Presence of a potential-sensitive Na+ transport across renal brush-border membrane vesicles from rats of the Milan hypertensive strain

Sodium transport was measured in brush-border membrane vesicles prepared from kidney cortex of the Milan hypertensive strain (MHS) rats and the corresponding normotensive controls. In the presence of an outwardly directed proton gradient, 22Na was transiently accumulated in the vesicles. When a transmembrane electrical potential was imposed across membrane vesicles, both the accumulation ratio and the initial uptake were increased, indicating the presence of an electrogenic pathway for sodium in these membranes. The potential-dependent sodium uptake was significantly higher in MHS rats. Kinetic analysis give simple Michaelis Menten curves in the presence and in the absence of a membrane potential. In both conditions Jmax was significantly increased in MHS rats, whereas Km was the same for the two rat strains. Sodium uptake was inhibited by amiloride at concentrations that inhibit Na+-H+ exchange. The presence of the higher, potential-sensitive, sodium uptake in MHS is in agreement with studies on renal physiology which support the hypothesis that an increase in tubular sodium reabsorption may be the primary cause for the development of hypertension in this rat strain.     

Relationships among alterations in renal membrane sodium transport,renin and aminopeptidase M activities in genetic hypertension

Rats of the Milan Hypertensive Strain (MHS) may be considered a useful model for understanding the genetic molecular mechanism underlying a primary form of hypertension in at least a subgroup of patients. Many differences between MHS and its normotensive control strain (MNS) were found at the organ, cellular and biochemical level. In the present investigation renal cell membrane proteins (BBMV) were analysed by two-dimensional electrophoresis and a difference between MHS and MNS was shown in a polypeptide of 32 kDa, subsequently identified as the C-terminal fragment of aminopeptidase M (APM). The activity of the enzyme was higher in MHS. Genetic relationships between this enzyme and the other biochemical cellular abnormalities of MHS, namely sodium transport in BBMV and renin activity in kidney cortex were investigated in MHS, MNS and in two inbred recombinant strains. This analysis showed that faster sodium transport, low kidney levels of renin and hypertension, but not differences in two-dimensional electrophoretic pattern and in aminopeptidase M activity, cosegregated in recombinant strains. These results are consistent with the hypothesis that the faster sodium transport can be considered a primary cellular abnormality responsible for hypertension in MHS and that the aminopeptidase difference is not involved in the cellular abnormalities.     

Effect of amiloride analogues on sodium transport in renal brush border membrane vesicles from Milan hypertensive rats.

The effect of ethylisopropyl-amiloride (EIPA) and phenamil on sodium uptake in renal brush border membrane vesicles from prehypertensive rats of the Milan strain (MHS) and their normotensive controls (MNS) was investigated. In the presence of both a membrane potential and a pH gradient a differential effect of EIPA and phenamil was evidenced between the two rat strains. In the absence of a pH gradient, but in the presence of a membrane potential, EIPA was about two-fold more potent than phenamil in inhibiting sodium transport in both rat strains, excluding the presence of epithelial sodium channels in our BBMV preparations. Taken together these results support the hypothesis that a structurally different Na+/H+ exchanger located on the brush border membrane may be involved in the increased tubular sodium reabsorption observed in vivo in hypertensive rats.     

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats

 Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na⁺,K⁺-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA⁺,K⁺-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting. Accepted: 23 June 1997     

A primary sodium pump gene of the moderate halophile Halobacillus dabanensis exhibits secondary antiporter properties

The primary sodium pump has been proved to be involved in Na(+) extrusion of bacteria. In our present study, a novel gene encoding a putative primary sodium pump was cloned from chromosomal DNA of moderate halophile Halobacillus dabanensis D-8 by functional complementation, which expression resulted in the growth of antiporter-deficient Escherichia coli strain KNabc in the presence of 0.2 M NaCl. The gene was sequenced and designated nap. The deduced amino acid sequence of Nap has 56% identity to NADH dehydrogenase of Bacillus cereus and 55% to NADH oxidase of Bacillus halodurans C-125. E. coli KNabc carrying nap exhibited resistance to uncoupler CCCP (carbonyl-cyanide m-chlorophenylhydrazone). Everted membrane vesicles prepared from E. coli KNabc carrying nap exhibited secondary Na(+)/H(+) antiporter activity, and nap also supported the growth of respiratory-deficient E. coli ANN0222 lacking NADH dehydrogenase. Based on these results, we proposed that Nap possessed both characteristics of secondary Na(+)/H(+) antiporter and primary sodium pump.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Dyslipoproteinemia in an inbred rat strain with spontaneous chronic progressive nephrotic syndrome.

Rats of the Milan Normotensive Strain (MNS) develop a dyslipoproteinemia that is associated with a spontaneous, age-dependent and slowly progressive nephropathy characterized by proteinuria and hypoalbuminemia (nephrotic syndrome). We assumed that the MNS strain might be a suitable model for studying the features of nephrotic dyslipoproteinemia and its relationship with proteinuria, hypoalbuminemia, and hepatic apolipoprotein production. Plasma lipoproteins were investigated in MNS rats at various ages (4-48 weeks) and in another rat strain (Milan Hypertensive Strain, MHS), genetically related to MNS but free of nephropathy, that was used as control. In MNS rats, abnormal proteinuria was detectable at 20 weeks and increased 2-fold up to 34 weeks with no reduction of plasma albumin (compensated stage). During this stage we found increased levels of plasma cholesterol (+ 34%), high density lipoprotein-1 (HDL1) (+ 73%), and HDL2 (+ 31%) that were positively correlated with proteinuria but not with plasma albumin. The later stage (34-48 weeks) (nephrotic stage) was characterized by a further increase of proteinuria, moderate hypoalbuminemia (- 25%), a 2-fold increase of plasma cholesterol, triacylglycerols, low density lipoprotein (LDL), and HDL1, and a 1.2-fold increase of HDL2. In this stage the levels of LDL, HDL1, and HDL2 were positively correlated with proteinuria, and negatively correlated with plasma albumin. The most striking change in apolipoproteins was a progressive increase of the relative content of apoA-I in HDL (in 48-week-old MNS rats the A-I/E ratio was 3-fold that found in MHS rats) that was associated with a similar increase of plasma apoA-I. None of these lipoprotein changes were observed in age-matched MHS rats. At the end of the compensated stage, the hepatic levels of A-I, B, A-II, and albumin mRNA were 5.3-, 3.5-, 1.3-, and 2.0-fold, respectively, those found in age-matched MHS rats. During the nephrotic stage, albumin mRNA continued to increase, whereas A-I, B, and A-II mRNAs decreased toward the levels found in age-matched MHS rats. Thus, nephrotic dyslipoproteinemia in MNS rats starts to develop in the compensated stage before the onset of hypoalbuminemia, is characterized by an early elevation of HDL1 + HDL2, and is associated with an increased content of hepatic mRNAs of some apolipoproteins, especially apoA-I. The slow progression of nephrotic syndrome with the long-standing proteinuria and no reduction in plasma albumin renders the MNS strain the most suitable animal model for the study of the effect of proteinuria on plasma lipoprotein metabolism.     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

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.     

Lipid composition and fluidity in the jejunal brush-border membrane of spontaneously hypertensive rats. Effects on activities of membrane-bound proteins

The lipid composition and fluidity of jejunal brush-border membrane vesicles (BBMV) have been studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The activities of both Na⁺-dependent D-glucose cotransport and Na⁺-H⁺ antiport have also been determined. A significant increase in the level of free cholesterol was observed in jejunal BBMV from SHR compared to WKY rats. Since phospholipid values did not change in either group of animals, a significant enhancement in the free cholesterol/phospholipid ratio was observed in SHR. A decrease in the levels of phosphatidylethanolamine together with an increase in the values of phosphatidylserine was observed in hypertensive rats. Although the content of phosphatidylcholine (PC) and sphingomyelin (SM) was not singificantly altered in SHR, the ratio PC/SM significantly increased in these animals when compared to WKY rats. The major fatty acids present in bursh-border membranes prepared from SHR and WKY rats were palmitic (160), stearic (180), oleic (181, n-9) and linoleic (182, n-6), and the fatty acid composition was not modified by the hypertension. A decreased fluorescence polarization, i.e., increased membrane fluidity, was observed in SHR, which was not correlated to the increased ratio of cholesterol/phospholipid found in the brush-border membrane isolated from these animals. These structural changes found in SHR were associated to an enhancement in both Na⁺-dependent D-glucose transport and Na⁺-H⁺ antiport activity in the jejunal BBMV of SHR.Abbreviations BBMV brush-border membrane vesicles - DPH 1,6-diphenyl-1,3,5-hexatriene - FC free cholesterol - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin - SHR spontaneously hypertensive rat - p steady-state fluoroscence polarization - r_s steady-state fluorescence anisotropy - WKY Wistar Kyoto     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.     

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ～70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats.

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.     

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.     

Sodium pump molecular activity and membrane lipid composition in two disparate ectotherms,and comparison with endotherms

Previous research has shown that the lower sodium pump molecular activity observed in tissues of ectotherms compared to endotherms, is largely related to the lower levels of polyunsaturates and higher levels of monounsaturates found in the cell membranes of ectotherms. Marine-based ectotherms, however, have very polyunsaturated membranes, and in the current study, we measured molecular activity and membrane lipid composition in tissues of two disparate ectothermic species, the octopus (Octopus vulgaris) and the bearded dragon lizard (Pogona vitticeps), to determine whether the high level of membrane polyunsaturation generally observed in marine-based ectotherms is associated with an increased sodium pump molecular activity relative to other ectotherms. Phospholipids from all tissues of the octopus were highly polyunsaturated and contained high concentrations of the omega-3 polyunsaturate, docosahexaenoic acid (22:6 (n–3)). In contrast, phospholipids from bearded dragon tissues contained higher proportions of monounsaturates and lower proportions of polyunsaturates. Sodium pump molecular activity was only moderately elevated in tissues of the octopus compared to the bearded dragon, despite the much greater level of polyunsaturation in octopus membranes. When the current data were combined with data for the ectothermic cane toad, a significant (P=0.003) correlation was observed between sodium pump molecular activity and the content of 22:6 (n–3) in the surrounding membrane. These results are discussed in relation to recent work which shows a similar relationship in endotherms.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

Solubilization and reconsitution of renal brush border Na+−H+ exchanger

Summary In order to permit future characterization and possible isolation of the Na⁺–H⁺ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pH_in 6.0, pH_out 8.0), the uptake of 1mm ²²Na⁺ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK _a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na⁺–H⁺ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.