Biochemical Studies of a New Class of Alcohol Dehydrogenase Inhibitors from Radix puerariae

Two potent, reversible inhibitors of human alcohol dehydrogenase; (ADH) isozymes were isolated from Radix puerariae (RP, commonly, known as kudzu root) and identified as the isoflavones daidzein and genistein. The 4-methoxy derivatives of daidzein (trivial name, for-mononetin) and genistein (biochanin A), minor constituents of RP, were also shown to be ADH inhibitors. All of these isoflavones inhibit 1 the human -γ₁γ₂-ADH isozyme competitively with respect to ethanol] and uncompetitively with respect to NAD⁺. A survey of more than 40 structurally related compounds revealed one more isoflavone (prunetin) and four flavones (7-hydroxyflavone, apigenin, galangin, and kaempferol) that inhibit ADH. The isoflavone inhibitors, however, are far more potent than the flavone inhibitors. Among the isoflavones studied, genistein is the most potent with K_i , = 0.1 μM toward γ₂γ₂- ADH. Human ADH isozymes differ in their sensitivity to these inhibitors in the order γ₂γ₂- , γ₁γ₁ > α₁α₁⁻,>⁻ > xx-ADH. These inhibitors, do not affect the β₁β₁ ,⁻ and β₂β₂ -ADH isozymes at concentrations as high as 20 μM. Rat and rabbit class I ADHs are also inhibited by these. isoflavone inhibitors. The 7-O-glucosyl derivatives of daidzein, genistein, formononetin, and biochanin A do not inhibit ADH, but are potent aldehyde dehydrogenase inhibitors.     

Potentiation of muscarinic and alpha-adrenergic responses by an analogue of guanosine 5''-triphosphate.

Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 microM) resulted in the full activation of both types of current. When 50-200 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 microM GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 microM). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate.     

Potentiation of inositol trisphosphate production by dexamethasone.

One of the mechanisms of glucocorticoid-induced hypertension has been thought to be the enhancement of vascular responsiveness to vasoconstrictors. In this regard, the effects of glucocorticoids on inositol trisphosphate production in vascular smooth muscle cells were studied. Angiotensin II and arginine vasopressin transiently increased inositol trisphosphate formation in a dose-dependent manner. Pretreatment with dexamethasone for 48 hours shifted the dose-response trisphosphate curves of angiotensin II- and arginine vasopressin-induced inositol trisphosphate production to the left, that is, it significantly reduced the half-maximal effective concentrations of angiotensin II (from 25 nM to 5 nM) and arginine vasopressin (from 50 nM to 25 nM). These effects of dexamethasone required a minimum of 12 hours of incubation; maximum effect was observed after 24 hours of treatment. A glucocorticoid antagonist, RU 38486, completely blocked these effects. To elucidate the interaction with prostaglandin, we used indomethacin, a potent inhibitor of prostaglandin synthesis. Treatment with indomethacin shifted the dose-response curves of angiotensin II- and arginine vasopressin-induced inositol trisphosphate production to the left. However, this shift was less than that seen after dexamethasone treatment. Indomethacin alone did not completely reproduce dexamethasone effects, and no additive effect between indomethacin and dexamethasone was observed. These results suggest, at least in part but not entirely, that the effects of dexamethasone depended on prostaglandin synthesis inhibition. We concluded that glucocorticoids altered the responsiveness of vascular smooth muscle cells to angiotensin II and arginine vasopressin through a glucocorticoid-specific receptor. These actions strongly support the mechanism by which the glucocorticoid induced hypertension through the increased sensitivity to vasoconstrictors.     

Potentiation of insulin release by glucose in man.

Glucose-induced potentiation of glucose-induced insulin release was quantitatively evaluated in 14 non-obese subjects with normal glucose tolerance but decreased insulin response, and in six non-obese patients with mild, adult-onset diabetes, by measuring the insulin responses to two consecutive glucose infusion tests, administered with 40 or 70 min interval. Enhancement of the second insulin response occurred in both groups. In low insulin responders, the dose-response relationship between blood glucose and plasma insulin was flatter and shifted to the right when compared to the control. Pretreatment with glucose increased strikingly the slope of this relationship, the responses now being within the normal range. The enhancement induced by glucose seems to be of multiplicative type. In mildly diabetic subjects, insulin response to glucose infusion was low and sluggish, only a minor initial response being observed. Pretreatment with glucose modified the profile of the insulin response, a clear-cut initial response of greater magnitude being obtained at least in some of the patients. The sensitivity of the islet to the potentiating action of glucose was higher in low insulin responders than in controls, the minimal glucose concentration needed to induce potentiations of the forthcoming response being much lower. The dose-response curve for the relationship between the blood glucose level of the preinfusion period and the percentual enhancement of the insulin response obtained at the second stimulation was, in low insulin response obtained at the second stimulation was, in low insulin responders, higher than and shifted to the left of the curve of the control subjects. In the group of diabetics, sensitivity for potentiation by glucose seemed not different from the controls.     

Potentiation of the ACTH response to metyrapone by L-dopa in the monkey.

The intravenous injection of L-Dopa (15 mg/kg) to monkeys (Macaca mulatta) failed to alter plasma concentrations of ACTH and of 11-deoxy-cortisol. When cortisol synthesis was blocked with iv metyrapone, potentiation of ACTH secretion by L-Dopa became apparent. Simultaneous injection of L-Dopa and metyrapone resulted in a marked increase in plasma ACTH from 93 +/- 18 pg/ml to 432 +/- 80 pg/ml, whereas plasma 11-deoxycortisol increased from 1.5 +/- 0.2 mug/100 ml to 14.6 +/- 1.0 mug/100 ml 90 min after treatment. Throughout the experiment the rise in ACTH and in 11-deoxycortisol following coadministration of L-Dopa and metyrapone was significantly (P less than 0.01) higher than that produced by metyrapone administration alone. The results suggest that acute administration of L-Dopa in monkeys enhances the response of ACTH to metyrapone. L-Dopa (or one of its metabolites) probably acts upon a noradrenergic or a dopaminergic system located in the hypothalamus to alter the release of hypothalamic corticotropin regulatory factor(s) and thereby enhance the release of ACTH.     

Potentiation of growth factor activity by exogenous c-myc expression.

The c-myc oncogene has been implicated in deregulation of cell growth in neoplastic cells and response to "competence-inducing" growth factors in normal cells. In the latter case, expression of c-myc has been shown to be associated with the transition from the G0 to the G1 phase of the cell cycle induced by platelet-derived growth factor (PDGF). In the work reported here, we have introduced the c-myc coding region, in a retroviral vector, into mouse and rat cells. We show that under conditions of anchorage-independent growth, constitutive c-myc expression increases the response of rodent cells to PDGF, as well as to other growth factors of both the competence-inducing and "progression" classes. These effects of the myc product are observed whether or not an exogenous ras gene has also been introduced into the same cells. Possible models for the influence of myc on growth responses are discussed.     

Potentiation of glucagon secretion by serotonin antagonists in man.

To study the possible implication of endogenous serotonin in the control of glucagon secretion in man, normal volunteers were subjected to alpha-cell stimulation before and after oral treatment with serotonin antagonists (cyproheptadine and methysergide) and with an inhibitor of serotonin synthesis (para-chlorophenylalanine, PCPA). After administration of cyproheptadine (16 mg daily, for two days) the glucagon responses to arginine (N=12) and to insulin-induced hypoglycemia (N=9) were more marked than in the control experiments (differences between maximal elevations: +165 pg/ml, P less than 0.0001, and +197 pg/ml, P less than 0.02, respectively). After methysergide treatment (9 mg daily, for two days), a potentiation of arginine-provoked glucagon secretion was also observed (+260 pg/ml, P less than 0.002; N=7). Similarly, after PCPA administration (2 g daily, for four days) the alpha-cell responsiveness to both aminogenic (N=12) and hypoglycemic (N=7) stimuli was enhanced (+108 pg/ml, P less than 0.05, and +164 pg/ml, P less than 0.05, respectively). Since glucagon secretion is potentiated by treatment with drugs which either antagonize serotonin action or inhibit its synthesis, the suggestion can be made that endogenous serotonin modulates alpha-cell function in man by acting as an inhibitor.     

Potentiation and inhibition of migration of human neutrophils by auranofin.

OBJECTIVES--As auranofin resembles some neutrophil activating sulphur containing compounds, it was decided to investigate whether it had activating effects on neutrophil migration in addition to the published inhibitory effects. METHODS--The Boyden chamber assay was used to determine the migration velocity of human neutrophils. The difference between chemotaxis and chemokinesis was established with a chequerboard assay. RESULTS--Low concentrations of auranofin stimulated human neutrophil migration; concentrations of auranofin higher than 1 mumol/l were inhibitory. Inhibitors of leukotriene formation, or of protein kinase C, had the same effect on auranofin induced potentiation of migration as on fMLP activated migration. Auranofin, at a concentration of 100 nmol/l, caused a transient increase in the cGMP level of neutrophils. The auranofin induced increase in migration was strongly inhibited by methylene blue and by LY83583, two inhibitors of cGMP accumulation. CONCLUSIONS--The auranofin induced enhancement of migration is partly due to a chemokinetic effect, but mainly due to a chemotactic effect. The potentiating effect of auranofin on migration is not specifically due to the ability of the drug to inhibit protein kinase C activity or to generate leukotrienes. These results suggest that the enhancement of neutrophil migration by low levels of auranofin is related to the enhancement of cGMP levels in neutrophils.     

Potentiation of antiherpetic activity of acyclovir by ribonucleotide reductase inhibition.

Compound A723U, a 2-acetylpyridine thiosemicarbazone, produced apparent inactivation of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase. Inactivation occurred after A723U formed a reversible complex with the enzyme and only while the enzyme was catalyzing the formation of deoxynucleotides. A723U inhibited HSV-1 replication at concentrations that were not toxic to the confluent host cells. Most importantly, A723U and acyclovir (ACV) were found to exhibit mutual potentiation of their antiviral activities. Subinhibitory concentrations of either compound greatly reduced the ED50 (median effective dose) of the other. Studies of the deoxynucleotide pool sizes and the levels of ACV triphosphate (ACV-P3) revealed that A723U not only significantly reduced the pool of dGTP but also increased the level of ACV-P3 in infected cells. The net result was an 80-fold increase in the ratio of ACV-P3 to dGTP. This should greatly facilitate the initial binding of ACV-P3 to HSV-1 DNA polymerase and probably accounts for the mechanism of potentiation.     

Effect of Scutellariae Radix extract on experimental dextran-sulfate sodium-induced colitis in rats

AIM： To investigate the effect of Scutellariae Radix extract （SRE） on ulcerative colitis （UC） in rats induced by dextran-sulfate sodium （DSS）. METHODS： Colitis was induced in male Sprague-Dawley （SD） rats （170-180 g） by 4% dextran sulfate sodium （DSS, wt/v, MW 54000） in drinking water for 8 d. The treated rats received 4% DSS and SRE orally （100 mg/kg per day）. Control rats received either tap water or SRE only. Macroscopic assessment which included body weight changes, fecal occult blood and stool consistency were determined daily. At the appointed time, the rats were sacrificed and the entire colons were removed. The colon length and the myeloperoxidase （MPO） activity were measured. The severity of colitis was graded by morphological and histological assessments. The ion transport activity of the colonic mucosa was assessed by electrophysiological technique. RESULTS： Rats treated with oral administration of 4% DSS regularly developed clinical and macroscopic signs of colitis. Treatment with SRE relieved the symptoms, including the reduction in body weight, shortening 2nd ulceration of the colon. Administration of SRE also significantly reduced the histological damage induced by DSS. Moreover, the Isc responses of the colonic mucosa to forskolin, were suppressed after the induction of colitis. The stimulated ion transport activity of DSS-rats treated with SRE displayed significant improvement in the secretory responsiveness. CONCLUSION： SRE was effective in treating acute DSS- induced ulcerative colitis, as gauged by reduced clinical disease, improved macroscopic and histological damage scores, and enhanced recovery of normal colonic secretory function.     

Potentiation of bleomycin toxicity by oxygen

The clinical usefulness of bleomycin is limited by pulmonary toxicity. Recent clinical observations indicate that the pulmonary toxicity is potentiated in patients who are exposed to elevated but nontoxic concentrations of O2. We studied the interactions of O2 and bleomycin using a murine model. Mice were divided into two groups. One group was continuously exposed to 40% O2 at 1 atmosphere while the other group breathed compressed room air. The elevated O2 concentration alone was not toxic to animals. Mice in each group received either 40 mg/kg of bleomycin or normal saline twice a week sc. Median survival of mice receiving bleomycin was shortened from 8.24 to 4.35 weeks when the animals were maintained on 40% O2 (P = 0.0001). We conclude that exposure to a nontoxic but elevated O2 concentration can potentiate the toxic effects of bleomycin.