to examine its clinical significance with respect to other clinical variables, erythrocyte Na+, K+-ATPase activity was measured in 51 patients with left ventricular ejection fractions <40% (coronary artery disease, n=26; dilated cardiomyopathy, n=25) and 24 control patients. Na+, K+-ATPase activity was lower in both coronary artery disease and dilated cardiomyopathy groups than control group even in the absence of digitalis use. There was a significant inverse correlation between Na+, K+-ATPase activity and plasma norepinephrine. The presence of non-sustained ventricular tachycardia was associated with a lower Na+, K+-ATPase activity in both groups with congestive heart failure without digitalis use than those without ventricular tachycardia. Plasma norepinephrine was higher in patients with non-sustained ventricular tachycardia than those without in the coronary artery disease group, but not in the dilated cardiomyopathy group. Na+, K+-ATPase activity may be helpful in predicting electrophysiologic instability in patients with heart failure.     

Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat

The effect of rat prolactin on rat renal Na+-K+-ATPase activity was investigated by a cytochemical technique. Rat prolactin caused stimulation of Na+-K+-ATPase activity only in the outer medulla of the kidney, and not in renal cortical structures. Peak enzyme activity in cultured rat renal segments occurred after tissue had been exposed to rat prolactin for 2 min, and the time of maximal stimulation did not vary with the concentration of prolactin. There was a curvilinear response in Na+-K+-ATPase activity over the rat prolactin concentration range, 0.04-40 ng/l, but higher prolactin concentrations caused inhibition of enzyme activity. Na+-K+-ATPase response was totally blocked by specific rat prolactin antiserum. Human prolactin had no consistent effect on rat medullary Na+-K+-ATPase activity. Addition of specific tri-iodothyronine and arginine vasopressin antisera to rat prolactin was without effect, confirming that the stimulatory action of rat prolactin on Na+-K+-ATPase was not due to contamination with these hormones which are known to stimulate this enzyme.     

Regression of renal hypertrophy and elevated renal Na+,K+-ATPase activity after insulin treatment in streptozotocin-diabetic rats

The effect of insulin treatment on the renal hypertrophy and elevated renal Na+,K+-ATPase activity in rats with streptozotocin (STZ)-induced diabetes was examined. Rats with STZ-diabetes of 6- to 8-week duration had significantly lower body weights, higher plasma and urinary glucose concentrations, greater urinary volumes, increased kidney weights, and increased kidney/body weights and protein/kidney weight ratios compared to those in saline-citrate-injected controls. Specific Na+,K+-ATPase activity per mg protein in both cortical and outer medullary kidney homogenates was significantly elevated in diabetic vs. control animals, as was total renal Na+,K+-ATPase activity. One week of insulin treatment returned elevated plasma glucose, urinary volume, the protein/kidney weight ratio, and cortical and outer medullary Na+,K+-ATPase activity per mg protein to control values. Kidney weights and kidney/body weight ratios of diabetic animals remained elevated, as did absolute total renal Na+,K+-ATPase activity. After 3 weeks of insulin treatment, kidney weight and total renal Na+,K+-ATPase activity in diabetic animals returned to control values, but body weights remained lower than those in the controls, resulting in continued elevation of kidney/body weight ratios in the diabetic animals. The concurrent regression of both renal hypertrophy and elevated Na+,K+-ATPase activity to normal levels after insulin treatment of STZ-diabetic animals implicates renal growth rather than a direct effect of insulin as the primary factor controlling elevation and regression of Na+,K+-ATPase activity in the diabetic kidney. This finding demonstrates that the effect of renal hypertrophy can outweigh the intrinsic effects of insulin on an important renal transport system and that this effect may be as important as lack of hormone in determining the renal physiological responses in the disease. It is suggested that the increased renal tubular Na+,K+-ATPase activity is a key component of the renal hypertrophy and hyperfunction seen in diabetes.     

Higher plasma Na+,K+-ATPase inhibitory activity in essential hypertensive patients

The ability of plasma to inhibit 86 rubidium uptake in rat aorta and to displace [3H]-ouabain from hog brain Na+,K+-ATPase was used as a measure of plasma Na+,K+-ATPase inhibitory activity in seven normotensive and eight hypertensive subjects. Rat aortae rings were incubated in oxygenated plasma containing 86 rubidium (2 microCi/mL) for 30 mins at 37 degrees C and uptake measured and expressed as mumol/kg wet weight/min. Plasma was extracted with a mixture of chloroform and methanol (2:1) and the extract separated by silicic acid column followed by thin layer chromatography and fractions assayed for ouabain displacement using digoxin as a standard. Total ouabain displacement was calculated as the sum of all fractions. There was a strong correlation between the two methods for total plasma Na+,K+-ATPase inhibitory activity (r = 0.761, P less than 0.01). There was a significant positive correlation between plasma Na+,K+-ATPase inhibitory activity and blood pressure in all subjects. Na+,K+-ATPase inhibitory activity was significantly higher in plasma of hypertensives by both methods (P less than 0.001). The increased Na+,K+-ATPase inhibitory activity in plasma from hypertensives was due to the nonesterified fatty acid, long chain acylcarnitine and diphosphatidylglycerol fractions.     

Inhibition of rat brain Na+,K+-ATPase activity by serum from patients with fulminant hepatic failure

Among the toxins accumulating in the circulation of patients with fulminant hepatic failure (FHF) are substances which inhibit leucocyte ouabain-sensitive sodium transport. A similar inhibition of brain Na+,K+-ATPase could lead to both coma and cerebral edema found in these patients which are associated with high mortality. In this study, we have investigated the effect of sera from FHF on normal rat brain Na+,K+-ATPase activity in vitro. Serum from patients with FHF significantly decreased the ouabain-sensitive Na+,K+-ATPase activity (13.58 +/- S.D. 2.60 mumoles Pi mg protein-1 hr-1) in the rat brain membrane preparation in vitro as compared to normal serum (20.33 +/- 3.24 mumoles Pi mg protein-1 hr-1, p less than 0.001). A final serum dilution of 1 in 40 was required to abolish the inhibition of Na+,K+-ATPase activity. Cerebrospinal fluid obtained at postmortem from FHF patients also contained the inhibitory substances. Serum from patients in coma due to decompensated chronic liver disease inhibited the Na+,K+-ATPase activity (17.25 +/- 1.37 mumoles Pi mg protein-1 hr-1), but this was less marked than with FHF serum. Hence, the inhibition of brain Na+,K+-ATPase by substances accumulating in the serum in FHF may be important in the pathogenesis of hepatic coma.     

Cytochemically assayable Na+,K+-ATPase inhibition by Milan hypertensive rat plasma

The ability of plasma from 3- and 9-week-old Milan hypertensive rats and their normotensive controls to inhibit Na+,K+-adenosine triphosphatase (ATPase) was studied using cytochemical bioassay techniques in fresh tissue. With a validated cytochemical bioassay that measures the capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase activity in guinea pig proximal tubules as an indication of their capacity to inhibit Na+,K+-ATPase, the mean glucose-6-phosphate dehydrogenase-stimulating ability of the plasma of the 9-week-old Milan hypertensive rats and their normotensive controls was 586.0 +/- 88 and 23.4 +/- 8.3 U/ml (n = 7; p less than 0.001), while that of the 3-week-old Milan hypertensive rats (before the main rise in arterial pressure) and their normotensive controls was 99.9 +/- 27.4 and 7.8 +/- 1.8 U/ml (n = 7; p less than 0.001). With the use of a semiquantitative cytochemical assay that measures Na+,K+-ATPase activity directly, plasma from the adult hypertensive rats had a much greater capacity to inhibit Na+,K+-ATPase than the plasma of the control rats. The significantly raised levels found in the young hypertensive rats before the main rise in arterial pressure are consistent with the hypothesis that the rise in the ability of plasma to inhibit Na+,K+-ATPase is due to an inherited renal difficulty in excreting sodium.     

Age-related oxidative inactivation of Na+, K+-ATPase in rat brain crude synaptosomes

The study was undertaken to examine the status of Na(+), K(+)-ATPase in aged rat brain and to verify if any alteration of this enzyme in aged brain could be related to an oxidative damage. The crude synaptosomes from rat brain were exposed in vitro to an oxidative stress in the form of a combination of Fe(2+) (100 microM) and ascorbate (2 mM) for up to 2 h when increased lipid peroxidation (nearly four-fold), extensive protein carbonyl formation and a marked decrease of Na(+), K(+)-ATPase activity (approximately 88%) were observed. All these changes were prevented by the presence of a chain-breaking anti-oxidant, butylated hydroxytoluene (0.2 mM), in the incubation mixture. When the same crude synaptosomal membranes from the young (4-6 months) and aged (18-22 months) rat brains were analysed, a significant reduction of Na(+), K(+)-ATPase activity (nearly 48%) along with significantly elevated levels of lipid peroxidation products and protein carbonyls could be detected in the aged animals in comparison to young ones. The latter data in combination with the results of in vitro experiments imply that the age-related decline of rat brain Na(+), K(+)-ATPase activity is presumably the consequence of an enhanced oxidative damage in aging brain     

Ovariectomy increases Na+, K+-ATPase, acetylcholinesterase and catalase in rat hippocampus

In the present work we investigated the effect of ovariectomy on Na+, K+-ATPase and acetylcholinesterase (AChE) activities in rat hippocampus. We also studied some parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), thiobarbituric acid-reactive substances (TBA-RS), as well as the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. Our hypothesis is that ovariectomy might cause alterations in essential enzyme activities necessary to brain normal functioning and that these chances could be caused by oxidative stress. Female adult Wistar rats were divided into three groups: (1) naive (control); (2) sham-operated; and (3) ovariectomized. Thirty days after ovariectomy rats were sacrificed. Results showed that rats subjected to ovariectomy presented a significant increase in Na+, K+-ATPase, AChE and CAT activities, but did not change the oxidative stress parameters studied when compared to sham or naive rats. Since ovariectomy mimics postmenopausal changes, our findings showing alteration in the activities of brain Na+, K+-ATPase, AChE and CAT may be related to problems in postmenopausal women.     

Peroxynitrite induced decrease in Na^＋, K^＋-ATPase activity is restored by taurine

AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed to ONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured. RESULTS: Different concentrations of ONOO- (100, 200, 500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO- to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.     

Erythrocyte ghost Na+,K+-ATPase and blood pressure

To examine the relationship between body mass index, blood pressure, and the Na+,K+-adenosine triphosphatase (ATPase) system, we measured the erythrocyte ghost Na+,K+-ATPase and the erythrocyte Na+ concentration in 120 blacks and 127 whites (136 males and 111 females). Blacks showed a 13.9% higher erythrocyte Na+ (7.63 +/- 0.19 vs 6.70 +/- 0.11 [SEM] mEq/L; p = 0.0001) and a 16.1% lower erythrocyte ghost Na+,K+-ATPase activity (140.3 +/- 4.2 vs 167.3 +/- 4.7 nmol inorganic phosphate/mg protein/hr; p = 0.0002) than whites. Male subjects demonstrated a 6.4% higher erythrocyte Na+ (7.35 +/- 0.17 vs 6.91 +/- 0.14 mEq/L; p = 0.043) and an 11.5% lower Na+,K+-ATPase activity (145.7 +/- 3.7 vs 164.7 +/- 5.5 nmol inorganic phosphate/mg protein/hr; p = 0.0015) than female subjects. Significant (p less than 0.001) negative correlations were identified for the systolic, diastolic, and mean blood pressure levels and the erythrocyte ghost Na+,K+-ATPase. These findings were complemented by positive correlations for the blood pressure levels and erythrocyte Na+ concentrations. The body mass index was negatively correlated with erythrocyte ghost Na+,K+-ATPase and it accounted for 6.7%, 5.6%, and 6.1% of the variabilities in the systolic, diastolic, and mean blood pressure levels, respectively. Variabilities of 1.4% systolic, 12.3% diastolic, and 11.1% in mean arterial pressure were attributable to the erythrocyte ghost Na+,K+-ATPase activity. Provided that findings in erythrocytes also reflect the relative status of the vascular smooth muscle cell Na+,K+-ATPase, the predisposition of black, male, and obese persons to hypertension may relate, among other factors, to a lower activity of this enzyme system, which results in an increased vascular tone.     

Erythrocyte Na+,K+-ATPase and nasal potential in pseudohypoaldosteronism

OBJECTIVES: Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disorder characterized by salt-wasting due to target organ unresponsiveness to mineralocorticoids. PHA1 comprises two clinically and genetically distinct entities; isolated renal and systemic forms. DESIGN: The aim of this study was to investigate red blood cell (RBC) Na+,K+-ATPase activity and nasal potential difference (PD) in two pairs of unrelated dyzygous twins; one with the systemic form of the disease (PHA1-S) and the second with the isolated renal form (PHA1-R). Total and ouabain-sensitive ATPase activities were measured spectrophotometrically by a method that couples ATP hydrolysis with NADH oxidation. Maximal PD and response to amiloride perfusion were evaluated by a standard technique. RESULTS: In the twins with PHA1-S, persistently low activity of RBC Na+,K+-ATPase was found during a 6-year follow-up. Normalization of plasma renin activity (PRA) and plasma aldosterone was observed at the end of the first year of life. Maximal nasal PD was low and there was no significant response to amiloride. In the twins with PHA1-R, RBC Na+,K+-ATPase activity was very low at the time of diagnosis and normalized at the age of 6-8 months. PRA reverted gradually to normal values, whereas aldosterone levels remained high during the 6 years of follow-up. Maximal nasal PD and response to amiloride were normal. CONCLUSIONS: The observed differences in RBC Na+,K+-ATPase activity and nasal PD response to amiloride between the two pairs of twins support the contention of different basic pathogenic mechanisms in the two forms of PHA1.     

cAMP regulates nitric oxide production and ouabain sensitive Na+, K+-ATPase activity in SH-SY5Y human neuroblastoma cells

Summary We investigated the relation between cyclic AMP (cAMP) and nitric oxide (NO) production, as well as the effect of NO on Na⁺ , K⁺-ATPase activity in the human neuroblastoma cell line SH-SY5Y. Two cAMP agonists, dibutyryl cAMP (DBC) and beraprost sodium (BPS), increased cAMP accumulation and NO production in a time and dose dependent manner at 50 mmol/l glucose. On the other hand, cellular sorbitol and myo-inositol contents and protein kinase C activity were not altered by DBC or BPS. A specific protein kinase A inhibitor, H-89, suppressed increases in nitrite/nitrate and cyclic GMP (cGMP) and protein kinase A activity stimulated by DBC or BPS. This finding suggests that cAMP stimulates NO production by activating protein kinase A via a pathway different from the sorbitol-myo-inositol-protein kinase C pathway. We observed that an NO donor, sodium nitroprusside, and an NO agonist, L-arginine, enhanced ouabain sensitive Na⁺, K⁺-ATPase activity at 50 mmol/l glucose. We also found that a nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), inhibited Na⁺, K⁺-ATPase activity at 5 mmol/l glucose, and partially suppressed the enzyme activity stimulated by DBC or BPS. The results of this study suggest that cAMP regulates protein kinase A activity, NO production and ouabain sensitive Na⁺, K⁺-ATPase activity in a cascade fashion. The results also suggest that protein kinase A at least partially regulates Na⁺, K⁺-ATPase activity without mediation by NO in SH-SY5Y cells. We speculate that cAMP and NO are two important regulatory factors in the pathogenesis of diabetic neuropathy. [Diabetologia (1998) 41: 1451–1458] Received: 17 November 1997 and in final revised form: 27 July 1998     

Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.     

The search for a hypothalamic Na+,K+-ATPase inhibitor

Accumulating experimental evidence suggests that natriuresis in response to intravascular volume expansion is promoted by an endogenous regulator of Na+,K+-adenosine triphosphatase (ATPase). Efforts to purify this substance by a number of laboratories have as yet been unsuccessful. The properties of partially purified inhibitors from plasma, urine, and tissue often fail to possess the characteristics thought to be consistent with those of a physiological regulator. These include potency (Ki of approximately 1 nM), reversibility of inhibition, specificity for Na+,K+-ATPase, and responsiveness to relevant physiological stimuli. Two rather different candidate substances, extracted from urine and hypothalamus, have been purified to a high degree. Neither is a peptide, and both are of low molecular weight and resistant to acid hydrolysis. The substance from urine is rather nonpolar and interacts with digoxin-specific antibodies, while that from hypothalamus is polar and does not appear to share epitopes with the cardiac glycosides. On the serosal surface of the toad urinary bladder, the hypothalamic substance causes a reversible inhibition of Na+ transport, inhibits rubidium uptake in red blood cells by acting on the membrane's exterior surface, inhibits binding of ouabain to purified Na+,K+-ATPase, and reversibly inhibits hydrolysis of adenosine 5'-triphosphate by the enzyme with a Ki of 1.4 nM. The hypothalamic inhibitor may be differentiated from ouabain by their respective ionic requirements for optimal inhibition of enzymatic activity, and although both ouabain and the hypothalamic inhibitor fix Na+,K+-ATPase in its E2 conformation, the hypothalamic inhibitor does not promote phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. Ionic requirements for inhibition also differentiate the hypothalamic inhibitor from vanadate ion, as does the inhibitor's activity in the presence of norepinephrine. Further enzymological and physiological studies will be facilitated by structural characterizations of the inhibitory substances and by the availability of a method to measure their concentrations in physiological fluids.     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Two ionophores (monensin and gramicidin) that carry Na+ into 3T3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing 86Rb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 86Rb+ uptake and cell Na+ and K+ over a range of monensin concentrations show that the activity of the Na+,K+ pump in 3T3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum rapidly enhances the rate of 22Na+ uptake and net Na+ entry when Na+ exit is inhibited by ouabain. At 0.3 microgram/ml, monensin increases the rate of net Na+ entry and activates the Na+,K+ pump by the same degree as serum. The stimulation of 86Rb+ uptake by serum or the ionophores has an absolute requirement for external Na+. Thus, serum appears to stimulate the Na+,K+ pump in quiescent 3T3 cells by increasing its supply of Na+.     

Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts

OBJECTIVE: Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. METHODS AND RESULTS: In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 muM followed by an 8-minute washout before 30 min of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cgamma1/protein kinase Cepsilon (PLC-gamma1/PKCepsilon) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-gamma1/PKCepsilon pathway, but also cardiac protection. This protection was also blocked by a PKCepsilon translocation inhibitor peptide (PKCepsilon TIP). CONCLUSION: Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-gamma1 and PKCepsilon.