The differential effects of tamoxifen and ICI 182,780 on the reduction of Na+/K+ ATPase activity and spontaneous oscillations by 17beta-estradiol

A prolonged treatment with 17beta-estradiol reduces the frequency of spontaneous oscillations and the Na+/K+ ATPase activity in rat uteri. Acute inhibition of Na+/K+ ATPase activity by a Na+/K+ ATPase inhibitor, ouabain, decreases the frequency of oxytocin-induced oscillations in uteri. Therefore, the purpose of this study was to examine whether the prolonged inhibition of Na+/K+ ATPase activity by 17beta-estradiol was estrogen receptor (ER)-dependent. The uterine explants from ovariectomized rats were cultured in vitro as our experimental model to compare the effect of two antiestrogenic compounds (ICI 182,780 and tamoxifen) on the Na+/K+ ATPase activity and the frequency of spontaneous oscillations. ATPase assay and a standard muscle bath apparatus were to measure the activity and the contraction. When compared with the control, a 2-day treatment with 17beta-estradiol in vivo or in vitro decreased the activity and the frequency. ICI 182,780 lowered the activity but tamoxifen did not. ICI 182,780 did not decrease the frequency but tamoxifen did. Even the reversal effects of these antiestrogenic compounds on the reduced activity and the frequency by 17beta-estradiol were different. Tamoxifen elicited a greater reversal effect on the reduced activity but ICI 182,780 did not. In contrast, ICI 182,780 elicited a greater reversal effect on the reduced frequency but tamoxifen did not. Prolonged inhibition of Na+/K+ ATPase activity by K+-free solution suppressed the frequency with the elevation of basal tension. Addition of KCl at lower concentrations (0.3-1.2 mM) induced oscillatory contraction after reducing the basal tension. As our data suggest, the prolonged effect of 17beta-estradiol may decrease uterine the activity through ER dependent and independent pathways. The reduction of uterine Na+/K+ ATPase activity by estrogens may increase the basal tension after each oscillatory cycle, which, in part, contributes to the reduced frequency of spontaneous oscillations.     

EGF modulated Na+/K+ ATPase in cultured fibroblasts

The behaviour of Na+/K+ ATPase during cell growth has been studied. Human cultured fibroblasts were used in the presence or absence of EGF. Sample and control cultures were stopped by gathering and washing the cells with tris buffer. Homogenates were tested for Na+/K+ ATPase activity by the method of incubating and for the -SH groups content (Ellman). Na+/K+ ATPase activity that slightly increases in the controls is strongly reduced by the addition of EGF. The behaviour shows evidence for a double mechanism of action: I) involvement of the cAMP system 2) decrease of the -SH group availability.     

Dexamethasone modulates the activity of the eel branchial Na+/K+ATPase in both chloride and pavement cells

In fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K+ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K+ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K+ATPase was dose-dependent in both environmental adaptations. Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K+ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions.     

Effect of amiodarone on membrane fluidity and Na+/K+ ATPase activity in rat-brain synaptic membranes

In rat-brain synaptic membranes at a fixed temperature (37 degrees C), amiodarone dose-dependently inhibits the Na+/K+ ATPase activity (IC50 approximately equal to 2.10(-5)M) and produces a linear increase in the degree of fluorescence depolarization (P) of 1,6-diphenylhexatriene embedded in the lipid matrix. Amiodarone has no effect on Mg++ ATPase and K+PNPase activity up to 3.10(-4)M. Studies carried out at different temperatures indicate that 10(-5)M amiodarone inhibits the Na+/K+ ATPase and decreases the lipid fluidity at all the temperatures studied (9 - 40 degrees C). The compound significantly displaces the temperature of transition observed around 20 degrees C in both Na+/K+ ATPase activity and lipid fluidity to 24 degrees C with no changes in slopes. The results suggest that part of the selective inhibition of Na+/K+ ATPase activity by amiodarone could be due to the effects of the drug on lipid dynamics.     

Epidermal growth factor up-regulates intestinal Na+/H+ exchange activity.

The present studies were designed to examine the regulation of Na+/H+ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na+/H+ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na+/H+ exchange activity without affecting vesicle size. The stimulation of Na+/H+ activity was the result of increasing Vmax of Na+/H+ (6.0 +/- 0.4 compared with 3.3 +/- 0.27 nmol/mg protein/5 sec, P < 0.01). Km values of the Na+/H+ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 +/- 3.0 vs 13.0 +/- 3.0, respectively). Na+/H+ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na+/H+ exchange activity. The results suggest that EGF stimulates Na+/H+ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway.     

The effects of captopril and losartan on erythrocyte membrane Na+/K(+)-ATPase activity in experimental diabetes mellitus

Diabetes mellitus induces a decrease in sodium potassium-adenosine triphosphatase (Na+/K(+)-ATPase) activity in several tissues in the rat and red blood cells (RBC) and nervous tissue in human patients. This decrease in Na+/K(+)-ATPase activity is thought to play a role in the development of long-term complications of the disease. Angiotensin enzyme inhibitors (ACEi) and angiotensin-II receptor antagonists (ARBs) reduce proteinuria and retard the progression of renal failure in patients with IDDM and diabetic rats. We investigated the effects of captopril and losartan, which are used in the treatment of diabetic nephropathy, on Na+/K(+)-ATPase activity. Captopril had an inhibitory effect on red cell plasma membrane Na+/K+ ATPase activity, but losartan did not. Our study draws attention to the inhibitory effect of captopril on Na+/K+ ATPase activity. Micro and macro vascular complications are preceeding mortality and morbidity causes in diabetes mellitus. There is a strong relationship between the decrease in Na+/K+ ATPase activity and hypertension. The non-sulphydryl containing ACEi and ARBs must be the choice of treatment in hypertensive diabetic patients and diabetic nephropathy.     

Atypical PKC-zeta and PKC-iota mediate opposing effects on MCF-7 Na+/K+ATPase activity

We demonstrated previously that in serum-starved MCF-7 breast cancer cell line, Ang II increased Na+/K+ATPase activity and activated the protein kinase C zeta (PKC-zeta) (Muscella et al., 2002 J Endocrinol 173:315-323; 2003 J Cell Physiol 197:61-68.). The aim of the present study was to investigate the modulation of the activity of the Na+/K+ATPase by PKC-zeta in MCF-7 cells. Here, using serum-starved MCF-7 cells, we have demonstrated that the effect of Ang II on the Na+/K+ATPase activity was inhibited by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-PS) and by high doses of GF109203X, inhibitor of PKCs. When MCF-7 cells, grown in 10% fetal bovine serum (FBS), were stimulated with Ang II a dose- and time-dependent inhibition of the Na+/K+ATPase activity was obtained. Under this growth condition we found that mRNAs for AT1, AT2, and for Na+/K+ATPase alpha1 and alpha3 subunits were unchanged; besides both the activity of the Na+/K+ATPase and the level of PKC-zeta also were unaffected by the serum. The atypical PKC-iota level (present in very low abundance in serum-starved MCF-7) was increased and Ang II provoked its translocation from the cytosol to plasma membrane. PKC-zeta was localized to the membrane, and upon Ang II treatment its cellular localization did not change. The Ang II-mediated decrease of the Na+/K+ATPase activity was inhibited by high doses of GF109203X but not by zeta-PS, thus indicating that such effect was not due to PKC-zeta activity. The treatment of cells with PKC-iota antisense oligodeoxynucleotides inhibited the effects of Ang II on the Na+/K+ATPase activity. Additionally, the effect of Ang II on Na+/K+ATPase activity was also blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, and by the actin depolymerizing agents, cytochalasin D. In conclusion, in MCF-7 cells Ang II modulates the Na+/K+ATPase activity by both atypical PKC-zeta/-iota. The effects of Ang II are opposite depending upon the presence of the serum-sensitive PKC-iota, with the inhibitory effect possibly due to the redistribution of sodium pump from plasma membrane to the inactive intracellular pool.     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Sodium/potassium adenosine triphosphatase alpha- and beta-subunit and alpha-subunit mRNA levels during mouse embryo development in vitro

Changes in sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content during preimplantation mouse embryo development were determined. Western blotting, using polyclonal antiserum against guinea pig Na+/K+ ATPase, was used to detect changes in Na+/K+ ATPase alpha- and beta-subunit content during mouse embryo development. Total RNA from mouse embryos was analyzed using Northern and slot blots hybridized with random-primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kb) in sheep and mouse kidneys and mouse embryos. Although Na+/K+ ATPase alpha-subunit mRNA content of mouse embryos increased 45-fold between Day 1 and Day 4 of development, Na+/K+ ATPase alpha-subunit content remained constant, and beta-subunit content increased 9-fold. The Na+/K+ ATPase alpha-subunit and alpha-subunit mRNA content did not increase in a similar manner. The results suggest that, in mouse embryos, blastocoel formation is not triggered by an increase in Na+/K+ ATPase alpha-subunit content. Changes in beta-subunit content may be important in regulating Na+/K+ ATPase activity and blastocoel formation.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

Effects of soybean lipoxygenase on Na+/K+-ATPase activity in vitro

Oxidized metabolites of polyunsaturated fatty acids produced by lipoxygenase are among the endogenous regulators of Na+/K+-ATPase. The direct effect of lipoxygenase on Na+/K+-ATPase activity was assessed in vitro using soybean lipoxygenase. Treatment of 4.2 microg/mL Na+/K+-ATPase (from dog kidneys) with 4.2 microg/mL of soybean lipoxygenase caused 20+/-2% inhibition of ATPase activity. A 10-fold increase in lipoxygenase concentration (41.6 microg/mL) led to 30+/-0.3% inhibition. In the presence of 12 microg/mL phenidone (a lipoxygenase inhibitor) and 15.4 microg/mL glutathione (a tripeptide containing a cysteine residue) inhibition of Na+/K+-ATPase activity was blocked and an increase in ATPase activity was observed. The presence of lipoxygenase enhanced the inhibition of Na+/K+-ATPase activity caused by 20 ng/mL ouabain (31+/-2 vs. 19+/-2) but had little or no effect with higher concentrations of ouabain. These findings suggest that lipoxygenase may regulate Na+/K+-ATPase by acting directly on the enzyme.     

Sodium/potassium adenosine triphosphatase alpha-subunit and alpha-subunit mRNA levels in early rabbit embryos

Sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content of rabbit embryos during preimplantation development were evaluated. Changes in Na+/K+ ATPase alpha-subunit content were detected with Western blotting using polyclonal antiserum against guinea pig Na+/K+ ATPase. Total RNA samples from rabbit embryos were analyzed by using Northern blots hybridized with random primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kilobases) in sheep kidneys and rabbit embryos. Between Day 4 and Day 6 of development, Na+/K+ ATPase alpha-subunit mRNA content increased 35-fold whereas Na+/K+ ATPase alpha-subunit content increased 22-fold. The similar increase in Na+/K+ ATPase alpha-subunit mRNA and alpha-subunit content in rabbit embryos suggests that Na+/K+ ATPase is partly regulated at the mRNA level during blastocyst expansion.     

PGE2 reduces net water and chloride absorption from the rat colon by targeting the Na+/H+ exchanger and the Na+ K+ 2Cl- cotransporter

An effect of PGE2 on water and chloride absorption was already established in a previous work. This study is an attempt to find the mechanism of action of the prostaglandin by investigating the involvement of three major transporters namely the Na+ -K+ ATPase, the Na+/H+ exchanger and the Na+ K+ 2Cl- cotransporter. Rats were injected with PGE2 and 15 min later, the colon was perfused in situ with Krebs Ringer buffer, and net water and chloride absorption were determined. When the involvement of the cotransporter and/or the exchanger was investigated, animals were injected with, respectively, furosemide and amiloride 10 min before PGE2. Superficial and crypt colonocytes were then isolated and the protein expression of the Na+ -K+ ATPase and the Na+ K+ 2Cl- was determined by western blot analysis. The effect of PGE2 on the pump activity in presence or absence of the transporters' inhibitors was also studied. PGE2 decreased net water and chloride absorption from the colon, increased the Na+ -K+ ATPase activity in superficial cells and reduced it in crypt cells. The prostaglandin was found to stimulate secretion in superficial cells by targeting the Na+ K+ 2Cl- symporter, and reduce absorption in crypt cells by targeting the Na+/H+ antiporter. Changes in the activity of the pump are secondary to changes in the activity of the other transporters.     

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.     

Cholinergic stimulation of the Na+/K+ adenosine triphosphatase as revealed by microphysiometry.

The activation of a wide range of cellular receptors has been detected previously using a novel instrument, the microphysiometer. In this study microphysiometry was used to monitor the basal and cholinergic-stimulated activity of the Na+/K+ adenosine triphosphatase (ATPase) (the Na+/K+ pump) in the human rhabdomyosarcoma cell line TE671. Manipulations of Na+/K+ ATPase activity with ouabain or removal of extracellular K+ revealed that this ion pump was responsible for 8.8 +/- 0.7% of the total cellular energy utilization by those cells as monitored by the production of acid metabolites. Activation of the pump after a period of inhibition transiently increased the acidification rate above baseline, corresponding to increases in intracellular [Na+] ([Na+]i) occurring while the pump was off. The amplitude of this transient was a function of the total [Na+]i excursion in the absence of pump activity, which in turn depended on the duration of pump inhibition and the Na+ influx rate. Manipulations of the mode of energy metabolism in these cells by changes of the carbon substrate and use of metabolic inhibitors revealed that, unlike some other cells studied, the Na+/K+ ATPase in TE671 cells does not depend on any one mode of metabolism for its adenosine triphosphate source. Stimulation of cholinergic receptors in these cells with carbachol activated the Na+/K+ ATPase via an increase in [Na+]i rather than a direct activation of the ATPase.     

Angiotensin II stimulates the Na+/H+ exchanger in human umbilical vein endothelial cells via AT1 receptor

Angiotensin II (Ang II) has an important role in cardiovascular regulation and in the control of electrolyte balance, and its role in the regulation of Na+ transcellular movements through its actions on the activity of Na+/K+ ATPase is well documented. We showed previously that human umbilical vein endothelial cells (HUVEC) express the Ang II type 1 (AT1) receptor, which mediates Ang II modulation of Na+/K+ ATPase activity (1). We here investigate the effects of Ang II on the activity of the Na+/H+ exchanger in HUVEC. When compared with controls, incubation of HUVEC for 20 min with different concentrations of Ang II provoked significant increases in Na+/H+ activity. The stimulation was dose dependent between 1 and 10 nM Ang II and varied with time of incubation up to 20 min. The maximal response, obtained with 10 nM Ang II after 20 min treatment, resulted in a 65% increment in Na+/H+ activity. Preincubation of HUVEC with 10 microM DuP753 blocked Na+/H+ activation by Ang II. These results suggest that the effects of Ang II on both the Na+/K+ ATPase and the Na+/H+ exchanger may increase the transendothelial flux of Na+ and are mediated by the AT1 receptor.     

Ouabain-insensitive Na+-ATPase of proximal tubules is an effector for urodilatin and atrial natriuretic peptide

In the present paper we studied the effect of urodilatin and atrial natriuretic peptide (ANP) on the proximal tubule Na+-ATPase and (Na+K+)ATPase activities. Urodilatin and ANP inhibit the Na+-ATPase activity but not the (Na+K+)ATPase activity. Maximal effect was observed at a concentration of 10(-11) M for both peptides. In this condition, the enzyme activity decreases from 10.8 +/- 1.6 (control) to 5.7 +/- 0.9 or 6.1 +/- 0.7 nmol Pi mg(-1) min(-1) in the presence of urodilatin or ANP, respectively. This effect was completely reversed by 10(-6) M LY83583, a guanylyl cyclase inhibitor, and mimicked by 10 nM cGMP. Furthermore, both ANP and urodilatin increase cGMP production by 33% and 49%, respectively. This is the first demonstration that it was shown that urodilatin and ANP directly modulate primary active sodium transport in the proximal tubule. The data obtained indicate that this effect is mediated by the activation of the NPR-A/guanylate cyclase/cGMP pathway.     

Mechanisms by which Li+ stimulates the (Na+ and K+)-dependent ATPase.

The addition of LiCl stimulated the (Na+ + K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg-ATP concentrations. Apparent affinities for Li+ were estimated at the alpha-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the beta-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent Km for Mg-ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the alpha-sites on the enzyme through which K+ decreases the apparent affinity for Mg-ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane Na+/K pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.