Ching-Chow Chen

Antiproliferative effect of esculetin on vascular smooth muscle cells: possible roles of signal transduction pathways

The effect of esculetin, a coumarin derivative with lipoxygenase inhibitor activity, on the proliferation response of cultured rabbit vascular smooth muscle cells was studied. Proliferation response was determined by the uptake of tritiated thymidine. Esculetin (10(-5)-10(-4) M) dose dependently inhibited the enhanced proliferation stimulated by 5% fetal calf serum. The structure-activity relationship of esculetin and eight other coumarin derivatives indicates that two adjacent phenolic hydroxyl groups at the C-6 and C-7 positions in the coumarin skeleton are necessary for the potent antiproliferative effect. The antiproliferative effects of other lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid (ETYA) and ketoconazole, were comparable to the effect of esculetin. However, esculetin exhibited the greatest maximal suppression. The enhanced releases of 12-hydroxyeicosatetraenoic acid (12-HETE), prostaglandin E2 and 6-keto-prostaglandin F1 alpha in the culture medium of smooth muscle cells stimulated by 5% fetal calf serum were significantly reduced by esculetin. Furthermore, the fetal calf serum-stimulated protein tyrosine kinase activity was reduced by esculetin (10(-5)-10(-4) M) in a dose-dependent manner. In contrast, the protein kinase C activity stimulated by phorbol-12-myristate-13-acetate was not affected by esculetin (10(-6)-10(-4) M). These results suggest that the antiproliferative effect of esculetin on vascular smooth muscle cells may be partly mediated through inhibition of protein tyrosine kinase and modulated by inhibition of lipoxygenase.

Possible mechanism of immunosuppressive effect of scoparone (6,7-dimethoxycoumarin).

The possible mechanism of the immunosuppressive effect of scoparone (6,7-dimethoxycoumarin) was investigated. Human peripheral blood mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin (PHA) and the proliferative response was determined from the uptake of tritiated thymidine. Scoparone (10(-6) to 3 x 10(-4) M) reduced the proliferative response in a dose-dependent manner. The proliferative response of mononuclear cells to mixed lymphocyte reaction was also reduced by scoparone (10(-5) to 10(-4) M). Interleukin-1, interleukin-2 production and interleukin-2 receptor expression were all reduced in the presence of scoparone. Scoparone (10 and 30 microM) significantly reduced the suppression elicited by the diabetogenic drug, alloxan (10 mM). The suppressive activity of scoparone was significantly reduced by quinacrine (a phospholipase A2 inhibitor), indomethacin (a cyclooxygenase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor). The levels of prostaglandin E2, prostaglandin F2 alpha, leukotriene B4 and 2,3-dinor-thromboxane B2 in culture medium of PHA-stimulated mononuclear cells, measured with an enzyme immunoassay, were elevated by scoparone treatment. We compared the effect of scoparone on the mononuclear cell response to genistein, a specific inhibitor of protein tyrosine kinase and demonstrated the non-additivity and cross-desensitization of the two compounds. Our results suggest that the immunosuppressive effect of scoparone may be exerted in part through inhibition of protein tyrosine kinase and release of arachidonic acid metabolites.

Decreased NA+-K+-ATPase activity and [3H]ouabain binding sites in various tissues of spontaneously hypertensive rats

Na+-K+-ATPase activity and [3H]ouabain binding were studied in cerebral cortex, kidney and heart isolated from spontaneously hypertensive rats (SHR) in both the prehypertensive (6 week old) and the hypertensive stages (14 week old). Na+-K+-ATPase activity of heart and kidney was found to be decreased by about 38 and 16% in the prehypertensive and hypertensive stages of SHR respectively; that of cerebral cortex decreased by 23.5% only in the hypertensive stages. Similar results were obtained by pretreatment of membranes with either 0.001% Triton X-100 or by increasing the K concentration from 4.7 to 12.7 mM in the Krebs solution. No significant differences in microsomal protein yield were noted between prehypertensive or hypertensive SHR and the age-matched WKY rats. The study of binding of [3H]ouabain to cerebral cortex, kidney and heart showed that the decreased Na+-K+-ATPase in hypertensive SHR was due to a 31.6, 21.8 and 41.3% reduction in the number of high affinity binding sites respectively, while the affinity constants (Kd) of ouabain binding sites on this enzyme in cerebral cortex, kidney and heart of the normotensive WKY rats were 26.5, 455.9 and 74.7 nM respectively and those from the hypertensive SHR were not altered. The plasma K concentration of the SHR in the prehypertensive and hypertensive stages was 4.07 and 4.13 mM, respectively, significantly less than that of the age-matched WKY rats. It appears that the decrease of plasma K and Na+-K+-ATPase activity in heart and kidney in SHR is derived from a genetic defect and may be related to the abnormal Na handling in this genetically hypertensive strain.

Possible biphasic changes of free radicals in ethylene glycol-induced nephrolithiasis in rats: FREE RADICALS IN NEPHROLITHIASIS OF RATS

Objective To evaluate the possible role of free radicals in nephrolithiasis in rats induced by ethylene glycol, and to examine the correlation between the urinary enzymes N‐acetyl‐β‐glucosaminidase (NAG), β‐galactosidase (GAL) and neutral endopeptidase (NEP), and free radical production.

Renal response during acute unilateral ureteral obstruction in rats

The early renal response to unilateral ureteral occlusion (UUO) and its mechanism have been extensively studied in dogs but seldom discussed in the most frequently used laboratory animals, rats. The acute phase of the renal response to UUO was studied in female rats weighing 190–236 g. We recorded the ureteral pressure and changes in renal parameters throughout 120 minutes of UUO in control (US, UUO + saline, n = 10), L‐arginine‐treated (UA, n = 10), and right‐nephrectomized rats (UO, UUO in one kidney, n = 9). Ureteral pressure increased in all three groups of rats after complete ureteral obstruction. The extent of the increase was not significantly different between US and UA rats but was significantly higher in the UO rats. In US rats, the cortical microvascular blood flow (CMVBF), measured by a laser Doppler flowmeter, declined significantly, from 321 ± 10 perfusion units (PU) to 260 ± 11 PU. The percentage of drop in CMVBF at 120 minutes of UUO was significantly greater in UO (25.7 ± 3.8 %) than in US (19 ± 2.1%) and in UA (14 ± 2%) rats. Acute UUO reduced the glomerular filtration rate (GFR) in US and UO rats, whereas L‐arginine attenuated this decrease. The excretion of nitrate/nitrite was increased after UUO. Giving NG‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME, 12 mg/kg/h) during UUO did not reduce CMVBF more severely. Western blot analysis of endothelial nitric oxide synthase expression in the renal cortex and medulla protein extracts revealed no differences between US and sham‐operated rats. Acute UUO did not lead to renal hyperemia in rats. Reduction of nitric oxide during UUO might contribute to the decrease of renal circulation during UUO. Neurourol. Urodynam. 20:125–137, 2001.

Mode of inhibitory action of melittin on Na+-K+-ATPase activity of the rat synaptic membrane

The effects of melittin from bee venom, cardiotoxin from Formosan cobra venom, and ouabain on Na+-K+-ATPase activity of the synaptic membrane isolated from rat cerebral cortex were studied. Melittin was the most potent in inhibiting Na+-K+-ATPase activity. Mg2+-ATPase was less susceptible than Na+-K+-ATPase to the inhibitory action of toxins. High K+ (30 mM) reversed the inhibitory action of melittin on Na+-K+-ATPase but did not affect that of cardiotoxin. A comparison between the effects of ouabain and melittin was studied, using double-reciprocal plots of Na+-K+-ATPase activity against K+. It was shown that both were competitive with K+ for binding to the K+ site. Moreover, a median-effect plot revealed that ouabain and melittin antagonized each other when inhibiting Na+-K+-ATPase. Phosphatidylcholine (PC) was the only one of the phospholipids tested capable of protecting Na+-K+-ATPase from the inhibitory action of melittin but not that of ouabain. However, the inhibitory action of cardiotoxin on this enzyme was decreased by phosphatidylserine and sphingomyelin, in addition to PC. All of these findings suggest that the melittin polypeptide potently inhibits Na+-K+-ATPase, possibly by binding to the K+ site.

The renal, cardiovascular and hemolytic actions in the rat of a toxic extract from the bile of the grass carp (Ctenopharyngodon idellus)

Grass carp bile, sometimes eaten by Chinese people, has previously been shown to induce acute renal failure in man and to result in the death of experimental mice and rats. A toxic extract from the bile acid fraction of grass carp bile (LD50 109 mg/kg) was injected into pentobarbitone anesthetized rats and an increase in urinary excretion of water and salts was produced. The bile extract also caused a prompt fall in systemic arterial blood pressure and cardiac output. It was also found, in vitro, that the bile salts caused a potent hemolysis. The hypothesis that hemoglobin is released from hemolysed red blood corpuscles and might be responsible for the long term renal functional damage is considered. Another alternative hypothesis is that the hypotension induced by the bile toxin may result in renal underperfusion and thus make the kidney more susceptible to the nephrotoxic action of the grass carp bile.

Effects of β-bungarotoxin and phospholipase A2 from Naja naja atra snake venom on ATPase activities of synaptic membranes from rat cerebral cortex

Abstract Non-neurotoxic phospholipase A 2 of Formosan cobra venom possessed higher hydrolytic activity on phosphatidylcholine vesicles and also had higher inhibitory action on Na + -K + -ATPase and Mg 2+ -ATPase of the rat synaptic membrane than neurotoxic β-bungarotoxin of Formosan Krait Venom. Na + -K + -ATPase was more susceptible than Mg 2+ -ATPase to the inhibitory action of toxins, especially in the presence of Triton X-100. The inhibition of ATPases by toxins followed the Michaelis-Menton equation. It is interesting that various phospholipids and ions influenced phospholipase A 2 and β-bungarotoxin inhibition of ATPases. Sphingomyelin antagonized phospholipase A 2 more profoundly than β-bungarotoxin, while egg lecithin had the reverse effect. Both phosphatidylethanolamine and phosphatidylserine protected Na + -K + -ATPase from the inhibitory action of phospholipase A 2 but not that of β-bungarotoxin. High K + (30mM) did not affect, while Ca 2+ (0.2mM) decreased, the inhibitory action of phospholipase A 2 on Na + -K + -ATPase; in contrast, high K + reversed, and Ca 2+ increased, that of β-bungarotoxin. These findings imply that phospholipase A 2 and β-bungarotoxin may have different substrate specificities and prefer different conformational states of the membrane for binding. This may explain, at least in part, why β-bungarotoxin is neurotoxic, while phospholipase A 2 is not.

THE EFFECT OF ALTHESIN, KETAMINE OR PENTOTHAL ON RENAL FUNCTION IN SALINE LOADED RATS

1. Rats were prepared with chronic cannulae in the carotid artery, jugular vein and urinary bladder; they were then kept 2 days to allow recovery from surgery.

Differentiation of renal Na(+)-K(+)-ATPase in control and streptozotocin-induced diabetic mice by G-protein acting toxins and phorbol esters.

The specific activity of Na(+)-K(+)-ATPase in the renal medulla and cortex of 50-day-old streptozotocin (STZ)-induced diabetic mice was increased 58% and 50%, respectively, as compared to controls. Km values of Na+ and K+ for this enzyme were unaltered, while that of ATP was decreased in diabetic mice. The Na(+)-K(+)-ATPase in control medulla and cortex was activated by both cholera and pertussis toxins, while this effect was abolished in diabetics. Since dibutyryl cAMP stimulates cortical Na(+)-K(+)-ATPase activity in control mice, the activation effect of cholera toxin on this enzyme might be due to its interaction with a Gs-protein and the persistent stimulation of adenylate cyclase activity, while the effect of pertussis toxin might be due to its masking of the inhibitory action of a Gi-protein on adenylate cyclase activity. However, the protein kinase C (PKC)-associated Na(+)-K(+)-ATPase might also be quiescent in diabetes, because the stimulating effect of phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on this enzyme was abolished in diabetic cortex. In addition, nicardipine and ouabain were found to have differential effects on this enzyme derived from control and diabetic mice.