Erythrocyte Na+/K+-ATPase and Membrane and Serum Lipid Profiles: As Related to Alcohol,Body Mass Index and Blood Pressure

Erythrocyte Na⁺/K⁺-pump activities have been measured in hypertensives, alcohol consumers and obese persons, but the results have been variously reported as decreased, increased or unchanged. We analyzed the relationships between erythrocyte Na⁺/K⁺-ATPase activities and the membrane and serum lipid profiles in 83 middle-aged men, to clarify the reasons for these inconsistencies. Increases in erythrocyte Na⁺/K⁺-ATPase activity related closely to decreases in cholesterol to phospholipid (C/P) ratio of the erythrocyte membrane. Decreases in the C/P ratio in turn related closely to elevations of serum triglycerides (TG) with increasing body mass index, and weakly to the volume of alcohol consumed. Thus, erythrocyte Na⁺/K⁺-ATPase activities depend largely on the membrane and serum lipid profiles as related to body weight and alcohol consumption.     

Reduction of erythrocyte (Na+-K+)ATPase activity in Type 1 (insulin-dependent) diabetic subjects and its activation by homologous plasma

Summary The (Na⁺-K⁺)ATPase and (Mg²⁺)ATPase activities of erythrocyte membranes of Type 1 (insulin-dependent) diabetic patients were found to be significantly reduced compared to matched controls (p < 0.005). On the contrary, erythrocyte Na⁺ and K⁺ contents were similar in diabetic patients and in normal subjects. When erythrocyte membranes from diabetic patients were incubated with their own plasma, a significant increase was observed in sodium-potassium ATPase activity (p < 0.005), whereas (Mg²⁺)ATPase activity was not affected. The plasma stimulatory effect showed saturation kinetics. Maximum average stimulation was 96% (±21.3). A similar stimulation pattern, although more limited in extent (maximum 48.3 % ± 12.2), was found when erythrocyte membranes from normal subjects were incubated with diabetic plasma. Normal plasma exhibited a modest stimulatory effect on erythrocyte (Na⁺-K⁺ATPase activity. Similar stimulatory effects by diabetic plasma were observed on a (Na⁺-K⁺) ATPase preparation from beef heart. It is proposed that diabetic plasma contains a specific (Na⁺-K⁺)ATPase activator in a higher concentration than normal plasma. This may explain why a normal cellular electrolyte content was found in diabetic erythrocytes in spite of a reduced Na⁺-K⁺ pump activity. Purification experiments indicate that the plasma activator is a protein with a molecular weight greater than 50,000. Both the (Na⁺-K⁺)ATPase activity and the stimulatory effect of diabetic plasma were not influenced by the metabolic control, since they did not correlate significantly with fasting blood glucose and daily insulin dosage. Moreover, no correlation was found with duration of diabetes or age at diagnosis of diabetes. It is suggested that the enzyme defect of erythrocyte membrane and the stimulation of (Na⁺-K⁺)ATPase activity by homologous plasma are early biochemical alterations in the course of diabetes mellitus.     

Basis of Rise in Intracellular Sodium in Airway Hyperresponsiveness and Asthma

The aim of this study was to investigate the basis of disturbances in sodium transport in asthma and in airway hyperresponsiveness without symptoms of asthma (asymptomatic AHR). We measured the intracellular sodium (Na_i); activity of Na⁺/K⁺-ATPase in unstimulated cells (resting activity) and in cell homogenate under optimal conditions (maximal activity); and sodium influx, in mixed leukocytes of 15 normal subjects, 12 subjects with asymptomatic AHR, and 26 asthmatics with or without active symptoms. Resting Na⁺/K⁺-ATPase activity was the same as sodium influx, consistent with homeostasis. Compared with normal subjects, those with asymptomatic AHR or asthma with controlled symptoms had a twofold increase in sodium influx and Na_i. Symptomatic asthmatics also had a twofold increase in sodium influx but a fourfold elevation of Na_i. Maximal Na⁺/K⁺-ATPase activity was reduced by half in symptomatic asthmatics compared with normal subjects. The reduction of maximal Na⁺/K⁺-ATPase activity was associated with a significant decrease in ATP turnover per Na⁺/K⁺-ATPase molecule but not number of Na⁺/K⁺-ATPase molecules per cell. In summary, airway hyperresponsiveness with or without asthma is associated with increased sodium influx and Na in leukocytes. Resting activity of Na⁺/K⁺-ATPase is also increased as a compensatory response to the increased sodium influx, but it is achieved at the expense of higher Na_i. Symptomatic asthma is additionally associated with reduction in maximal activity of Na⁺/K⁺-ATPase, resulting in reduced capacity to handle the increase in sodium influx and consequent severe elevations in Na_i.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

Effect of hypermethioninemia on some parameters of oxidative stress and on Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in hippocampus of rats

In the present study we investigated the effect of chronic administration of methionine, a metabolite accumulated in many inherited pathological conditions such as methionine adenosyltransferase deficiency and homocystinuria, on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), catalase activity and total thiol content, as well as on Na⁺,K⁺-ATPase activity in rat hippocampus. For chronic treatment, rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that chronic hypermethioninemia significantly increased TBARS, decreased Na⁺,K⁺-ATPase activity but did not alter catalase and total thiol content. Since chronic hypermethioninemia altered TBARS and Na⁺,K⁺-ATPase activity at 12 h after methionine administration, we also investigated the effect of acute administration of this amino acid on the same parameters studied after chronic methionine administration. For acute treatment,29-day-old rats received one single injection of methionine (2.68 μmol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that rats subjected to acute hypermethioninemia presented a reduction of Na⁺,K⁺-ATPase activity and an increase in TBARS when the animals were killed at 3 and 12 h, but not at 1 h, after methionine administration. These data indicate that hypermethioninemia increases lipid peroxidation which may, at least partially, explain the effect of methionine on the reduction in Na⁺,K⁺-ATPase activity. If confirmed in human beings, our findings could suggest that the induction of oxidative stress and the inhibition of Na⁺,K⁺-ATPase activity caused by methionine might contribute to the neurophysiopathology observed in patients with severe hypermethioninemia.     

Kinetics of Red Cell Na+ and K+ Transport in Prague Hypertensive Rats

Kinetics of ouabain-sensitive, furosemide-sensitive (FS), bumetanide-sensitive (BS) and -resistant Na⁺ and K⁺ transport were studied in erythrocytes of Prague hypertensive rats (PHR) and Prague normotensive rats (PNR). Maximal transport rates (V_max) and apparent affinities for either intracellular Na⁺ or extracellular K⁺ (replaced by Rb⁺) were determined in red cells in which Na⁺ content varied around the physiological range and that were incubated in Na⁺ media. No major differences between PHR and PNR were disclosed in the kinetics of ion transport mediated by the Na⁺-K⁺ pump or BS inward Na⁺-K⁺ cotransport. FS Rb⁺ uptake was higher (due to a greater V_max) in red cells of PHR as compared to PNR. In cells with a lowered Na⁺ content this elevation of FS Rb⁺ uptake was largely due to an augmented K⁺-Cl^? cotransport which exhibits a low affinity for Rb⁺_o and is blocked by 1 mM furosemide but not by 10 μM bumetanide. Red cells of PHR and PNR strains did not differ in either Na⁺ or Rb⁺ leaks. A slight increase of red cell Na⁺ content in PHR was evaluated in terms of the pump-leak concept. The present study did not reveal any obvious kinetic abnormalities of red cell cation transport the presence of which in tissues involved in blood pressure regulation would favor the development or the maintenance of genetic hypertension in PHR.     

Body Fluid Variations and Endogenous Digitalis-Like Compounds During Chronic NaCl Loading in Wistar Rats

Circulating digitalis-like compounds have been proposed to be raised in volume expanded hypertension and to participate in Na⁺ homeostasis. We have investigated the temporal relationships between the activity of these circulating digitalis-like compounds, blood pressure and body fluid volume variations during a chronic NaCl load in the Wistar rat.

Characteristics of salt-loaded rats were compared to those of weight-matched controls. At one week, when extracellular fluid volume (ECFV) was elevated, the capacity of plasma extracts to inhibit the Na⁺K⁺ATPase activity begun to rise. At two weeks, ECFV remained elevated, and plasma volume, blood pressure and the activity of plasma digitalis-like compounds increased. After 13 weeks, the continuous rise in plasma digitalis-like activity and in blood pressure was accompanied by the return of body fluid volumes towards control values. These changes in plasma digitalis-like activity, body fluid volumes, and systolic blood pressure during a high NaCl diet are compatible with the proposed role of circulating digitalis-like compounds as natriuretic and hypertensive factors.     

Activities of Transport Adenosine Triphosphatases in Erythrocyte Membranes of Healthy and Hypertensive Subjects

The Na⁺, K⁺-ATPase activity in the erythrocyte membrane was measured in 25 untreated essential hypertensive patients and 25 age-matched healthy normotensive subjects. In addition, the Ca²⁺, Mg²⁺-ATPase activity was measured in 20 hypertensive and 25 age-matched normotensive subjects. The Na⁺, K⁺-ATPase activity of healthy Chinese measured in this study was similar to the data reported in a Dutch study. We therefore could not support a theory which speculated an ethnic influence on Na⁺, K⁺-ATPase activity. Both Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activities were slightly lower in hypertensive patients as compared with those in normotensive subjects, but the differences were not statistically significant. We concluded that the transport ATPase activities were not impaired in the erythrocyte membranes of hypertensive patients.     

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)- and Mg <Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-ATPase activities of adult rat hypothalamus and cerebellum

Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na⁺, K⁺)- and Mg²⁺-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25μg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (−23%, p < 0.001) and the Na⁺, K⁺-ATPase activities (−26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (−17%, p < 0.001) and Na⁺, K⁺-ATPase (−27%, p < 0.001). Mg²⁺-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. In conclusion: neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na⁺, K⁺-ATPase activities. The decreased (by the THs) Na⁺, K⁺-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.     

Ouabain-Like and Non-Ouabain-Like Factors in Plasma of Patients with Essential Hypertension

Circulating inhibitor of Na⁺, K⁺-ATPase and ouabain-like immuno-reactivity were studied in patients with essential hypertension. In the plasma of patients, two types of Na⁺, K⁺-ATPase inhibitors (ouabain-like and non-ouabain-like inhibitors) and ouabain-like immunoreactivity were detected. Ouabain-like inhibitor was clearly detected at a low KC1 concentration (0.1 mM) in the assay buffer, and non-ouabain-like inhibitor was detected at a high KC1 concentration (10 mM). The plasma level of ouabain-like inhibitor correlated significantly with that of ouabain-like immunoreactivity (p<0.001) and with a mean blood pressure (p<0.01). The plasma level of non-ouabain-like inhibitor was not correlated with the levels of either ouabain-like immunoreactivity or mean blood pressure. The level of plasma ouabain-like inhibitor did not correlate with that of plasma non-ouabain like inhibitor. Both ouabain-like inhibitor and ouabain-like immunoreactivity in the plasma of patients with essential hypertension were significantly higher than those in normotensive subjects, but the plasma level of non-ouabain-like inhibitor in patients with essential hypertension was not higher than that in normotensive subjects. These results suggest that the plasma from patients with essential hypertension contains ouabain-like factor(s) which is important to mantain the high blood pressure.     

Myocardial (Na+k+)-Atpase Activity in Daul Salt-Sensitive and Resistant Rats

Vascular (Na⁺K⁺)-punp activity (ouabain-sensitive ⁵⁶Rb⁺uptake) and myocardial (Na⁺K⁺)-ATPase activity are reduced in. animals with various forms of low renin, experimental hypertersion. On the other hand, vascular (Na⁺, K⁺)-puinp activity is increased in Dahl salt-sensitive relative to resistant rats (a genetic model of hypertension), regardless of salt intake or blood pressure and it is also increased in Dahl salt-sensitive rats on high salt (3% NaCl) relative to low salt (0.4% NaCl) diets. It has been suggested that this increase in vascular (Na⁺K⁺)-pump activity may be secondary to an increase in the vascular sarcolemmal permeability to Na⁺in these salt-sensitive rats. In the present study, (Na⁺K⁺)-ATPase activity of left ventricular microsomal fractions, was increased in Dahl salt-sensitive relative to resistant rats on low salt diets; however, this difference disappeared when these salt-sensitive and resistant rats were placed on high salt diets. In contrast, mvocardial (Na⁺K⁺)-ATPase activity was decrease in Dahl salt-sensitive rats on high relative to low salt diets. Evidence that this decrease in (Na⁺K⁺)-ATPase activity is not secondary to myocardial hypertrophy in the hypertensive salt-sensitive rats, and mechanisms by which decreased cardiovascular (Na⁺K⁺)-pump activity, increased sarcolemmal permeability or both, might contribute to elevated blood pressure, are discussed.     

Isoform-specific alterations in cardiac and erythrocyte Na ,K-ATPase activity induced by norepinephrine

Background: Myocardial Na⁺,K⁺-ATPase activities are decreased in congestive heart failure because of an increase in plasma norepinephrine levels, but it is difficult to monitor the activities in the clinical setting.Methods and Results: This study investigated whether erythrocyte Na⁺,K⁺-ATPase activity can reflect myocardial enzyme activity and whether isoform-specific alterations occur in the presence of catecholamine. Na⁺,K⁺-ATPase activity was measured by the colorimetric method by using the left ventricular myocardium and erythrocytes prepared from eight rabbits given norepinephrine for 7 days and from eight control rabbits that received saline. The protein levels of total catalytic subunit and α₁ - or α₃-isoform of Na⁺,K⁺-ATPase were determined by Western blot analysis. Na⁺,K⁺-ATPase activity was lower in both myocardium and erythrocytes from norepinephrine-treated rabbits than control rabbits (P < .01 and P < .01, respectively). There was a close correlation in Na⁺,K⁺-ATPase activity between myocardium and erythrocytes (r = .963). Total catalytic subunit protein level was lower in myocardium from norepinephrine-treated rabbits than control rabbits, but the α₁-isoform level was similar between the two groups. The α₃-isoform level was lower in norepinephrinetreated rabbits than control rabbits. In erythrocytes, α₁-isoform was lower in norepinephrinetreated rabbits than control rabbits.Conclusions: Na⁺,K⁺-ATPase activity in myocardium could be reflected in erythrocyte membrane, although there was a difference in isoform-specific regulation between the two.     

Assay of a Circulating Sodium Pump Inhibitor in Patients with Essential Hypertension and Normotensive Subjects

The plasma levels of a sodium pump inhibitor (Na⁺PI) were measured by a modified method of Hamlyn et al, using dog kidney Na⁺, K⁺-ATPase. When the level of Na⁺PI was expressed as the % inhibition of the enzyme and compared with that of a control solution, it was found to be 9.0 ± 0.7% in 43 untreated patients with essential hypertension. This was significantly higher than 5.0 ± 0.4% for 56 normotensive subjects (p < 0.01). Male patients with essential hypertension showed the highest mean value of 10.5 ± 1.1%, disclosing an apparent sex difference in the patient group (p< 0.01). Only in female patients was there a significant positive correlation between the inhibitor's level and the mean blood pressure (r = 0.649, p < 0.01). These results provided additional evidence for increased Na⁺ PI in the plasma of patients with essential hypertension, which might bear an important role in the pathogenesis of the disease.     

Platelet Na+,K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria

Thein vitro effects of phenylalanine or alanine alone or combined on Na⁺,K⁺-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na⁺,K⁺-ATPase activity by 20–50% [F(4,25)=11.47; p<0.001]. Alanine had no effect on Na⁺,K⁺-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na⁺,K⁺-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na⁺,K⁺-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.     

Plasma Na+-K+ATPase Inhibitory Activity in Normal And Hypertensive Subjects: Relationship To Intracellular Electrolytes and Blood Pressure

The ability of plasma extracts to inhibit Na⁺-K⁺ATPase in vitro (P.I.A.) was tested in 20 normotensives, 10 without (F-) and 10 with (F+) familial hypertension, in 20 borderlines (BL) and in 21 essential hypertensives (EH). In these subjects we also measured intralymphocytic sodium (ILSC) and potassium (ILKC) content, P.R.A. urinary aldosterone and Na⁺(Na_u⁺), and blood pressure. P.I.A. of EH, BL and F+ subjects was significantly higher than that of F-. 60% of EH and BL and 40% af F+ had P.I.A. values greater than the high est found in F-. P.I.A. was significantly related to mean blood pressure (r=0.63), to ILSC (r=0.56), to ILKC (r=-0.56), to ILSC/ILKC ratio (r=0.71) and to Na_u⁺(r=0.39) but not to P.R.A. or aldosterone. These data demonstrate that plasma extracts from young subjects prone to hypertension may inhibit sodium pump and that this inhibitor may affect blood pressure by altering the Na⁺/K⁺intracellular ratio.     

In vitro insulin action on different ATPases of erythrocyte membranes in normal and diabetic rats

Summary Thein vitro effect of porcine insulin on Na⁺+K⁺-, Ca²⁺- and Mg²⁺-ATPases of the rat erythrocyte membrane of normal and alloxan-induced diabetic rats was investigated. Na⁺+K⁺- and Ca²⁺-stimulated enzyme activities were significantly decreased in diabetic rats in comparison to normal animals. The specific activities of both these ATPases in the latter group were markedly reduced on pre-incubating the ghosts with insulin. Similar treatment of the erythrocyte membranes of diabetic animals, however, resulted in a significant increase of these activities. These qualitatively different effects of the hormone in the two groups increased progressively with hormone concentration and duration of pre-incubation. Mg²⁺-stimulated ATPase activity was not significantly affected in diabetes or by insulin.     

Alterations of Na/K-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na⁺/K⁺-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na⁺/K⁺-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na⁺/K⁺-ATPase α1 subunit. However, it did increase ouabain-sensitive ⁸⁶Rb⁺ uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na⁺/K⁺-ATPase. While depletion of caveolae increases the pumping function of Na⁺/K⁺-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.     

Effect of ouabain on hemodynamics in acute volume expanded hypertensive dogs

Summary To evaluate possible roles of endogenous Na⁺–K⁺-ATPase inhibitors in vasoconstricted blood pressure elevation produced by acute volume expansion, we administered ouabain (Na⁺–K⁺-ATPase inhibitor) intravenously (30 g/kg) for 10 min to dogs, 3 h after volume expansion with dextran in lactated Ringer's solution (20 ml/kg, for 1 h). Acute volume expansion resulted in the elevation of blood pressure associated with an increase in cardiac output. In some dogs the blood pressure remained elevated with gradual increase in total peripheral resistance (Group I) or with sustained high cardiac output (Group II), and in other dogs (Group III) it returned to the control level. Ouabain administration elevated the blood pressure and total peripheral resistance in these groups and sham dogs which did not have volume expansion. And these effects of ouabain were not correlated with the degree of blood pressure or vasoconstriction produced by volume expansion. Thus, it is not likely that endogenous Na⁺–K⁺-ATPase inhibitors increased to produce vasoconstricted hypertension after acute volume expansion.     

Na+-mediated coupling between AMPA receptors and KNa channels shapes synaptic transmission

Na⁺-activated K⁺ (K_Na) channels are expressed in neurons and are activated by Na⁺ influx through voltage-dependent channels or ionotropic receptors, yet their function remains unclear. Here we show that K_Na channels are associated with AMPA receptors and that their activation depresses synaptic responses. Synaptic activation of K_Na channels by Na⁺ transients via AMPA receptors shapes the decay of AMPA-mediated current as well as the amplitude of the synaptic potential. Thus, the coupling between K_Na channels and AMPA receptors by synaptically induced Na⁺ transients represents an inherent negative feedback mechanism that scales down the magnitude of excitatory synaptic responses.     

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.