Cyclic stretch decreases TRPC4 protein and capacitative calcium entry in rat vascular smooth muscle cells.

We investigated whether cyclic stretch affects TRPC4 or TRPC6 expression and calcium mobilization in cultured vascular smooth muscle cells. In aortic and mesenteric smooth muscle cells isolated from male Sprague-Dawley rats, TRPC4 expression was decreased after 5 h stretch and remained suppressed through 24 h stretch. After removal of the stretch stimulus, TRPC4 expression recovered within 2 h. Stretch did not affect TRPC6 expression. Stretch also decreased capacitative calcium entry, while agonist-induced calcium influx was increased. Similar results were obtained in primary aortic smooth muscle cells. TRPC4 mRNA levels were not decreased in response to mechanical strain. TRPC4 downregulation was also achieved by increasing extracellular calcium and was attenuated by gadolinium and MG132, suggesting that TRPC4 protein is regulated by intracellular calcium concentration and/or the ubiquitin-proteasome pathway. These data suggest that stretch-induced downregulation of TRPC4 protein expression and capacitative calcium entry may be a protective mechanism to offset stretch-induced increases in intracellular calcium.

Mechanical stretch regulates TRPC expression and calcium entry in human myometrial smooth muscle cells.

Stretch is known to stimulate myometrial hyperplasia and hypertrophy in early pregnancy and uterine contraction at term. We propose that transduction of the stretch signal involves alteration of intracellular calcium signalling, including changes in transient receptor potential canonical (TRPC) isoform expression. The aim of the present study was to investigate the effect of prolonged mechanical (tonic) stretch in vitro on human myometrial smooth muscle cell calcium signalling and TRPC expression. Cells were cultured from myometrial biopsies, obtained from women undergoing elective Caesarean section at term, grown on Flexiplates and subjected to 25% tonic mechanical stretch for 1, 4 and 14 h. Time-matched control cells were not stretched. Mechanical stretch (14 h) increased basal calcium entry and cyclopiazonic acid (CPA)-induced calcium/Mn(2+) entry (P < 0.05) in Fura-2 loaded cells. The calcium selectivity of CPA-thapsigarin induced inward currents, measured by patch clamp electrophysiology, was also increased in stretched cells compared with control cells (P < 0.05). Real time PCR and Western blot data demonstrated that TRPC3 and TRPC4 mRNA and TRPC3 protein expression were increased by stretch (P < 0.05), respectively. These data support the hypothesis that uterine stretch modulates uterine growth and contractility in pregnancy via alterations in calcium signalling.

Effect of short-term cyclic stretch on sodium pump activity in aortic smooth muscle cells.

We previously demonstrated that expression of both the alpha1- and alpha2-subunits of Na+-K+-ATPase is elevated after a 2- to 4-day cyclic stretch in aortic smooth muscle cells. In this study, we determined the effect of short-term (2-30 min) cyclic stretch on the activity of the Na pump and investigated possible mechanisms that may be involved in the action of stretch. Na pump activity was significantly increased above the baseline activity between 2 and 30 min of stretch. This effect of stretch was reversible within 1 h. Intracellular Na was also elevated at corresponding time points. Blocking the entry of Na with Gd and amiloride did not affect the stretch-induced increase in Na pump activity. Inhibition of protein kinase A (PKA) activity attenuated the effect of stretch on the Na pump. Furthermore, inhibition of polymerization of actin and phosphatidylinositol 3-kinase (PI3K) activity prevented the action of stretch on Na pump activity. We conclude that the stimulation of the Na pump in response to cyclic stretch requires the integrity of the actin cytoskeleton as well as the activity of PI3K, which has a role in intracellular vesicular trafficking. PKA may also be involved in this effect of stretch on Na pump.

Effect of cyclic stretch on alpha-subunit mRNA expression of Na+-K+-ATPase in aortic smooth muscle cells.

We previously demonstrated that protein expression of both alpha(1)- and alpha(2)-catalytic subunits of the Na+-K+-ATPase is elevated after a 2- to 4-day chronic cyclic stretch regimen in cultured aortic smooth muscle cells (ASMC). In the present study, we investigated whether cyclic stretch affects mRNA expression of the alpha-isoforms of the Na+-K+-ATPase. Using a stretch apparatus, rat ASMC were cyclically stretched 10 or 20% of their length for 1, 3, or 6 h. alpha-Isoform mRNA levels were measured using Northern analysis. A 3-h 10% stretch had no significant affect on mRNA expression for either isoform, but a 20% stretch increased mRNA of both isoforms approximately twofold. Whereas a 6-h 20% stretch increased alpha(1) mRNA by 3.3-fold, alpha(2) was not affected any further. Actinomycin D blocked the stretch-induced stimulation of mRNA expression of both alpha-subunits. In conclusion, cyclic stretch stimulates the mRNA expression of both alpha(1)- and alpha(2)-subunits of Na+-K+-ATPase. The sensitivity of the two genes to the degree and duration of stretch is different. The stretch-induced increase of mRNA may be a result of increased transcription.

Effect of mechanical strain on expression of Na+,K+-ATPase alpha subunits in rat aortic smooth muscle cells.

This article reviews related studies from the authors' laboratory, which focus on the regulation of vascular Na+,K+-ATPase in hypertension. Earlier studies, including the authors', suggested that Na-pump activity in cardiovascular tissues is subject to regulation during hypertension; most of these studies report a stimulation of the vascular enzyme during established stages of hypertension. To test hypothesis that in vascular smooth muscle, strain resulting from elevated pressure may be a signal initiating a cascade of events leading to increased expression of Na+,K+-ATPase, the authors used cell culture and the Flexercell Strain Unit to apply cyclical stretch to rat aortic smooth muscle cells (ASMC) for several days. These studies demonstrated that mechanical strain induces the upregulation of both the alpha-1 and alpha-2 subunits of Na+,K+-ATPase. Mechanisms underlying these changes appear to involve a transient increase in intracellular sodium entering the cell through stretch-activated channels. Calcium entering the cell via L-type channels did not affect stretch-induced upregulation of the alpha isoforms. In addition, protein kinase C inhibition resulted in inhibition of the Na-pump during stretch, but not under nonstretch conditions. The authors conclude that the stretch component of vascular pressure upregulates the Na+,K+-ATPase catalytic subunits. Intracellular sodium may be a signal for this regulation. In addition, phosphorylation by PKC may be important in stretch-induced short-term regulation of the vascular Na-pump.

Effect of Na+ on Na+,K+-ATPase alpha-subunit expression and Na+-pump activity in aortic smooth muscle cells.

In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na+ and upregulates the alpha-1 and alpha-2 isoform expression of Na+,K+-ATPase, and that the increase of intracellular Na+ and upregulation of the alpha-2 isoform expression are blocked by Gd3+, which blocks entry of ions (including Na+) through stretch-activated channels. The present study was designed to investigate the role of intracellular Na+ in Na+,K+-ATPase regulation by increasing intracellular Na+ with chronic ouabain treatment. In parallel experiments, we measured Na+,K+-ATPase alpha isoform expression, Na+-pump activity and intracellular Na+ in cultured aortic smooth muscle cells after treatment with two concentrations of ouabain for various time periods. Treatment with 100 nM ouabain resulted in a significant elevation in intracellular Na+ after 1 (21%) and 2 h (12%), but the value returned to baseline after 12 h. Both alpha-1 and alpha-2 subunits of Na+,K+-ATPase were significantly upregulated after 1 through 4 days. Na+-pump activity was also stimulated, and the time course of this effect closely followed protein expression. At 200 microM of ouabain, the effects on intracellular Na+, isoform expression and Na+-pump activity at earlier time points (1 h through 1 day) were similar to those with 100 nM treatment, but prolonged treatment (2 and 4 days) resulted in an accumulation of intracellular Na+ and inhibition of the isoform expression and Na+-pump activity, possibly due to general dysfunction of the cells as a result of chronic exposure to high concentrations of ouabain. We conclude that elevated intracellular Na+ can serve as a signal to mediate the alpha isoform upregulation and the regulatory process requires less than one day.

Role of Na+ and Ca2+ in stretch-induced Na(+)-K(+)-ATPase alpha-subunit regulation in aortic smooth muscle cells.

This study was designed to test the role of Na+ and Ca2+ entry in the stretch-induced Na(+)-K(+)-ATPase alpha 1- and alpha 2-isoform upregulation observed in our previous studies. We measured intracellular Na+ in cyclically stretched rat aortic smooth muscle cells, with or without gadolinium treatment, for various durations and performed Western blotting to analyze the effects of stretch and the calcium channel blocker isradipine on the expression of alpha-isoforms. Intracellular Na+ was elevated significantly after 1- and 2-h stretch, but returned to baseline after 1-, 2-, and 4-day stretch. This increase in intracellular Na+ was blocked by gadolinium. Both alpha 1- and alpha 2-isoforms were upregulated after either 2 or 4 days of cyclical stretch. Isradipine had no apparent effect on stretch-induced upregulation on either alpha-isoform, thus suggesting that Ca2+ entry through L-type channels is not involved in the stretch-induced upregulation. We therefore conclude that a transient intracellular Na+ elevation during stretch may serve as a signal to mediate the alpha 1- and alpha 2-isoform upregulation.

Alterations in alpha subunit expression of cardiac Na+,K+-ATPase in spontaneously hypertensive rats: effect of antihypertensive therapy.

The alpha-2 subunit abundance of Na+,K(+)-ATPase in the rat heart has been reported to be reduced in several induced hypertensive models. To determine whether this reduction also occurs in a genetic model of hypertension, we studied expression of the alpha subunits in left ventricles of spontaneously hypertensive rats (SHR), and normotensive Wistar-Kyoto (WKY) and Sprague-Dawley rats using Western blotting and quantitative dot-blotting analysis with monoclonal antibodies. While the alpha-1 subunit was not affected in any of the strains, a significant reduction of the alpha-2 subunit expression was noted in 19-week-old SHRs, but not in age-matched WKY and Sprague-Dawley rats, supporting the hypothesis that elevated arterial pressure may differentially downregulate the alpha-2 subunit in the rat heart. To further test this hypothesis we designed experiments in which hypertensive rats were treated with the antihypertensive agents hydralazine and nifedipine. Both agents effectively normalized the blood pressure in the SHRs with no significant effect on the blood pressure in the WKY and Sprague-Dawley rats. The alpha-2 subunit in SHRs treated with hydralazine and nifedipine showed a 63.3% (n = 6, P < 0.05, analysis of variance and Fischer's test) and a 27.4% increase, respectively, over the hypertensive SHR controls, although the reversal effect of nifedipine did not quite reach significance. The alpha-1 subunit expression was not affected by any of the drug treatments. No effect of either of the drugs on the alpha-1 or alpha-2 subunit was observed in the WKY or Sprague-Dawley rat groups. These data support our hypothesis that the alpha-2 subunit may be a pressure-sensitive isoform of the cardiac Na+,K(+)-ATPase and that high blood pressure is, directly or indirectly, responsible for the reduction of the alpha-2 subunit protein expression.

Effects of ouabain on vascular reactivity.

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the alpha-subunit of Na+,K(+)-ATPase inhibiting the Na(+)-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the alpha-subunit of Na+, K+ -ATPase inhibiting the Na+ -pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells.

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.

Regulation of Na+,K+-ATPase alpha-subunit expression by mechanical strain in aortic smooth muscle cells.

We have previously demonstrated that vascular sodium pump activity is stimulated in several rat models of hypertension. In addition, others have reported an upregulation of mRNA for the Na+,K+-ATPase alpha1-subunit in hypertension. To test the effect of sustained, cyclic, stretch-relaxation stimuli on the expression of alpha1- and alpha2-subunits of Na+,K+-ATPase in vascular smooth muscle cells, we used the Flexercell strain unit to stretch rat aortic smooth muscle cells for several days on a collagen-coated silicone elastomer substratum. Six-second cycles of stretch-relaxation were applied to obtain 10% average surface elongation (22% maximum) for 4 days. Control cells were not stretched but were grown on a similar surface. The effect of Gd3+, a blocker of stretch-activated channels, was also investigated. At the end of 4 days, protein expression of alpha1- and alpha2-subunits was determined by Western blot analysis. Intensity of the bands for alpha1- and alpha2-subunits was quantified with the use of a computerized image analyzer. In the stretched cells, both the alpha1- and the alpha2-subunit protein-band intensities were significantly increased compared with those of the non-stretched cells. Treatment with 50 micromol/L Gd3+ during the application of stretch prevented the upregulation of alpha2-expression but not that of alpha1-expression. Sodium pump activity, the functional counterpart of Na+,K+-ATPase, was inhibited as a result of stretch; Gd3+ had no effect on this variable. Our results suggest that in vascular smooth muscle, stretch may be a signal for the upregulation of both the alpha1- and alpha2-isoforms. However, a differential response of the two isoforms to the blocker of stretch-activated channels implies involvement of different mechanisms. This alteration in protein expression is not reflected in the function of the enzyme.

Regulation of Na(+)-pump activity by dopamine in rat tail arteries.

We investigated the effect of dopamine on the vascular Na+-pump activity in isolated rat tail artery sections. Effect of dopamine on vascular tone was also assessed using a perfused tail artery preparation. Dopamine inhibited the Na+-pump activity in isolated rat tail arteries in a dose-dependent manner. Both SKF-38393 HCl, a selective dopamine D1 receptor agonist, and quinpirole HCl, a selective dopamine D2 receptor agonist inhibited the Na+-pump activity. The inhibition of the Na+-pump activity. The inhibition of the Na+-pump by dopamine was accompanied with a transient increase in the vascular tone. SKF-38393, but not quinpirole produced a sustained increase in the vascular tone. Tissues preincubated simultaneously with SCH-23390 HCl, a selective dopamine D1 receptor antagonist, and sulpiride, a selective dopamine D2 receptor antagonist, prevented the dopamine inhibition of the Na+-pump activity. Pertussis toxin blocked the Na+-pump inhibition produced by the dopamine D1 receptor agonist but not by the dopamine D2 agonist. Similarly, the dopamine D1 receptor but not dopamine D2 agonist increased the rate of phosphoinositide hydrolysis in rat tail artery sections. Our results indicate that dopamine inhibition of the Na+-pump is mediated by a pertussis toxin-sensitive mechanism and may be coupled to the activation of the phospholipase C system in rat tail arteries. The modulation of the Na+-pump by dopamine may contribute to the vascular tone.

Regulation of Na+,K(+)-ATPase alpha-subunit isoforms in rat tissues during hypertension.

We investigated the regulation of the protein expression of the alpha isozymes of Na+,K(+)-ATPase in reference to the enzyme activity in the heart, brain and skeletal muscle of rats during deoxycorticosterone acetate (DOCA)-salt hypertension. Treatment of rats with DOCA and salt for 28 days produced a significant increase in systolic blood pressure compared to the control groups which remained normotensive. Rats treated with DOCA expressed greater amounts of the immunoreactive alpha-1 isoform than untreated controls in whole heart membranes. However, the DOCA-induced increase in the alpha-1 isoform did not occur during DOCA-salt hypertension. There was a parallel change in the enzyme activity of the Na+,K(+)-ATPase and the protein expression of the alpha-1 isoform as a result of these treatments. We have also demonstrated that the hearts of DOCA-salt hypertensive rats expressed less of the alpha-2 isoform compared to the controls. We could not detect any alteration in the alpha-1 and alpha-2 isoforms of the skeletal muscle and alpha-1, alpha-2 and alpha-3 isoforms of the whole brain Na+,K(+)-ATPase during salt or DOCA treatments alone or DOCA-salt hypertension. Furthermore, the Na+,K(+)-ATPase activity was unaltered in these tissues during these treatments. In conclusion, cardiac Na+,K(+)-ATPase alpha-subunit protein expression appears to be regulated during DOCA-salt hypertension. In the skeletal muscle and brain, tissues not subjected directly to increased pressure, this regulation of the Na+,K(+)-ATPase was not apparent.

Ouabain amplifies contractile responses to phenylephrine in rat tail arteries in hypertension.

Ouabain, a cardiac glycoside, binds to the alpha-subunits of Na+, K(+)-ATPase and inhibits Na+ pump activity. It has been proposed that endogenous ouabain, by inhibiting vascular Na+, K(+)-ATPase, can increase vascular resistance and thus may contribute to hypertension. One of the consequences of inhibition of the membrane Na+ pump is enhanced responsiveness of vascular smooth muscle to vasopressor substances. The purpose of the present study was to determine whether ouabain can enhance the responsiveness of the vasculature in hypertension. In the present study 100 microM ouabain enhanced the contractile response elicited by phenylephrine in isolated, perfused tail arteries from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The enhanced contractile response was more pronounced in the arteries of the SHR. We demonstrated that this concentration of ouabain inhibits the Na+ pump activity, measured as ouabain-sensitive 86Rb uptake, by about 65%, in isolated tail arteries. We conclude that ouabain can sensitize the vascular smooth muscle to the effects of vasopressor substances and this effect is more pronounced in genetically hypertensive rats. Endogenous ouabain may contribute to the pathophysiology of hypertension by enhancing vascular tone.

A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells.

The putative mannose receptor (MR), previously implicated in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle (ASM) cells, was studied to determine its properties. Specific binding of the mitogenic neoglycoprotein, mannosylated bovine serum albumin (Man-BSA) to ASM cells was saturable, with an apparent Kd = 5.0 x 10(-8) M. Cell-bound ManBSA-colloidal gold conjugate was localized by electron microscopy to clathrin-coated pits on the cell surface, and was found to undergo internalization to endosomes; this was inhibitable by weak bases and swainsonine, that also inhibited ligand-induced mitogenesis. The ASM-MR, isolated by mannose-affinity chromatography, had the same apparent molecular mass as the macrophage (Mø) MR (M(r) = 175 kD), and was immunoprecipitated by an anti-MøMR immune serum. This antiserum blocked 125I-labeled-ManBSA binding to intact ASM cells, stimulated mitogenesis, and immunolocalized the ASM-MR in cytoplasmic vesicles compatible with endosomes. A monoclonal antibody directed against the MøMR also reacted with the ASM-MR; like the polyclonal antibodies, it stimulated mitogenesis as effectively as beta-hexosaminidases. These data indicate that the ASM-MR shares a number of functional and structural properties with the MøMR and suggest that similar receptors may have different main functions in different cells.

Rat vascular tissues express all three alpha-isoforms of Na(+)-K(+)-ATPase.

The catalytic alpha- and smaller glycosylated beta-subunits of the membrane transport enzyme Na(+)-K(+)-adenosinetriphosphatase (ATPase) occur in different molecular forms, alpha 1, alpha 2, alpha 3, beta 1, and beta 2. The catalytic alpha 1-, alpha 2-, and alpha 3-subunits of the enzyme have varying affinities for digitalis and exist in different tissues with unique distribution patterns. In this report we document for the first time the existence of alpha 1-, alpha 2-, alpha 3-subunit proteins (all approximately 97.5 kDa) in cultured rat aortic smooth muscle cells and rat tail arteries. In addition to the three molecular forms of the alpha-protein we detected a minor band at approximately 68-kDa position in aortic smooth muscle cells, which may correspond to the truncated alpha 1-protein reported earlier.

Effect of hypothalamic lesions on interaction of centrally administered ANF and the circulating sodium-pump inhibitor.

We investigated the possible central interaction of atrial natriuretic factor (ANF) and an endogenous Na+, K(+)-ATPase (Na-pump) inhibitor in normal rats. Release of an endogenous Na-pump inhibitor associated with deoxycorticosterone acetate-salt hypertension may be regulated in the anteroventral third ventricle (AV3V) area of the CNS. We reported earlier that bolus injection of synthetic 26-amino acid ANF (Arg101-Tyr126, 6 micrograms/250 g rat) into the lateral brain ventricle (ICV) promotes the appearance in the plasma of a Na-pump inhibitor in rats. To determine whether the AV3V area of the brain is involved in the ICV effect of ANF, we introduced electrolytic lesions in this area. This treatment abolished the appearance of the Na-pump inhibitor after intraventricular injection of ANF. To further localize the area and the pathways involved in the interaction of ANF and the Na-pump inhibitor, we produced bilateral medial coronal knife cuts designed to transect the medially coursing pathway through the periventricular tissue of the AV3V region between the level of the medial preoptic area and the anterior hypothalamic nuclei. These knife cuts also abolished the appearance of the Na-pump inhibitor after ICV injection of ANF. Our data to date indicate that centrally administered ANF promotes the appearance of a Na-pump inhibitor in the plasma. A central site of interaction between ANF and the Na-pump inhibitor appears to be the AV3V area and a medial pathway coursing caudally from the AV3V region.

Vascular sodium pump activity kinetics in early and advanced stages of deoxycorticosterone-salt hypertension in rats.

We determined the kinetic properties--the maximal velocity, Vmax, and the half-maximal activating concentration of K+, km values--of the vascular sodium pump in rats 6, 28, and 50 days after deoxycorticosterone and sodium chloride (DOC-salt) or vehicle treatment. Tail arteries from six or eight rats from each treatment group were pooled, and Na-pump activity was measured in a Krebs medium containing varying K+ (plus 86Rb+) concentrations (0.25-10 mM). Na-pump activity was plotted as a function of [K + 86Rb]. Data were fit to a two-site model to calculate Vmax and km values. Systolic blood pressures were normal after 6 days but high after 28 and 50 days of DOC-salt treatment. No difference in kinetic parameters existed between the treatment and control groups 6 and 50 days after DOC-salt treatment. After 28 days, Vmax was significantly elevated compared with controls; km was not affected. Thus, stimulation of the vascular Na-pump during established hypertension is due to an increase in the maximal velocity of ouabain-sensitive uptake of K+.

Centrally administered ANF promotes appearance of a circulating sodium pump inhibitor.

We have previously reported that release of an endogenous Na pump inhibitor, a putative natriuretic hormone associated with many hypertensive states, may be regulated by the central nervous system (CNS). The atrial natriuretic factor (ANF), which causes diuresis, vasorelaxation, and inhibition of aldosterone and vasopressin release, has been documented in the CNS as well as the atria and peripheral blood. Our experiments investigate the possible interaction of ANF with a circulating specific inhibitor of the Na pump in normal rats. The Na pump inhibitory capacity of plasma from rats previously injected with either [Arg101-Tyr126]ANF or vehicle into the lateral cerebral ventricle was measured in cultured aortic smooth muscle cells (ASMC) using 86Rb uptake methodology. Plasma from rats injected with ANF inhibited the Na pump activity (18 +/- 4%, n = 4 experiments) in ASMC compared with plasma obtained from rats injected with the vehicle. The nonspecific 86Rb uptake was not affected by the ANF treatment. Plasma obtained from rats injected intravenously with ANF did not affect the Na pump activity in the ASMC. Furthermore, ANF did not directly affect Na pump activity in ASMC when added to the culture dishes in vitro. These findings raise the possibility that central ANF regulates the release of circulating Na pump inhibitor.