Dysfunction of calcium handling by smooth muscle in hypertension

Dysfunction of ion handling, including binding and fluxes (passive and active transport) of physiologically important ions such as potassium, sodium, calcium, and magnesium, by vascular smooth muscle cell membranes has repeatedly been reported to be associated with the pathophysiology of hypertension. The specific purpose of this review is to summarize and evaluate the evidence for alterations of calcium ion (Ca2+) handling by vascular smooth muscle in various forms of hypertension in the animal model on the basis that regulation of cytoplasmic Ca2+ concentration is a complex and yet vitally important process for a normal function of vascular smooth muscle and that derangement of such a regulation may result in excessive retention of cytoplasmic Ca2+, contribute toward increase of total peripheral resistance, and ultimately lead to elevation of blood pressure. Emphasis is placed upon the consideration of the usefulness of the subcellular membrane fractionation technique in studies of binding and transport of Ca2+ by vascular and nonvascular smooth muscle membranes from genetic as well as experimental hypertensive rats. The limitations of the interpretation of data using such an approach are also considered. Decreased active transport of Ca2+ across isolated plasma membrane vesicles from large and small arteries occurs in several but not all forms of hypertension. This membrane abnormality also occurs in nonvascular smooth muscles and other tissues or cells not confined to the cardiovascular system in genetic hypertension, but not in experimental hypertension. A hypothesis of general membrane defects in spontaneous hypertension is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of cellular calcium metabolism as primary cause of hypertension

In the pathogenesis of hypertension, the importance of intracellular calcium is increasing. Clinical and experimental studies of essential hypertension indicate a pathological increase of intracellular Ca2+ in this disease. In the past, changes in cellular Na+ and its transport mechanisms were considered the triggering factors and Na+-Ca2+ exchange was attributed a decisive influence on intracellular homeostasis. Recently, a reduced Ca2+-binding capacity of the cellular membrane was observed in hypertension, which could have been due to a defect of the Ca2+-ATPase or its control. It is therefore necessary to establish the hypothesis that changes in the cellular Ca2+ metabolism associated with an increase in the intracellular Ca2+ concentration may be the primary cause of hypertension. Disorders of Na+ transport can also be traced to the increase in intracellular Ca2+ and were thus a consequence but not the cause of the increased intracellular Ca2+ concentration.     

牛磺酸与心脏及其疾病的研究进展

<正> 牛磺酸(Taurine,Tau.2-氨基乙磺酸)为体内一种β-氨基酸,属于非蛋白质氨基酸。主要分布在兴奋性较高的组织如神经系统、肌肉组织、视网膜及血小板中。近年来研究认为牛磺酸不仅参与合成胆汁酸、调节渗透压、阻断神经冲动的功能,还有抗氧化及维持膜稳定性等方面作用。自从     

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.     

Impairment of capping in lymphoblastoid cell lines of Duchenne patients indicates an intrinsic cellular defect

Summary Duchenne muscular dystrophy (DMD) is a lethal sex-linked degenerative disorder of the muscle in man. Generalized cell membrane abnormalities seem to be involved in the pathogenesis of the disease; in particular, the impairment of lymphocyte capping capacities has been repeatedly confimed. To clarify whether capping impairment is a consequence of factors related to the activity of the disease or an expression of an intrinsic cellular defect, we have investigated the capping capacities of DMD EBV-transformed cell lines. The results indicate a significant impairment of capping capacity in cultured cell lines, providing evidence for an intrinsic cell deficiency in DMD.     

Multiple Alterations in Metabolite Uptake in a Mutant of Neurospora crassa

A temperature-conditional lethal mutant of Neurospora crassa, un-t (55701), was resistant to neutral amino acid analogues by virtue of a decreased ability to transport these analogues and their natural congeners across the cell membrane. The uptake of acidic, but not basic, amino acids was also impaired, as was the uptake of potassium ions. After preincubation above the tolerated temperature, the ability to take up a still wider variety of metabolites was greatly reduced. Protoplasts of the mutant were more osmotically fragile than those of wild type. The possibility that the mutant has a generalized membrane defect is discussed.     

Metabolic stress in isolated mouse ventricular myocytes leads to remodeling of t tubules

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents (I(K1,tail)), and inward rectifier potassium current (I(K1)) "inactivation". At room temperatures and in the absence of Mg(2+) ions in pipette solution, the amplitude of I(K1,tail) measured ~10 min after the establishment of whole cell configuration was reduced by ~18%, but declined nearly twofold in the presence of 1 mM cyanide. At ~35°C I(K1,tail) was essentially preserved in intact cells, but its amplitude declined by ~85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg(2+) ions played protective role at all temperatures. Decline of I(K1,tail) was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of I(K1) inactivation, a marker of depletion of t-tubular K(+). The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced I(K1,tail), as well as the inactivation of inward I(K1). Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.     

Coexisting independent sodium-sensitive and sodium-insensitive mechanisms of genetic hypertension in spontaneously hypertensive rats (SHR)

Some essential hypertensive patients and genetic hypertensive rat strains have less than the normal levels of Mg2+ tightly bound to the plasma membranes of their erythrocytes and other cells, i.e., the magnesium binding defect (MgBD). This binding defect appears to cause increased passive permeability of the membrane to Na+ and thereby its increased intracellular concentration, particularly if the Na+-extrusion enzyme systems of the cell are also defective. The Na+-Ca2+ exchange system in the cell membrane exports Na+ and imports Ca2+, increasing the tone of the smooth muscle cell and thus producing hypertension (HTn). This HTn is Na+-sensitive. Evidence supporting this postulate was obtained by determining the intraerythrocyte total concentrations of Na+, Ca2+, K+, and Mg2+ in two strains of spontaneously hypertensive rats (SHR and SS/Jr rats, having the MgBD together with the other requisites of the Na+-sensitive pathway) and their respective controls (WKY and SR/Jr rats, in which this complete pathway is absent). The Na+ and Ca2+ concentrations in the hypertensive rats were increased, and that of K+ was decreased. The concentrations of these cations were very similar in the two hypertensive strains. The level of membrane tightly bound Ca2+ in SHR erythrocyte membranes was significantly higher than those in the other three rat strains, which were not statistically different from each other. These results support previously reported evidence of the existence of a novel HTn-generating mechanism in the SHR rat, in which the intracellular Ca2+ concentration is increased as the result of the enhanced diffusion of this ion into the cell and the accompanying deficiency of the Ca2+ extrusion enzyme systems. This pathway is therefore Na+-insensitive, i.e., Ca2+-sensitive.     

The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation

The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

Activation of cell membrane potassium conductance by mercury in cultured renal epithelioid (MDCK) cells

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)-cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1 microM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by approximately 0.2 microM. Higher concentrations of mercury ions (greater than 10 microM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 microM, 1 microM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 microM, but not 1 microM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin- or pertussis toxin-sensitive G-proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.     

Intercellular communication and tissue growth: VIII. A genetic analysis of junctional communication and cancerous growth

Summary Normal, proliferating cells are interconnected at their junctions by membrane channels through which molecules can pass from cell to cell (Loewenstein, W.R. 1966.Ann. N.Y. Acad. Sci. 137:708). A channel-competent, normally growing cell (human fibroblast) was hybridized with a channel-incompetent cancer cell (mouse L-1d cell), and the segregant hybrid clones were analyzed in a genetic approach to the question of whether the junctional membrane channels are instrumental in transmission of growth-controlling molecular signals. The channel competence of the human parent was characterized by the ability to transfer small inorganic ions (electrical coupling) and fluorescein, and the growth patterns of this cell, by growthin vitro to low saturation densities and nontumorigenicity in immuno-suppressed hosts. The mouse parent cell had the opposite characteristics. The early hybrid generations (which still had a large part of each parent chromosome complement) were of two classes: one class resembled the human parent cell in channel competence,in vitro growth pattern, and low tumorigenicity within 26 days; the other class presented an intermediate expression of channel competence characterized by transfer of small inorganic ions but not of fluorescein. As the hybrid generations lost human chromosomes, there was segregation of several biochemical and morphological traits, but no segregation of channel competence and normal growth traits. Among the segregants were 22 clones which had reverted to the channel-incompetent trait of the mouse parent. In every case, reversion to the channel defect went hand in hand with reversion to the growth defect, just as, in the early-generation hybrids, correction of the channel defect went hand in hand with correction of the growth defect. Thus, the human genetic factor that corrects the channel defect of the mouse parent cell seems closely linked, if not identical, with that correcting the growth defect. This genetic correlation encourages us in the belief that the channel defect may be an etiological factor in this particular cancer form.     

Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome

Cardiolipin (CL) and phosphatidylglycerol (PG) are the major polyglycerophospholipids observed in mammalian tissues. CL is exclusively found in the inner mitochondrial membrane and is required for optimal function of many of the respiratory and ATP-synthesizing enzymes. The role of CL in oxidative phosphorylation is, however, not fully understood and although reduced CL content leads to aberrant cell function, no human disorders with a primary defect in cardiolipin metabolism have been described. In this paper we present evidence that patients with the rare disorder X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) have a primary defect in CL and PG remodeling. We investigated phospholipid metabolism in cultured skin fibroblasts of patients and show that the biosynthesis rate of PG and CL is normal but that the CL pool size is 75% reduced, indicating accelerated degradation. Moreover, the incorporation of linoleic acid, which is the characteristic acyl side chain found in mammalian CL, into both PG and CL is significantly reduced, whereas the incorporation of other fatty acids into these phospholipids is normal. We show that this defect was only observed in Barth syndrome patients' cells and not in cells obtained from patients with primary defects in the respiratory chain, demonstrating that the observed defect is not secondary to respiratory chain dysfunction. These results imply that the G4.5 gene product, which is mutated in Barth syndrome patients, is specifically involved in the remodeling of PG and CL and for the first time identify an essential factor in this important cellular process.     

SOME BIOCHEMICAL PROPERTIES OF CHINESE HAMSTER CELLS SENSITIVE AND RESISTANT TO ACTINOMYCIN D

A graded series of drug-resistant Chinese hamster sublines has been examined for biochemical changes accompanying resistance to actinomycin D. The most highly resistant subline, DC-3F/AD X, is maintained at 10 µg/ml of the antibiotic. It was shown that over 250 times more actinomycin D is required to inhibit RNA synthesis in this subline than in the parental DC-3F line. The DC-3F/AD X subline was also shown to have a somewhat reduced capacity to transport uridine as compared to parental cells. Sensitive cells took up over 50 times more tritiated antibiotic than the most resistant cells, as determined in a 1-h assay. Uptake of actinomycin D was shown to be temperature-dependent in both resistant and sensitive cells and was not influenced by various metabolic inhibitors. Resistance could not be explained by a rapid uptake and release of the antibiotic, as demonstrated in efflux experiments, or by its metabolism. In addition, highly resistant cells which are cross-resistant to puromycin were shown to have a reduced capacity to take up labeled puromycin. These studies provide further evidence indicating that the mechanism of resistance to actinomycin D is reduced permeability to drug and suggesting that cell membrane alteration accounts for resistance to both actinomycin D and puromycin.     

Intramuscular lipid oxidation and obesity

There is an accumulating amount of evidence indicating that lipid oxidation is depressed in the skeletal muscle of obese individuals. Decrements in fatty acid oxidation (FAO) have been reported with obesity in models ranging from whole body measurements to isolated skeletal muscle preparations as well as in myotubes raised in culture. This reduction appears to be associated with a depression in the activities of enzymes involved in various steps of lipid oxidation, which subsequently partitions lipid entering the cell toward storage. The defect in FAO in skeletal muscle may be critical in relation to health, as a reduction in the capacity for lipid oxidation could directly or indirectly contribute to the insulin resistance commonly evident with obesity. Although less characterized, a decrement in FAO has also been linked with weight gain, which suggests that this characteristic may be an integral aspect leading to the obese state. In terms of intervention, weight loss does not seem to correct the defect in FAO with obesity. This review will provide evidence supporting a reduction in muscle FAO with obesity.     

Characteristics of the Na+-K+-cotransport system in the erythrocyte membrane of patients with hypertension and symptomatic (renal) hypertension

The data on erythrocyte membrane permeability for Na and K ions, obtained in the studies of Na+-K+ cotransport in erythrocytes of 38 patients with essential hypertension, stage I and II, 9 patients with borderline hypertension and 12 patients with symptomatic (renal) hypertension are reviewed. The data demonstrate that Na+-K+ cotransport in Na+ loaded and K+-depleted erythrocytes under the effect of P-chlormercuribenzoate was considerably reduced in patients with essential hypertension and borderline hypertension than in the control group. No deviations from the normal Na+-K+ cotransport were observed in renal hypertension. Disturbances of erythrocyte membrane permeability have been also revealed in practically healthy subjects (15 cases) with family history of hypertension.     

Lipid rafts in lymphocyte activation and migration

Functional polarization of leukocytes is a requisite to accomplish immune function. Immune synapse formation or chemotaxis requires asymmetric redistribution of membrane receptors, signaling molecules and the actin cytoskeleton. There is increasing evidence that compartmentalization of the plasma membrane into distinct lipid microdomains is pivotal in establishing and maintaining leukocyte polarity. Specific rafts assemble into large-scale domains to create plasma membrane asymmetries at specific cell locations, thus coordinating temporally and spatially cell signaling in these processes. In this review we discuss the roles of lipid rafts as organizers of T lymphocyte polarity during cell activation and migration.     

An electrophysiological study of the non-junctional membrane of epidermal cells in Tenebrio molitor

The contribution of K and Cl to the membrane potential of the epidermal cells of the recently-ecdysed larva of the mealworm was examined. The ionic basis for the membrane potential is complex. Although increasing the external K level depolarized the cell membrane, the relationship obtained suggests that ions other than K contribute largely to the recorded membrane potential. In particular, exposing the cells to K concentrations below the normal level of 40 mM has only slight effects on membrane potential, irrespective of whether K is lowered by direct substitution with Na or under conditions in which Na and Cl levels are held constant. Increasing the external Cl levels from 4 mM to 154 mM while holding K and Na levels constant resulted in a 10 mV hyperpolarization. The slight hyperpolarizing effects of high external Cl could be mimicked by citrate, but not by acetate, the latter drastically hyperpolarizing the cell membrane at levels of K that normally maintain a reduced membrane potential. External Na has little effect on the membrane potential at normal physiological levels of K, but may depolarize the cell at low K levels. The results suggest that several inorganic ions, and possibly organic acids, participate in generating the membrane potential of the epidermal cell. The passive ionic properties of non-junctional epidermal membrane and muscle membrane appear to the similar in this insect.The electrical resistance on the non-junctional membrane is highly dependent on the external K level, and can be reduced by three orders of magnitude by increasing external K from 1 mM to 120 mM. The resistance of the junctional membrane remains constant over this range of external K concentrations.     

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.