Search PubMed⌕ Search

Biomedical subjects

C Y Kwan

Publications and source records attributed to C Y Kwan.

At least 163 records · Page 9Linked to original sources
PubMed1988

Mechanism of inhibition by alloxan of ATP-driven calcium transport by vascular smooth muscle microsomes.

The direct in vitro effects of alloxan on the Ca2+ handling by microsomal membranes isolated from dog mesenteric arteries were investigated. Preincubation of the vascular muscle microsomal membranes with alloxan showed a suppressive effect on both binding of Ca2+ (in the absence of ATP) and ATP-driven Ca2+ transport. Such an inhibition was time dependent, dose dependent, and temperature dependent. ATP-driven Ca2+ transport was much more susceptible to the inhibitory action of alloxan than Ca2+ binding under all experimental conditions examined. Alloxan inhibited ATP-driven Ca2+ transport at a comparable level over the entire period of Ca2+ uptake, but had no significant effect on the efflux of Ca2+ from preloaded microsomal membranes. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via its action on the Ca-pump protein rather than the membrane permeability to Ca2+. Catalase and mannitol but not superoxide dismutase partially protected against such as inhibition by alloxan. The possible involvement of H2O2 mediating the inhibitory action of alloxan was further supported by the finding of a similar in vitro inhibitory effect of H2O2 on the ATP-driven Ca2+ transport by the vascular smooth muscle microsomes.

Adenosine Triphosphate↗
PubMed1988

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium.

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.

Animals↗
PubMed1988

Morphology and contractile properties of smooth muscle cells isolated from the dog carotid artery.

Collagenase and elastase treatment was used to isolate vascular smooth muscle cells from canine carotid artery. Their structure and function were compared to those in situ. Morphological studies showed that these cells when relaxed in situ were 120-133 micron mean length, connected by numerous typical gap junctions, covered by a basal lamina and like other smooth muscles in structure. After isolation, the median length of single cells was 82 micron. There was structural evidence of some contraction and the basal lamina was absent, but many structures were preserved. Cell clumps of 2-15 cells were often found; cells in such clumps often appeared to be all relaxed or all contracted. Isolated single cells contracted to KCl elevation or to norepinephrine up to 49 or 37% of initial length, EC50 values for contraction by norepinephrine and KCl were 0.4 microM and 40 mM, respectively; norepinephrine maximum contraction was about 35% less than that for KCl. Lightly loaded spirally cut strips from carotid artery were also studied. EC50 values for norepinephrine and KCl were 4 microM and 40 mM and unaffected by removal of the endothelium. Again, maximum contractions to norepinephrine were less than those to KCl. Contraction speeds were similar for isolated cells and intact strips. However, relaxation of maximally contracted isolated cells did not occur within 10 min. We conclude that canine carotid artery smooth muscle cells can be isolated with little structural or functional damage. The large number of gap junctions between cells and the tendency for cells to be isolated in small groups connected by gap junctions suggests that these cells would be useful for study of cell-to-cell coupling between arterial muscle cells.

Animals↗
PubMed1988

Alpha-2 adrenoceptors on nerves and muscles of rat uterus.

To test the hypothesis that functional postsynaptic alpha-2 adrenoceptors exist in rat myometrium, we examined whether specific binding sites for [3H]rauwolscine were present on microsomal membranes from myometrium of nonpregnant, day 16 pregnant and delivering rats and whether an alpha-2 adrenoceptor agonist has functional effects. The myometrium of rats at term undergoes a physiological denervation, confirmed in this study by ultrastructural examination of uterine samples and by [3H]saxitoxin binding studies. Binding sites for rauwolscine of similar Kd (11-15 nM) were present in all groups of myometrium and were localized on plasma membranes. There was no significant change in the density of rauwolscine binding sites in membranes from day 16 animals compared to nonpregnant ones, but a significant fall (38%) from these values at term. Strips of uterine circular or longitudinal muscle from nonpregnant, day-16 or day-22 pregnant rats failed to respond to the selective alpha-2 agonist, BHT-920, in the presence of propranolol; i.e., BHT-920 neither caused contraction nor inhibited contractions induced by oxytocin. BHT-920 did not affect the inhibitory effects of isoproterenol which were antagonized by propranolol. However, it antagonized contractions to norepinephrine in the presence of propranolol with a pKB value of 5.6 to 5.7. These contractions were phentolamine-sensitive. BHT-920 displaced rauwolscine from binding to all groups of myometria (IC50 = 2 to 3 x 10(-6) M) and displaced prazosin (Kd = 0.65 nM) from binding to myometria of nonpregnant rats (IC50 value congruent to 2 x 10(-4) M). Phentolamine also displaced rauwolscine from binding (IC50 = 2 x 10(-8) M). 5-Hydroxytryptamine displaced rauwolscine from binding only at higher concentrations (IC50 greater than 10(-5) M). We conclude that binding sites for alpha-2 adrenoceptor antagonists in myometrial microsomes were located primarily on smooth muscle plasma membrane. A smooth muscle alpha-2 adrenoceptor agonist appeared to occupy a site on muscle with the same affinity as it displayed toward rauwolscine binding site and competitively inhibited effects of an alpha-1 agonist. Our data suggest that alpha-2 adrenoceptor binding sites may exist on smooth muscle without coupling to contractile function, but their occupancy competitively prevents occupancy of alpha-1 agonist receptor activation sites.

5'-Nucleotidase↗
PubMed1987

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin.

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.

Animals↗
PubMed1987

Elastin and elastase-like enzyme change in aorta of rat with malignant hypertension.

In order to study the role of elastin in arteries with respect to hypertension and hypertensive arterial disease, aortic elastin content and elastase-like enzyme activity were examined and compared in stroke-prone spontaneously hypertensive rats (SHRSP), which show malignant hypertension, and Wistar-Kyoto normotensive rats (WKY). The elastin content was lower, whereas the elastase-like activity was higher at 20 weeks of age in SHRSP than in WKY, so that the aortic elastin/enzyme ratio of SHRSP was lower than that in WKY. These differences were not found at 6 weeks of age (prehypertensive stage). For SHRSP anti-hypertensive treatment resulted in lowering the elastase-like activity and in increasing the elastin content in comparison to untreated animals. The subcellular distribution of the elastase-like activity closely correlated with that of 5'-nucleotidase activity, a plasma membrane marker enzyme. The results indicate involvement of a smooth muscle plasmalemmal elastase-like enzyme in vascular connective tissue metabolism in health and possibly also its participation in hypertensive arterial diseases.

Aging↗
PubMed1987

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution.

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.

Acid Phosphatase↗
PubMed1987

Comparative studies of acid and alkaline phosphatase activities in nonvascular smooth muscle membranes.

Acid and alkaline phosphosphatase activities of subcellular fractions isolated from rat gastric muscle and vas deferens by differential centrifugation, sucrose density gradient and cation-induced aggregation methods were studied using p-nitrophenyl phosphate as the substrate. Alkaline phosphatase and a large portion of acid phosphatase activities were found to be of plasmalemmal origin. Acid and alkaline phosphatase activities were different in the effect of Mg2+, fluoride, vanadate, EDTA and resistance to heat inactivation suggesting that these two phosphatase activities were not expressed by the same enzyme.

Acid Phosphatase↗
PubMed1987

High resolution 23Na-nuclear magnetic resonance study of stroke-prone spontaneously hypertensive rat erythrocytes.

The intracellular Na+ content of washed erythrocytes from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) was measured by a high resolution 23Na-nuclear magnetic resonance (NMR) technique using a non-permeant aqueous shift reagent, dysprosium triethylenetetramine hexaacetic acid, Dy(TTHA)3-. The initial intracellular Na+ of freshly isolated and washed erythrocytes was very low (approximately 5 mmol/l) and increased progressively with prolonged incubation in isotonic salt solution at 37 degrees C. There was no significant difference in the erythrocyte Na+ concentration between SHRSP and WKY over the entire period of measurement, nor was any difference detected in their osmotic fragility or total cellular volume, although the osmotic fragility decreased with incubation time. The high energy phosphate metabolites were also studied in the same erythrocytes by 31P-NMR. The level of intracellular ATP decreased with incubation at 37 degrees C but showed no difference between the SHRSP and WKY samples. Inclusion of 1 mmol/l ouabain in the incubation medium substantially retarded the breakdown of intracellular ATP and resulted in a concomitant increase in intracellular Na+. However, neither the ouabain-sensitive nor the ouabain-insensitive component of Na+ influx altered in SHRSP erythrocytes compared with WKY erythrocytes in paired experiments. Our results do not support the hypothesis that altered Na+ transport, resulting in an increase in erythrocyte Na+ concentration, is associated with spontaneous hypertension.

Animals↗
PubMed1987

Neurotensin-metabolizing peptidases in rat fundus plasma membranes.

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.

Amino Acid Sequence↗
PubMed1987

Subcellular fractionation of the longitudinal smooth muscle/myenteric plexus of dog ileum: dissociation of the distribution of two plasma membrane marker enzymes.

The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain-sensitive, potassium-stimulated p-nitrophenyl phosphatase (K+-pNPPase)], [3H]saxitoxin binding, and 5'-AMPase, was studied in the subcellular fractions prepared from the homogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+-pNPPase activity and [3H]-saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+-pNPPase and [3H]saxitoxin binding, but not 5'-AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+-pNPPase activity and [3H]saxitoxin binding, the 5'-AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on density gradient resulted in copurification of 5'-AMPase but not K+-pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane-bound enzymes, such as high-affinity Ca2+-ATPase, Mg2+-ATPase, and Ca2+-ATPase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5'-AMPase activity is concentrated in the smooth muscle plasma membranes.

4-Nitrophenylphosphatase↗
PubMed1987

In vitro inhibition of calcium binding by alloxan and of calcium transport by isolated vascular smooth muscle microsomes.

The primary purpose of this study was to investigate the possible direct toxic effect of alloxan on the Ca2+ handling by microsomal membranes isolated from rat mesenteric arteries. It was found that preincubation of the vascular muscle microsomal membranes with alloxan had a suppressive effect on both binding of Ca2+ (in the absence of ATP) and ATP-supported Ca2+ transport. Such an inhibition was time, dose, pH, and temperature dependent. ATP-supported Ca2+ transport was more susceptible to the inhibitory action of alloxan than Ca2+ binding. Unlike alloxan, another commonly used diabetogenic drug, streptozotocin, was not effective in causing such an in vitro inhibition of Ca2+ handling.

Alloxan↗
PubMed1987

Beta adrenoreceptor in vascular smooth muscle with special reference to subcellular localization and number of binding sites.

The binding of monoiodinated cyanopindolol (ICYP) to membranes from canine and rat aortas, mesenteric arteries and mesenteric nerves has been studied. Binding of ICYP was shown by differential centrifugation and sucrose gradient purification as well as digitonin treatment to be directly related to the content of arterial muscle plasma membrane assessed by marker enzymes. The Kd values obtained were all in the range of 10 to 30 pM but maximum binding site (Bmax) values varied depending upon the extent of contamination by nonplasmalemmal membranes. The more purified plasma membrane fractions have Bmax many times higher than those reported in the literature. Although purified membrane fractions all had ICYP binding characterized by a single binding site with similar Kd values, binding to some crude fractions was complex. In addition, plasma membranes for mesenteric nerves, removed carefully by dissection in our study, were shown to contain ICYP binding sites with a similar Kd and Bmax values to those in arterial muscle membrane. Using arterial muscle purified membranes after removal of nerves by dissection, Bmax values from mesenteric arteries were demonstrated to be much larger than those from aorta in both species. Although the necessity of purifying the membranes depends on the objectives of a given experiment, our result strongly suggest that measurement of changes in receptor densities requires an analytical approach which provides plasma membrane-enriched fractions derived from a single cell type and purified to a known and comparable degree, especially when changes in density related to experimental or disease processes are being investigated.

Animals↗
PubMed1986

Properties of potassium activated p-nitrophenyl phosphatase of plasma membranes isolated from rat stomach muscle.

The plasma membrane-enriched fraction isolated from smooth muscle of rat gastric fundus was found to contain a substantial level of potassium-stimulated p-nitrophenylphosphatase activity (K-pNPPase), and its subcellular distribution closely resembled that of other plasma membrane enzyme markers. The kinetic profile of K-pNPPase and its sensitivity toward ouabain and vanadate confirmed the identification of this activity with the partial reaction of the sodium pump. The specific activity of K-pNPPase and its sensitivity to ouabain was significantly increased in the presence of saponin, indicating that part of this activity is latent when assayed on native membrane preparation. K-pNNPase was sensitive to the presence of calcium ions in the assay medium. The Ca2+-inhibition of K-pNNPase was accompanied by increased sensitivity of the enzyme to ouabain. On the other hand, calmodulin and Ca antagonists had no effect on K-pNPPase activity nor its sensitivity to calcium.

4-Nitrophenylphosphatase↗
PubMed1986

Cation-induced aggregation of membrane vesicles isolated from vascular smooth muscle.

Cations stimulated aortic muscle membrane aggregation with increasing potency according to their effective charge, e.g., K+ less than Mg2+ less than La3+, and the stimulation is reciprocally related to the apparent affinity for these cations. Divalent metal ion-induced membrane aggregation showed a dependence on the ionic radius, being optimal for Cd2+. Polyvalent cation-induced membrane aggregation was reversibly suppressed by high ionic strength as well as by metal ion chelators, irreversibly inhibited by the cross-linking agent glutaraldehyde, and enhanced by increasing concentrations of ethanol and increased temperature of the medium. When the pH is lowered below 6.0, membrane aggregation progressively increased with a concomitant decrease in cation-induced aggregation. The patterns of aggregation of microsomal membranes and further purified plasma membranes were almost identical whereas the aggregation of the heterogeneous mitochondrial membrane-enriched fraction was distinctly different in the initial rate of aggregation, its pH dependence, and metal ion concentration dependence. Our results indicate that cation-induced membrane aggregation can also be used to isolate a plasma membrane-enriched fraction from vascular smooth muscle.

Animals↗
PubMed1986

High affinity Ca-binding to smooth muscle plasma membrane: inhibition by cations.

The effects of Mg2+, Na+ and K+ on the pH-dependent high affinity passive Ca-binding to sarcolemmal enriched membrane fractions from the smooth muscles of rat myometrium and gastric fundus were examined at 1 uM Ca2+-concentration. For the myometrial plasma membranes, these ions inhibited the passive Ca-binding with the following IC50 values: Mg2+: 1.75 mM; Na+: 19 mM; K+: 233 mM. Similarly, for the gastric fundus membranes, the IC50 values were Mg2+:1.6 mM, Na+:46 mM and K+:67 mM.

Animals↗
PubMed1986

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).

Adenosine Triphosphate↗