Sergei N. Orlov

Altered permeability of the erythrocyte membrane for sodium and potassium ions in spontaneously hypertensive rats

SummaryPermeability of the erythrocyte membrane for sodium and potassium ions was studied in 8–10-week old spontaneously hypertensive rats (SHR, Kyoto Wistar strain), normotensive Wistar and Sprague-Dawley rats.The rate constant of Na/Na exchange was considerably greater in the SHR than in the normotensive Wistar and Sprague-Dawley rats. This difference remained the same in the rats adrenalectomized 7 days prior to the experiment. The maximum difference in the constants was found when the sodium pump was blocked by ouabain.The accumulation of42K in the erythrocytes of the SHR (the sodium pump being blocked) took place at a considerably slower rate, and the K+ washout into a potassium-free medium was faster than in the normotensive Wistar and Sprague-Dawley rats.These results seem to indicate a higher permeability of the SHRs erythrocyte membrane for Na+ and K+ions, as compared to normotensive Wistar and Sprague-Dawley strains. It is suggested that the increased permeability of the erythrocyte membrane for Na+ and K+ in the SHR may reflect a more widespread cell membrane defect, which could serve as a general cause for activating the mechanisms maintaining high blood pressure.

Decrease of calcium binding by the red blood cell membrane in spontaneously hypertensive rats and in essential hypertension

Ca binding in the red blood cell (RBC) membrane of spontaneously hypertensive rats (SHR) and of patients with essential hypertension was studied. Under conditions of physiological concentration of free Ca in the incubation medium of RBC the outer part of the membrane binds 393±32 and 435±30 nmole of Ca per ml of RBC in rats and humans, respectively, without essential differences in the amount of Ca in hypertensive individuals as compared to the normotensive controls.The membrane of red blood cell ghosts (RBCgh) at concentrations of free Ca corresponding to its intracellular concentration binds 4.28±0.39 and 3.53±0.15 nmole of Ca per mg of protein of RBCgh in rats and humans, respectively. This part of membrane-bound Ca pool (most probably related to the inner part of the red blood cell membrane) is reduced by 48% in SHR and by 28% in patients with essential hypertension as compared to normotensive controls.It is suggested that the decrease of Ca binding ability of the RBC membrane in both types of hypertension studied may be a pattern of a more widespread cell membrane defect.

Inversion of the Intracellular Na+/K+Ratio Blocks Apoptosis in Vascular Smooth Muscle at a Site Upstream of Caspase-3

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in the majority of cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition of the Na+,K+ pump in VSMC by ouabain or a 1-h preincubation in K+-depleted medium attenuated apoptosis triggered by serum withdrawal, staurosporine, or okadaic acid. In the absence of ouabain, both DNA degradation and Caspase-3 activation in VSMC undergoing apoptosis were insensitive to modification of the extracellular Na+/K+ ratio as well as to hyperosmotic cell shrinkage. In contrast, protection of VSMC from apoptosis by ouabain was abolished under equimolar substitution of Na+ o with K+ o , showing that the antiapoptotic action of Na+,K+ pump inhibition was caused by inversion of the intracellular Na+/K+ ratio. Unlike VSMC, the same level of increment of the [Na+] i /[K+] i ratio caused by a 2-h preincubation of Jurkat cells with ouabain did not affect chromatin cleavage and Caspase-3 activity triggered by treatment with Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results show for the first time that similar to cell proliferation, maintenance of a physiologically low intracellular Na+/K+ratio is required for progression of VSMC apoptosis.

Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na+]i/[K+]i Ratio in Cultured Vascular Smooth Muscle Cells

Abstract— Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na+]i/[K+]i-responsive signaling pathway that may play an important role in the regulation of programmed cell death in VSMCs.

Mechanisms of cell volume regulation and possible nature of the cell volume sensor

In animal organisms, cell volume undergoes dynamic changes in many physiological and pathological processes. To protect themselves against lysis and apoptosis and to maintain an optimal concentration of intracellular enzymes and metabolites, most animal cells actively regulate their volume. In the present review, we shortly summarize the data on ion transport mechanisms involved in regulatory volume decrease (RVD) and regulatory volume increase (RVI) with an emphasis on unresolved aspects of this problem such as: (i) how cells sense their volume changes; (ii) what signals are generated upon cell volume alterations; and (iii) how these signals are transferred to the ion transport systems executing cell volume regulation.

Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase-3

Intracellular signaling pathways that are involved in protection of vascular smooth muscle cells (VSMC) from apoptosis remain poorly understood. This study examines the effect of activators of cAMP/cGMP signaling on apoptosis in non-transfected VSMC and in VSMC transfected with c-myc (VSMC-MYC) or with its functional analogue, E1A-adenoviral protein (VSMC-E1A). Serum-deprived VSMC-E1A exhibited the highest apoptosis measured as the content of chromatin and low molecular weight DNA fragments, phosphatidylserine content in the outer surface of plasma membrane and caspase-3 activity (ten-, five-, four- and tenfold increase after 6 h of serum withdrawal, respectively). In VSMC-E1A, the addition of an activator of adenylate cyclase, forskolin, abolished chromatin cleavage, DNA laddering, caspase-3 activation and the appearance of morphologically-defined apoptotic cells triggered by 6 h of serum deprivation. In non-transfected VSMC and in VSMC-MYC, 6 h serum deprivation led to ∼six- and threefold activation of chromatin cleavage, respectively, that was also blocked by forskolin. In VSMC-E1A, inhibition of apoptosis was observed with other activators of cAMP signaling (cholera toxin, isoproterenol, adenosine, 8-Br-cAMP), whereas 6 h incubation with modulators of cGMP signaling (8-Br-cGMP, nitroprusside, atrial natriuretic peptide, L-NAME) did not affect the development of apoptotic machinery. The antiapoptotic effect of forskolin was abolished in 24 h of serum deprivation that was accompanied by normalization of intracellular cAMP content and protein kinase A (PKA) activity. Protection of VSMC-E1A from apoptosis by forskolin was blunted by PKA inhibitors (H-89 and KT5720), whereas transfection of cells with PKA catalytic subunit attenuated apoptosis triggered by serum withdrawal. The protection of VSMC-E1A by forskolin from apoptosis was insensitive to modulators of cytoskeleton assembly (cytochalasin B, colchicine). Neither acute (30 min) nor chronic (24 h) exposure of VSMC to forskolin modified basal and serum-induced phosphorylation of the MAP kinase ERK1/2. Thus, our results show that activation of cAMP signaling delays the development of apoptosis in serum-deprived VSMC at a site upstream of caspase-3 via activation of PKA and independently of cAMP-induced reorganization of the cytoskeleton network and the ERK1/2-terminated MAPK signaling cascade.

Quantitative Founder-Effect Analysis of French Canadian Families Identifies Specific Loci Contributing to Metabolic Phenotypes of Hypertension

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

Membrane Reserves and Hypotonic Cell Swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o−, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.

Ca2+‐dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca2+‐dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca2+ for the elevation of intracellular Ca2+ concentration ([Ca2+]i) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca2+‐dependent ATP release. We found that ATP release does not directly involve Ca2+ influx from extracellular spaces, but depends entirely on Ca2+ mobilization from intracellular stores. The [Ca2+]i response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)‐insensitive stores and a superimposed rapid spike due to Ca2+ release from TG‐sensitive endoplasmic reticulum (ER) Ca2+ stores. The latter could be abolished by hydrolysis of extracellular triphospho‐ and diphosphonucleotides with apyrase; blocking P2Y2/P2Y6 receptors of A549 cells with suramin; blocking UDP receptors (P2Y6) with pyridoxal phosphate 6‐azophenyl‐2′,4′‐disulfonic acid (PPADS); emptying TG‐sensitive stores downstream with TG or caffeine in Ca2+‐free extracellular solution; or blocking the Ca2+‐release inositol 1,4,5‐triphosphate receptor channel of the ER with 2‐aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca2+]i spike results from the autocrine stimulation of IP3/Ca2+‐coupled P2Y, predominantly P2Y6, receptors, accounting for ∼70% of total Ca2+‐dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress‐induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtained during the last two decades show that spontaneously hypertensive rats, an acceptable experimental model of primary human hypertension, possess increased activity of both ubiquitous and renal cell-specific isoforms of the Na+/H+exchanger (NHE) and Na+-K+-2Cl-cotransporter. Abnormalities of these ion transporters have been found in patients suffering from essential hypertension. Recent genetic studies demonstrate that genes encoding the β- and γ-subunits of ENaC, a renal cell-specific isoform of the Na+-K+-2Cl-cotransporter, and α3-, α1-, and β2-subunits of the Na+-K+pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis of the homology of genome maps, several other genes encoding these transporters, as well as the Na+/H+exchanger and Na+-K+-2Cl-cotransporter, can be predicted in QTL related to the pathogenesis of hypertension. However, despite their location within QTL, analysis of cDNA structure did not reveal any mutation in the coding region of the above-listed transporters in primary hypertension, with the exception of G276L substitution in the α1-Na+-K+pump from Dahl salt-sensitive rats and a higher occurrence of T594M mutation of β-ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities of systems involved in the regulation of their expression and/or function. Further analysis of QTL in F2hybrids of normotensive and hypertensive rats and in affected sibling pairs will allow mapping of genes causing abnormalities of these regulatory pathways.