Sergio Salardi

HYPERTENSION-ASSOCIATED POINT MUTATIONS IN THE ADDUCIN ALPHA AND BETA SUBUNITS AFFECT ACTIN CYTOSKELETON AND ION TRANSPORT

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

Evidence for an interaction between adducin and Na+-K+-ATPase: relation to genetic hypertension

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na+-K+-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na+-K+-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na+-K+-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na+-K+-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E2 (K) → E1 (Na) or E2(K) ⋅ ATP → E1Na ⋅ ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human α- and β-adducins stimulate Na+-K+-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na+-K+-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na+-K+-ATPase, which does not contain adducin, added adducin stimulates the Na+-K+-ATPase activity of microsomes only about one-half as much as that of purified Na+-K+-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na+-K+-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

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.

Na+/K+/Cl--cotransporter mediated Rb+ fluxes in membrane vesicles from kidneys of normotensive and hypertensive rats

This paper describes experiments to examine Rb+ fluxes via the Na+/K+/Cl- cotransporter in membrane vesicles from renal outer medulla of three strains of rat: (A) Wistar (B) Milan hypertensive (MHS) and normotensive (MNS), and (C) Sabra salt-sensitive hypertensive (SBH) and salt-resistant (SBN). Initially, Na(+)-dependent furosemide- or bumetanide-inhibited 86Rb+ fluxes were characterised using Wistar rat microsomes. The latter were partially purified on a metrizamide cushion, and assay conditions were optimized for use with microsomes from the other rats. The major result is that in microsomes from adult Milan hypertensive (MHS) rats the rate of the Na+/K+/Cl(-)-cotransporter mediated 86Rb flux at sub-saturating concentrations of Rb, appears to be significantly greater than in the normotensive (MNS) controls. The effect reflects an increased apparent Rb affinity of the cotransporter in MHS microsomes. There is no difference in maximal rate or in the apparent Na+ activation affinity of the 86Rb+ flux. In addition bumetanide appears to be a somewhat more effective inhibitor in MHS compared to MNS microsomes. The 86Rb+ flux result is compatible with a previous finding that in red cells, Na+/K+ -cotransporter mediated fluxes are increased in MHS compared to MNS. It supports the notion that the Na+/K+/Cl(-)-cotransporter in in both red cells and kidney is a genetic marker for hypertension. It is of interest that apparently more than one Na+ transport system is affected in MHS hypertensive kidneys (a) the Na+/K+/Cl- cotransporter in the thick ascending limb of Henle and (b) the Na+/H+ exchanger and/o a conductive Na(+)-pathway in brush-border membranes from proximal tubule. It is conceivable that in the hypertensive animals a common regulatory pathway (e.g., phosphorylation) or protein (e.g., cytoskeleton) is affected along the length of the nephron. In Sabra SBH and SBN rat microsomes, no difference was found for the 86Rb+ flux via the Na+/K+/Cl- cotransporter (or via a K+ channel).

Molecular cloning of an adducin-like protein : evidence of a polymorphism in the normotensive and hypertensive rats of the Milan strain

Differences genetically associated with the development of hypertension in a strain of genetically hypertensive rat (MHS) were described in ion transport across erythrocyte membranes compared to normotensive control (MNS). Antibodies against the MNS ghost proteins were raised in the MHS, producing an immunoreaction against a 105 KDa protein later identified as adducin. A clone coding for a portion of mouse adducin was isolated with these antibodies. Using this clone, overlapping cDNA clones coding for a 63 KDa adducin-like protein were isolated. A family of related mRNAs of about 3500, 3800, 4200 nt, was found to be present in spleen, kidney and heart tissues. Similar mRNAs and an additional tissue specific 8000 nt mRNA are present in brain. All mRNAs seem to be generated by alternative splicing from the transcript of a single gene. An interesting polymorphism, a Gln to Arg substitution, was detected in the carboxiterminal area of rat adducin 63.

SODIUM TRANSPORT KINETICS IN ERYTHROCYTES AND INSIDE-OUT VESICLES FROM MILAN RATS

This paper describes the kinetics of the Na(+)-K+ pump and the Na(+)-K(+)-Cl- cotransport in sodium-loaded erythrocytes and of the Na(+)-K(+)-Cl- cotransport in erythrocyte inside-out vesicles (IOV) from Milan hypertensive (MHS) and normotensive (MNS) rats in order to evaluate the possible role of intracellular factor(s). In intact erythrocytes, no difference was detectable in the Na(+)-K+ pump kinetics between the two strains while the apparent affinity (Km) of Na(+)-K(+)-Cl- cotransport for internal sodium was significantly greater and the maximal rate of sodium transport lower in MHS when compared with MNS rats. IOV, which are depleted of cytoskeleton, showed a bumetanide-sensitive potassium- and chloride-dependent sodium uptake. However, the erythrocyte differences in Na(+)-K(+)-Cl- cotransport activity and the Km between strains disappeared in IOV, suggesting tha the altered sodium transport of MHS erythrocyte might be due to some intracellular factor or a membrane skeleton protein abnormality.

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.

Genetic and experimental hypertension in the animal model-similarities and dissimilarities to the development of human hypertension

In this article, we present the results we have obtained from experimental and genetic models of human essential hypertension, in order to investigate those findings relevant to understanding the time course and the mechanisms underlying the human disease. With experiments on the renal artery constriction in the conscious dog, we have shown that a kidney lesion can produce a form of hypertension not different, in the established phase, from the essential one and that the onset of this form follows a phasic pattern during which the initial stages are crucial for understanding the mechanisms leading to hypertension. We also consider a rat model (MHS) that spontaneously develops a form of hypertension very similar to the human disease. In this model, we have demonstrated by a kidney cross-transplantation experiment and functional studies that the kidney is responsible for the rise in blood pressure and that the organ dysfunction is probably due to a primary abnormality in ion handling of the cell membrane. This cellular alteration, detected both in MHS erythrocytes and in their kidney proximal tubular cells, should be the cause for the higher rate of kidney Na+ reabsorption observed in the MHS. Comparing this animal model with, at least, a subgroup of humans prone to develop hypertension or already hypertensive, it is possible to detect a series of similarities in the kidney function, hormonal pattern, and cellular function of the two species that allows us to argue that the MHS is a suitable model from which to draw conclusions relevant to the pathogenesis of essential hypertension in some humans.

Characteristics of a ouabain-like factor from Milan hypertensive rats

Ouabain-like factor (OLF) has been extracted from hypothalamus and adrenal glands of the ox and rats of the Milan hypertensive strain (MHS) and their normotensive controls (MNS). OLF was identified by its ability (a) to inhibit ouabain-sensitive 86Rb uptake into human erythrocytes, (b) to displace [3H]ouabain binding, and (c) to inhibit purified dog kidney Na-K-ATPase. Rat and bovine OLF have similar characteristics. Those that are close to ouabain are (a) ligand conditions for maximal inhibitory activity, (b) high-performance liquid chromatography retention time, (c) reversibility of inhibitory activity on Na-K-ATPase, (d) reduced Na-K-pump inhibitory activity by K+, (e) high affinity for Na-K-ATPase, and (f) no activity on Ca(2+)-ATPase. OLF does not resemble ouabain because the capacity of OLF to inhibit ouabain low-affinity Na-K-ATPase isoform is greater than that of ouabain. The yield of OLF is greater from MHS than MNS hypothalamic and adrenal extracts. These findings represent the first direct evidence that a higher amount of OLF is present in tissues from genetically hypertensive rats than from their inbred normotensive controls, maintained under the same dietary and environmental conditions. This further supports previous observations on the role of OLF in the pathogenesis of MHS hypertension.

Characterization of erythrocyte adducin from the Milan hypertensive strain of rats

Previous studies showed that erythrocytes from the Milan hypertensive strain of rats (MHS) are smaller and have a faster Na-K cotransport when compared with their normotensive controls (MNS). These characteristics are determined within the stem cell, are genetically associated with hypertension and are similar to other renal tubular cell abnormalities more directly involved in the development of hypertension in MHS. The difference in volume is maintained in ghost membranes, while the difference in transport is abolished in inside-out vesicles. Ghosts and cytoskeletons contain a 105-kilodalton protein already characterized by immunoblotting. This protein has been identified with erythrocyte adducin by several criteria, including binding to calmodulin and protein kinase C, phosphorylation and full immunological cross-reactivity with human adducin. Since only MHS rats immunized with MNS erythrocyte cytoskeletons produce anti-adducin antibodies, we suggest an immunogenic structural difference in adducin from the two strains, and an involvement of this difference in the alteration of Na-K cotransport observed.