C.Y. Kwan

An Analytical Approach To The Preparation and Characterization of Subcellular Membranes From Canine Mesenteric Arteries

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.

Alterations of elastin and elastase-like activities in aortae of diabetic rats

The elastin content of the aortic muscle and the elastase-like activity of the extracts of aortic muscle were studied in spontaneously diabetic BB rats and in rats made diabetic by a single bolus i.v. injection of alloxan. In both modes of diabetes, the total alkaline-insoluble aortic elastin content was significantly reduced in diabetic rats compared to that in the corresponding control rats. This reduction in aortic elastin was also accompanied by a consistent increase in the elastase-like activities of the aortic extracts prepared from the same tissues. Such a reciprocal relationship between aortic elastin content and elastase-like activity has previously been reported in rats with malignant hypertension. Since the rats used in this study were not hypertensive, the altered elastin metabolism observed in this work is likely to be a manifestation of diabetic disease and may in part account for the vascular changes associated with diabetes mellitus.

Membrane fractionation of canine aortic smooth muscle: Subcellular distribution of calcium transport activity

Various subcellular membrane fractions were isolated from dog aortic smooth muscle by conventional differential centrifugation followed by isopycnic centrifugation on a sucrose density gradient. These subcellular fractions were characterized by membrane marker enzyme activities, morphological features and the electrophoretic patterns on a sodium dodecyl sulfate polyacrylamide gel. Our results showed that the microsomal membrane fraction isolated by differential centrifugation was very heterogeneous and contained substantial amount of plasma membranes which could be further enriched as a light density fraction on the sucrose density gradient. The subcellular distribution of Ca2+ binding in the absence of ATP and Ca2+ transport in the presence of ATP closely paralleled the distribution of plasma membrane markers. The ATP-supported Ca2+ transport was inhibited by several Ca2+ ionophores, enhanced by inorganic phosphate and oxalate ions and cosedimented toward higher density in a continuous source density gradient with plasma membrane marker enzyme activity in the presence of digitonin. Our present work strongly suggests that plasma membrane is the predominant component of microsomal fraction and responsible for most, if not all, of the azide-insensitive ATP-supported Ca2+ accumulation.

Instability of malondialdehyde in the presence of H2O2: Implications for the thiobarbituric acid test

The content of thiobarbituric acid-reactive material (primarily malondialdehyde) is frequently used to estimate the extent of lipid peroxide formation. How-ever, malondialdehyde is unstable in the presence of millimolar concentrations of hydrogen peroxide. This observation considerably limits the applicability of the thiobarbituric acid test, as hydrogen peroxide is known to be formed in a number of lipid peroxidation-promoting systems. The instability of malondialdehyde in the presence of hydrogen peroxide seems to account for the inconsistent outcomes in studies relating the manipulations of intermediate H2O2 levels to the initiation of lipid peroxidation.

PERTURBATION BY LYSOPHOSPHATIDYLCHOLINE OF MEMBRANE PERMEABILITY IN CULTURED VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL CELLS

Lysophosphatidylcholine (LPC), a major component of oxidized low-density lipoprotein found in atherosclerotic arterial walls, has been shown to have insignificant effect on arterial contraction but cause an impairment of endothelium-dependent relaxation (EDR). The aim of this study was to compare the degree of LPC-induced perturbation in the plasma membrane of cultured aortic smooth muscle cells (SMC) and endothelial cells (EC). In contractility studies phenylephrine (PE) elicited a sustained contraction and a subsequent addition of acetylcholine (ACh) caused an almost complete relaxation. Preincubation of endothelium-intact aortic rings with LPC did not significantly affect PE-elicited contraction but substantially inhibited ACh-triggered relaxation. Such inhibition by LPC was both concentration- and time-dependent. LPC also inhibited relaxation triggered by extracellular ATP and cyclopiazonic acid. Exposure of cultured EC to LPC (30 microM) resulted in an elevation of [Ca2+]i with a lag period of some 25 min. Following [Ca2+]i elevation, addition of Ni2+ resulted in a rapid entry of this ion into the cell. In addition, fura-2 leak-out was observed. Exposure of cultured SMC to 30 microM LPC also resulted in [Ca2+]i elevation and Ni2+ entry. However, LPC did not cause fura-2 leak-out in SMC. Also, LPC raised [Ca2+]i at a slower rate in SMC than in EC. Our results suggest that the plasma membrane of EC is more susceptible to LPC-induced derangement than that of SMC. This may contribute in part to the selective impairment of EDR by LPC.

Inhibition of endothelium-dependent vascular relaxation by tetrandrine

The effects of tetrandrine, a Ca2+ antagonist of bis-benzylisoquinoline alkaloid origin, on endothelium-dependent and -independent vascular responsiveness were investigated in perfused rat mesenteric artery. In endothelium-intact preparations pre-contracted with 3 microM phenylephrine and fully relaxed by 0.3 microM acetylcholine tetrandrine caused a rapid transient contraction. In endothelium-denuded preparations, tetrandrine caused only vasorelaxation of phenylephrine-contraction. The biphasic effect of tetrandrine in acetylcholine-relaxed preparations could also be mimicked by sequential applications of atropine/tetrandrine or N(G)-nitro-L-arginine-methylester (L-NAME)/tetrandrine, but atropine or L-NAME alone caused only vasoconstriction. This tetrandrine-induced transient vasoconstriction was also observed in preparations relaxed with ATP, histamine or thapsigargin (TSG), but not those relaxed with A23187, sodium nitroprusside or nifedipine. The present results suggest that tetrandrine, in addition to its known inhibitory effects on vascular smooth muscle by virtue of its Ca2+ antagonistic actions, also inhibits NO production by the endothelial cells possibly by blockade of Ca2+ release-activated Ca2+ channels.

Abnormal Biochemistry of Subcellular Membranes Isolated from Nonvascular Smooth Muscles of Spontaneously Hypertensive Rats

Microsomal fractions enriched in plasma membranes and endoplasmic reticulum were isolated from stomach fundus and vas deferens from age-matched Okamoto spontaneously hypertensive (SHR) rats and corresponding Kyoto-Wistar normotensive rats (KWR). Alterations of several enzyme activities and Ca2+ accumulation of the isolated microsomal fraction from these nonvascular smooth muscles provide direct evidence of abnormal smooth muscle membrane biochemistry in SHR. Decreased Ca2+ accumulation in the presence of but not in the absence of adenosine triphosphate by the microsomal fractions of both fundus and vas deferens from SHR is consistent with previous findings using plasma membranes from vascular smooth muscles from SHR and cannot be explained in terms of adaptation induced by elevation of blood pressure in SHR. Defective Ca2+ handling now observed in both vascular and nonvascular smooth muscles from hypertensive animals not only provides a cellular basis for the altered reactivity and contractility of smooth muscles observed in SHR, but also supports the hypothesis that spontaneous hypertension is associated with a generalized widespread alteration in smooth muscle membrane fraction.

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+.

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.

Isolation of Plasma Membranes from Rat Mesenteric Veins: A Comparison of Their Physical and Biochemical Properties with Arterial Membranes

Plasma membranes were isolated from smooth muscles of rat mesenteric veins. The plasma membrane fraction is relatively pure according to its morphological and enzymatic characteristics. The membrane distribution, enzymatic activities, as well as calcium accumulation by the plasma membrane fraction from venous smooth muscle were compared to those from arterial smooth muscle. The isolated venous smooth muscle plasma membranes formed primarily closed vesicles which were capable of accumulating Ca2+ in the presence of ATP suggesting active transport of Ca2+ across the membrane vesicles. Evidence obtained from several approaches by studying the effect of A23187, phosphate ions and hypotonic shock on the Ca2+ accumulation in the presence of ATP revealed that there is an active transport of Ca2+ across isolated vascular smooth muscle membrane vesicles in addition to binding of Ca2+. However, venous smooth muscle plasma membrane fraction appears to be different from arterial smooth muscle plasma membrane fraction in its low activity of alkaline phosphatase, greater Ca2+ binding and lower Ca2+ transport. These and previous studies show that the plasma membrane of vascular muscles may play an important role in the steady state regulation of cellular calcium concentration during excitation-contraction coupling, especially in small arteries and veins.

Dual effects of extracellular Ca2+ on cardiotoxin-induced cytotoxicity and cytosolic Ca2+ changes in cultured single cells of rabbit aortic endothelium

The effects of extracellular Ca2+ on cytotoxicity induced by cardiotoxin (CTX), isolated from Chinese cobra venom, were investigated in cultured rabbit aortic endothelial cells (RAECs). In Hanks buffered saline solution (HBSS) containing 1.2 mM Ca2+, CTX (1-30 microM) caused cell necrosis and cell death in a concentration-dependent manner, as determined by trypan blue exclusion test performed after a 20-min CTX treatment. The concentration of CTX that caused 50% cell death was about 6.5 microM. CTX (10 microM)-induced RAEC damage was also evident but less prominent in Ca2+-free medium and almost completely prevented in medium containing 7-10 mM Ca2+. Therefore, Ca2+ appears to provoke CTX-induced injury at physiological concentrations, but protects against it at high concentrations. The protection of RAECs from CTX-induced injury could also be achieved by high concentrations of Ni2+ and Mg2+. Using the fura-2 fluorescence technique to measure the cytosolic free Ca2+ concentration ([Ca2+]i) of single RAEC, it was shown that in 1.2 mM Ca2+-containing HBSS, treatment of RAECs with 10 microM CTX for 7-35 min resulted in a tremendous and irreversible [Ca2+]i elevation, suggestive of cell membrane damage and extracellular Ca2+ entry. Ni2+ could also enter the cytosol of these damaged RAECs. However, there was no [Ca2+]i elevation or Ni2+ entry in RAECs that were preincubated in HBSS containing 7 mM Ca2+ or Ni2+ before CTX exposure. In RAECs protected with 7 mM Ca2+, the intracellular Ca2+ signals triggered by 100 microM extracellular ATP or 10 microM bradykinin in CTX-treated groups were similar to those in the untreated control groups. Taken together, the results indicate that high extracellular Ca2+ concentrations protected RAECs from CTX-induced injury, and preserved the ability of CTX-treated RAECs to generate Ca2+ signals in response to physiological stimuli.