Chiu-Yin Kwan

An improved procedure for the isolation of plasma membranes from rat mesenteric arteries.

Abstract A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.

Subcellular membrane properties in vascular and non-vascular smooth muscles of Dahl hypertensive rats.

Subcellular membrane fractions were isolated from mesenteric arteries and vas deferens of salt-resistant and salt-sensitive Dahl rats on low-salt (0.4% NaCl) high-salt (8.0% NaCl) diets. Only the salt-sensitive Dahl rats on the high-salt diet developed sustained high blood pressure (BP) after 5 weeks of the high-salt diet. Protein contents, membrane associated enzyme activities, calcium ion (Ca2+) binding and ATP-dependent CA2+ transport were compared in fractions isolated from all four groups. No obvious changes were observed, except for minor enhancement in magnesium ion (Mg2+)- and CA2+ ATPase activities of mesenteric arterial membranes isolated from salt-sensitive Dahl rats on high-salt diet compared to those from other groups of rats. The membrane fractions from vas deferens of salt-sensitive Dahl rats on the high-salt diet, on the other hand, showed decreased ATP-dependent Ca2+ transport compared to those from salt-sensitive Dahl rats on the low-salt diet. No difference was observed in membrane fractions isolated from salt-resistant Dahl rats on high-salt diet compared to those on low-salt diet. The significance of these observations are discussed in relation to the findings previously obtained from corresponding smooth muscle tissues of spontaneous hypertensive rats (SHR).

Endothelium-independent vasorelaxation by leonurine, a plant alkaloid purified from Chinese motherwort

Leonurine, a plant alkaloid present in Chinese motherwort, induced concentration- dependent and endothelium-independent relaxation of phenylephrine (PE)- pretreated rat aortic arterial rings. The IC50 values for leonurine were 86.4+/-10.4 and 85.9+/-17.2 microM in the presence and absence of endothelium respectively. It inhibited the responses of aortic smooth muscle to PE in Ca2+ free medium containing 100 microM EGTA, suggesting a possible action on the release of intracellular Ca2+. Leonurine is not a specific alpha-adrenoceptor blocker, since it also caused a concentration-dependent inhibition of vascular contractile responses to KCl with an IC50 value of 96.4+/-13.4 microM, suggesting that leonurine also blocks the L-type Ca2+-channel. In addition, leonurine relaxed the aortic contraction induced by prostaglandin F2alpha (PGF2alpha). These inhibitory effects of leonurine were reversible and did not affect the resting tension. In conclusion, these findings suggest that leonurine is an effective inhibitor of vascular smooth tone, probably acting by inhibiting the Ca2+ influx and the release of intracellular Ca2+.

Effects of papaya seed extract and benzyl isothiocyanate on vascular contraction

To investigate their potentially toxic effects on mammalian vascular smooth muscle, pentane extracts of papaya seeds and the chief active ingredient in the extracts, benzyl isothiocyanate (BITC), were tested for their effects on the contraction of strips of dog carotid artery. BITC and the papaya seed extract caused relaxation when added to tissue strips that had been pre-contracted with phenylephrine (PE). Incubation of the tissue with papaya seed extract or BITC caused inhibition of contraction when the strips were subsequently contracted with KCl or PE. This relaxation and inhibition of contraction did not appear to be endothelium-dependent, as endothelium-denuded rings showed the same degree of relaxation or inhibition of contraction in response to the preparations/drugs as those with the endothelium intact. The effects of both BITC and the extract were irreversible, i.e., the tissue did not recover to normal contractile ability after extensive washing. Exposure of the tissue to the papaya seed extract caused slower relaxation of the tissue, compared to controls, both after contraction with PE and subsequent addition of carbachol (CCh), and after contraction with KCl and then washing. Calcium imaging studies using cultured endothelial cells showed strong influxes of Ca2+ into the cells in response to addition of the papaya seed extract. We conclude that these extracts, when present in high concentration, are cytotoxic by increasing the membrane permeability to Ca2+, and that the vascular effects of papaya seed extracts are consistent with the notion that BITC is the chief bio-active ingredient.

A NOVEL IN VITRO ENDOTHELIUM‐DEPENDENT VASCULAR RELAXANT EFFECT OF APOCYNUM VENETUM LEAF EXTRACT

1. In the present study, a novel in vitro vascular relaxant effect of Apocynum venetum leaf extract (AVLE; also called ‘Luobuma’), obtained from a traditional Chinese medicinal herb with known antihypertensive effects, is reported in isometric contraction studies of rat aorta and superior mesenteric artery. At low concentrations (0.3–10 µg/mL), AVLE had no effect on the resting tension of either blood vessel and caused relaxation in agonist‐precontracted vessels with functionally intact endothelium.

Tetrandrine inhibits Ca2+ release-activated Ca2+ channels in vascular endothelial cells.

The effects of tetrandrine (TET), a Ca2+ antagonist of bis-benzylisoquinoline alkaloid origin, on cultured single bovine pulmonary artery endothelial cells were examined using fluorescence ratio imaging and whole-cell attached patch-clamp techniques. Thapsigargin (TSG, 100 nM), a selective endoplasmic reticulum Ca2+-ATPase pump inhibitor known to induce the release of nitric oxide (NO) from vascular endothelial cells via a Ca2+-dependent manner, caused a rapid elevation of cytosolic Ca2+ concentration, which was inhibited by 30 microM TET. In whole-cell patch-clamp study using the same vascular endothelial cells, addition of 100 nM TSG caused a significant enhancement of depolarization-evoked Ca2+-dependent, outward K+ currents, which could also be abolished by 30 microM TET. The present results demonstrate directly that TET, 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 through blockade of Ca2+ release-activated Ca2+ channels.

Alpha-adrenoceptors in dog mesenteric vessels--subcellular distribution and number of [3H]prazosin and [3H]rauwolscine binding sites.

Binding of the α-adrenergic antagonists [3H]prazosin and [3H]rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 ± 0.1 nM for [3H]prazosin and 5.85 ± 0.85 nM for [3H]rauwolscine in arteries, and 0.87 ± 0.4 nM for [3H]prazosin and 6.6 ± 1.5 nM for [3H]rauwolscine in veins. In veins, the maximum number of binding sites for [3H]rauwolscine was higher than that for [3H]prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for [3H]prazosin was higher than that for [3H]rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of [3H]saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on α2-adrenoceptors. This study thus identified two distinct populations of [3H]prazosin and [3H]rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of α2- compared to α1-adrenoceptor binding sites is required for a contractile response.

Early structural changes in precapillary vessels in hypertension and their relationship to functional changes.

This presentation has reviewed evidence from our laboratory that both structural and functional changes participate in the initiation and maintenance of hypertension in spontaneously hypertensive rats (SHR). Structural changes are present in the muscular arteries of the mesenteric and renal vasculature at 3- to 5-week-old SHR as compared with WKY. The major structural change in SHR arteries was increased cross-section area with increased thickness of the media owing to hyperplasia of smooth muscle; lumen sizes were interchanged. Later, at 10-12, 21, and 28 weeks of age, there was further increase in medial thickness owing to hyperplasia, and some hypertrophy and changes in elastic arteries also became evident. Increases in medial thickness of elastic arteries included hypertrophy as well as hyperplasia. Changes in lumen diameter were never observed in arteries fixed in a relaxed state at physiological flow rates. In addition, a deficit in Ca handling (decreased ATP-dependent Ca2+ accumulation) was observed in plasma-membrane vesicles from mesenteric arteries of SHR prior to and after the development of hypertension. It persisted when hypertension was reversed by hydralazine in SHR. It was present in various forms of experimental hypertension. It was present whenever hypertension was present and disappeared with normalization of blood pressure by withdrawal of the stimulus. The Ca-handling deficit was found in several nonarterial tissues and may be a generalized genetic defect in SHR. It was always accompanied by increased alkaline phosphatase activity of plasma membranes, and this was suggested to reflect the smooth-muscle hyperplasia occurring simultaneously. A model of the initiation and maintenance of hypertension based on medial thickening and deficient Ca handling as primary, interacting causes of genetic hypertension is proposed.

Pramanicin, an antifungal agent, raises cytosolic Ca2+ and causes cell death in vascular endothelial cells

The effects of a newly discovered antifungal agent, pramanicin, on cytosolic Ca(2+) and cell viability of cultured bovine pulmonary artery endothelial cells and on endothelium-dependent relaxation of dog carotid arterial rings were investigated by digital dynamic fluorescence ratio imaging and morphological and contractility studies, respectively. Pramanicin 100 microM, previously shown to cause maximal endothelium-dependent and NO-mediated vascular relaxation, induced a small transient elevation of cytosolic Ca(2+) concentration in Ca(2+)-free medium; subsequent introduction of 1 mM Ca(2+) caused a steady, nonsaturating increase of Ca(2+), which could be brought down to the basal level by the addition of EGTA. At the single cell level, the elevation of cytosolic Ca(2+) initiates from the cell periphery and progresses toward the central region. When added to the plateau phase of phenylephrine-induced contraction, pramanicin induced a slow endothelium-dependent relaxation, which could be reversed with the NO synthase inhibitor, L-NOARG. When preincubated with vascular tissue, pramanicin resulted in an irreversible loss of endothelial function characterized by the lack of carbachol-induced relaxation. Pramanicin caused cell injury characterized by plasmalemmal bleb formation, leading to cell death characterized by Trypan blue staining of the nuclei in cultured vascular endothelial cells in a concentration- and time-dependent manner. Such pramanicin-induced cell death was not associated with Ca(2+)-mediated or NO-mediated mechanisms. The time course of Ca(2+) elevation corresponds with that of pramanicin-induced relaxation of precontracted arterial rings, whereas the time course of endothelial cell death corresponds to that of pramanicin-induced loss of endothelial function as assessed by carbachol-induced relaxation. The pramanicin analogue, PMC-A, a by-product of the biosynthesis of pramanicin, in which the epoxy group is replaced by a CC bond, caused little endothelial-dependent relaxation, but it was able to cause endothelial cell dysfunction, albeit to a lesser extent compared to pramanicin, suggesting a role of the epoxy group in pramanicin for its vasorelaxant effect.

Alterations in beta-adrenoceptors and polyploidy in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats and Wistar-Kyoto rats

Objective: The relationship between the number of β-adrenoceptors and polyploidy in cultured aortic smooth muscle cells derived from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were examined. Design: The number of β-adrenoceptors, the percentage of multinucleated cells and the incidence of polyploidy from cultured smooth muscle cells derived from SHR and WKY rats aged 3-4, 10-12 and 28-30 weeks were measured. The effect of passaging of the cells on the expression of β-adrenoceptors and polyploidy on cultured smooth muscle cells from both SHR and WKY rats was also investigated. Methods: Receptor binding experiments were carried out using [125]-monoiodocyanopindolol with osmotically lysed cultured aortic smooth muscle cells to investigate the properties of vascular β-adrenoceptors in SHR and WKY rats. The proportion of polyploid smooth muscle cells was determined by frequency distribution analyses of Feulgen DNA microdensitometric measurements. Results: The incidence of polyploid smooth muscle cells was consistently higher in cells cultured from SHR than in those from WKY rats in all three age groups, with a positive correlation between polyploidy and age in SHR. Furthermore, in all three age groups the number of β-adrenoceptor binding sites was also higher in cultured smooth muscle cells from SHR than in those from WKY rats. There was no significant difference in the receptor affinity. The increase in β-adrenoceptor number was associated with an increase in polyploidy, and both of these changes were positively correlated both with the age of the rats from which these cells were derived and with the number of passages. Conclusions: Under cell culture conditions the expression of P-adrenoceptor density increases with the number of passages in both SHR and WKY rats. Smooth muscle cells derived from older SHR and WKY rats have a greater propensity to develop polyploidy. This trend is significantly accelerated in cultured smooth muscle cells derived from SHR compared with those from WKY rats, suggesting a premature ageing process. These findings suggest that, in cultured smooth muscle cells from SHR and WKY rats, β-adrenoceptors may influence the expression of polyploidy.