Number Of Pump Sites Research Articles

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 ★π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 ★π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 ★π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 ★π= 0.5 and π= 2 ★π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton.

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (pi) from its normal value (pi = 1) to one-half (pi = 0.5), but also in muscles that are returned to pi = 1 after equilibration in pi = 2 medium. The equilibration in pi = 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+-K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 microM). Under those conditions, the pi = 1 pi = 0.5 transfer produced a 43 % increase in pump sites, while the pi = 2 pi = 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 microM) and intermediate filaments by acrylamide (4 mM) did not alter the hypotonic effect. Likewise, genistein (100 microM), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 microM) and latrunculin B (10 microM) reduced the increase in Na+ efflux induced by pi = 1 pi = 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by pi = 1 pi = 0.5 and pi = 2 pi = 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Regulation of Na+pump expression by vascular smooth muscle cells

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.

Renal Na,K-ATPase in genetic hypertension.

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Sodium pump inhibition in sarcolemma from ischemic hearts

Ischemic myocardial cells lose K+ and accumulate Na+. The role of the Na+/K(+)-pump in these changes was investigated by measuring both Na+/K(+)-ATPase activity and Na+ pumping in highly purified sarcolemmal vesicles from rabbit hearts made globally ischaemic for 1 h compared to non-ischemic controls. Purification of the sarcolemma was similar for control, 31 +/- 8-fold, and ischemia, 38 +/- 10-fold. The fraction of intact inside-out vesicles, in which Na+ pumping could be measured, was also the same for control, 60 +/- 16%, and ischemic, 56 +/- 8% as measured by 3H-ouabain binding in the presence and absence of detergent. Scatchard analysis of ouabain binding revealed a 26% increase in binding sites in ischemia compared to control. The Na+/K(+)-ATPase in the inside-out vesicles, measured as monensin-stimulated activity, was not affected by ischemia: 22 +/- 9 v 21 +/- 9 mumol Pi mg -1 h -1 for control and ischemic respectively. However, the initial velocity of ATP-dependent Na+ pumping into inside-out vesicles, assayed by subsequent exchange of Na+i for 45Ca2+ by the Na(+)-Ca2+ exchanger present in the vesicles was inhibited in ischemia. At 18 mM Na+o the velocity for control vesicles was 2.4 +/- 0.2 nmol mg -1 s -1 compared to 1.1 +/- 0.1 for ischemia vesicles. Passive sarcolemmal Na+ permeability was unchanged after 1 h of ischemia. The large reduction in Na+ pumping with unchanged Na+/K(+)-ATPase suggests uncoupling of the Na+/K(+)-pump in ischemia and a decreased ability to extrude Na+ despite the increase in number of pump sites in the sarcolemma.

Sarcoplasmic reticulum Ca2+ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes.

Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were measured simultaneously with indo 1 and a Ca(2+)-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes (approximately 10 mg/mL protein). In the presence of 25 mumol/L ruthenium red and 10 mmol/L oxalate, the Km for Ca2+ uptake by the SR was approximately 250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary to inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 mumol/kg wet wt for rabbit and 54.6 mumol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to < 150 nmol/L from a peak value > 10 mumol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in < 15 seconds and reduced the uptake rate by 95% in 5 seconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypo-osmotic stimulation of active Na+ transport in frog muscle: Apparent upregulation of Na+ pumps

The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na+ transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in (i) active K+ influx, (ii) passive Na+ and K+ fluxes, and (iii) the number of 3H-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (pi) to one-half of its normal value (pi = 0.5) produced the following effects: (i) an increase in active K+ influx on the order of 160%, (ii) a 20% reduction in Na+ influx and K+ permeability (PK), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na+ pump is not caused by an increased leak of either Na+ (inward) or K+ (outward). It is unlikely that the stimulation of active Na+ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na+ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance pi = 2 to pi = 1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na+ and K+ fluxes were not significantly altered by hypertonicity (pi = 2).

Apparent upregulation of Na+,K+ pump sites in SHR skeletal muscle with reduced transport capacity.

Slow-twitch, oxidative skeletal muscles in SHR exhibit several physiological defects, including a reduced ability to maintain force during high frequency repetitive stimulation (1). Muscle fatigue may be produced by one of a variety of factors acting at different levels of the neuromuscular system. Several lines of evidence, however, suggest that SHR soleus fatigues more rapidly than WKY soleus because SHR muscles allow more K+ to accumulate in the extracellular space during repetitive muscle activity. An increase in extracellular K+ can lead to a failure in the generation or conduction of muscle action potentials. Comparison of the compound action potentials recorded from SHR and WKY muscles during repetitive stimulation provided evidence for a decrease in excitability of SHR soleus. Since the K+ released from muscle fibers during exercise is returned to the fiber principally via the activity of the Na+, K+ pump, the increase in extracellular K+ in SHR muscle may reflect a decrease in pump capacity. Measurements including intracellular K+ and Na+ content at rest, the level of hyperpolarization produced by the addition of epinephrine and insulin to SHR soleus and the post-exercise recovery of resting membrane potentials all appear to indicate that Na+, K+ pump capacity is reduced in SHR soleus muscles. Nonetheless, ouabain binding studies show a significantly greater number of pump sites in SHR muscles. The data suggest that Na+ pump activity is decreased in SHR soleus muscles without an apparent reduction in either the number of pump sites or in pump binding affinity.

Properties of the Na+-K+ pump in human red cells with increased number of pump sites.

We studied the Na+/K+ pump in red cells from an obese human subject (MAJ) in which the number of pumps/cell was 10-20 times higher than normal. Through measurements of the kinetic properties of several modes of operation of the Na+/K+ pump we determined that the pumps in MAJ cells are kinetically normal. In the presence of adequate metabolic substrate the maximum rates of Na+ pumping and lactate production saturated at 60 and 12 nmol/1 cell per h, respectively. Under physiological conditions pump and "leak" Na+ fluxes were similar in MAJ and normal cells. Since internal Na+ was lower in MAJ than in normal cells (Nai+ approximately 2 and 8 mmol/1 cell, respectively), we conclude that the reduction in cell Na+ allows the Na+/K+ pump in MAJ cells to operate at lower fraction of maximum capacity and to compensate for the increased number of pumps.

The kidney in potassium depletion. I. Na+-K+-ATPase activity and [3H]ouabain binding in MCT.

The effect of potassium depletion on renal Na+-K+-ATPase was studied in rats. K depletion produced a striking, time-dependent increase in Na+-K+-ATPase activity of the outer medullary collecting tubules (inner stripe; MCTis). After 3 wk on the K-free diet, when the urine was almost potassium-free, Na+-K+-ATPase activity in MCTis was over fourfold higher than in control animals (2,964 +/- 185 vs. 645 +/- 108 pmol X mm-1 X h-1). Repletion of potassium restored enzyme activity to base line within 7 days (t1/2 = 3.8 days), which corresponds to the catabolic rate of the renal enzyme, suggesting the cessation of enhanced synthesis that took place during K deprivation. Changes in Na+-K+-ATPase activity and aldosterone levels during both K depletion and repletion occurred in opposite directions and were therefore independent of each other. [3H]Ouabain binding to intact MCTis, reflecting the number of pump sites on the basolateral membrane, was similar in K-depleted and control animals; in contrast, tubule permeabilization that exposes additional pump units to the ligand, unmasked a nearly fourfold increase in [3H]ouabain binding (50.0 +/- 6.8 vs. 13.2 +/- 1.7 fmols X mm-1) in K-depleted rats, comparable to the increment in Na+-K+-ATPase activity. These results show that K depletion leads to a marked increase in Na+-K+-ATPase activity of MCTis, and suggest that the new enzyme units are located at a ouabain-inaccessible site in the intact tubule, i.e., either in an intracellular compartment or at the luminal membrane, where they may be involved in potassium reabsorption.

Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes.

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.

Designing optimal strategies for contaminated groundwater remediation

The problem of locating pumps and setting pump rates to most effectively stabilize and remove a plume of contaminated groundwater at a hazardous waste site is examined. Nonlinear optimization methods are combined with convective-disperisve transport simulation in a unit response matrix type of optimization formulation. Constraints are used which guarantee that the contaminant plume is removed by limiting the concentrations at nodal points in the domain at a future time. Additional constraints explicitly require that concentrations not increase in the area outside the initial plume boundary. The effectiveness of alternative formulations are examined by performing numerical experiments using a hypothetical aquifer. The experiments show that computational costs are dominated by the repeated simulations required for computation of constraint gradients and are proportional to the number of pump sites under consideration. This characteristic of the formulation and algorithm used, limits the use of the approach to problems where the number of potential pump sites is relatively small.

Effect of chronic surgical denervation of dog heart on myocardial Na+-K+ pump.

The effect of chronic denervation of the myocardium on myocardial Na+-K+ pump activity and numbers of pump sites was studied in dogs. Right ventricular biopsies were taken immediately before and 4 wk after chronic surgical denervation, when depletion of norepinephrine is complete. Na+-K+ pump activity was assayed by ouabain-sensitive 86Rb uptake into ventricular tissue slices. The number of pump sites was measured by [3H]ouabain binding in ventricular tissue slices. Ouabain-sensitive Rb uptake was increased after denervation by an average of 147%. [3H]-ouabain binding at a concentration of 1.25 X 10(-6) M increased by an average of 27.6% after denervation. Acutely applied norepinephrine (2 X 10(-7) M) stimulated ouabain-sensitive Rb uptake both before and after denervation. The stimulation was greater after denervation, indicating supersensitivity. The cause of these changes is not known.

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line.

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.

Estimates of Na+-K+ pumping in intact canine iliac arteries.

Estimates of Na+ pumping capacity were made using Na+-loaded canine iliac arteries. Ouabain-sensitive uptake of 204Tl or 86Rb was used to measure near-maximal pump rates and [3H]ouabain binding to measure the number of pump sites. Compared with Rb+, Tl+ had the higher affinity for the pump and showed better signal-to-noise characteristics. Maximal uptakes were 0.545 mumol . g-1 . min-1 for Rb+ and 0.40 mumol . g-1 . min-1 for Tl+. Specific ouabain binding (Kd: 28.62 +/- 0.58 nM) was inhibited by external K+, Tl+, and Rb+ and a maximal binding of 51.6 pmol/g wet weight translated into 3.2 X 10(13) sites per gram wet weight. Using these values, the maximal values of K+ transported per pump site per minute lie between 7752 and 10562. If each activation of the pump moves 2K+, the turnover rates could lie between 3876 and 5281 per minute.

Rubidium uptake of mononuclear leukocytes from normotensive and borderline hypertensive first degree relatives to patients with essential hypertension.

Uptake of 86Rubidium of mononuclear leukocytes (MNL) was used as a measure of cellular sodium-potassium pump activity. 86Rb-uptake was determined with the pump stimulated mainly from inside the cells by sodium as well as with a combined stimulation from inside by sodium and from outside by Rb. In the first case there was an increased pump activity in MNL from borderline hypertensive offspring of hypertensive patients (BHO), and this may be related to an increased number of pump sites observed previously (10). Estimation of maximal pump activity (Vmax) of MNL suggested that Vmax of each pump site in MNL from BHO may be decreased compared to control value of MNL from healthy normotensive subjects.

Ca 2+ capacity and uptake rate in skinned fibers of myodystrophic muscle

Parameters related to the capacity and the rate of uptake of calcium ions by the sarcoplasmic reticulum were measured in skinned extensor digitorum longus fibers of control and myodystrophic mice. Single fibers were isolated and skinned in a relaxing solution and mounted on a force transducer and apparatus for changing the bathing solution ( T = 25°C). To test the capacity of the sarcoplasmic reticulum, fibers were placed in a solution for maximal loading and then moved to a test solution in which the major anion in the relaxing solution, gluconate, was replaced by chloride. In the resulting contractures, the means of the forces produced by 10 control and myodystrophic fibers were not significantly different. The conclusion is that the capacities of sarcoplasmic reticulum for calcium in control and myodystrophic fibers are equivalent. To test the rate of loading of sarcoplasmic reticulum, 11 control and myodystrophic fibers were depleted of calcium with caffeine and EGTA. Then they were placed in a solution with pCa = 5.5, and the delay before a contracture began was recorded. The delay was the time required for the sarcoplasmic reticulum to load calcium and attain a threshold for calcium-induced calcium release. The mean delay for the control fibers was significantly less than the mean delay in myodystrophic fibers. The disparity of loading times probably reflected a difference in the activities of the calcium pumps or a difference in the number of pump sites; 5 μ M valinomycin did not significantly alter the loading times of either type or fiber.

The sodium pump in opossum vascular smooth muscle

1. 1. Ouabain-sensitive Rb + uptake and [ 3H]ouabain binding were used to measure rates of Na + pumping and the number of pump sites, respectively, in thoracic aortae from opossums. 2. 2. From the number of Rb + ions pumped per site per minute, estimates of pump turnover have been made. 3. 3. Values obtained are comparable to those of other species (see Table 1).

Insulin regulation of Na/K pump activity in rat hepatoma cells.

Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by 3H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of 22Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.

Ouabain binding, Na+-K+-ATPase activity, and 86Rb uptake of canine arteries

In a previous report [Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H598-H603, 1983] we measured potassium-induced relaxation of canine femoral and renal arteries as an index of sodium pump function and concluded that it may not be an accurate measure. In this report, the sodium pumps of femoral and renal arteries were examined using three separate approaches to compare sodium pump function more directly. The number of pump sites in subcellular fractions was measured using [3H]ouabain binding. No differences were observed (femoral artery 14.5 +/- 4.8 pmol/g wet wt; renal artery 17.4 +/- 6.5). Similarly, there was no difference in ouabain-inhibitable Na+-K+-ATPase activity in NaI-treated microsomal fractions of these arteries (femoral artery 3.10 +/- 0.65 mumol Pi X mg-1 X h-1; renal artery 3.42 +/- 0.23). Finally, sodium pump function was measured using the ouabain-inhibitable 86Rb uptake method. The rate of ouabain-inhibitable 86Rb uptake was found to be identical for the two arteries, as were the dissociation constants of the two arteries for rubidium. However, in the presence of 3.5 microM norepinephrine, the rate of 86Rb uptake in the renal artery was greater than that of the femoral artery. We conclude that, under resting conditions, the sodium pumps of femoral and renal arteries are identical, whereas in the presence of norepinephrine, the renal artery sodium pump is not rate limiting with respect to contractility. These data have significant implications with regard to the use of potassium-induced relaxation as an index of sodium pump activity.