The (+)- and (−)-gossypols potently inhibit human and rat 11β-hydroxysteroid dehydrogenase type 2

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC₅₀) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC₅₀ was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.     

Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa

Gossypol, a known antispermatogenic agent, was found to effectively inhibit the highly purified boar sperm proacrosin-acrosin proteinase enzyme system by irreversibly preventing the autoproteolytic conversion of proacrosin to acrosin and reversibly inhibiting acrosin activity. The agent appears to prevent the self-catalyzed by not the acrosin-catalyzed activation of proacrosin. In additional experiments, brief exposure of human semen to concentrations of gossypol, which did not visibly alter spermatozoal motility or forward progression, was found to irreversibly inhibit the conversion of proacrosin to acrosin although the activity of the nonzymogen acrosin was not decreased, and also to prevent the human spermatozoa from penetrating denuded hamster oocytes. Gossypol inhibition of proacrosin conversion to acrosin closely paralleled the decline in oocyte penetration. Racemic (+/-) gossypol was equally as effective as the enantiomer (+) gossypol. The results suggest that the inhibition of proacrosin conversion to acrosin is a mechanism by which gossypol exerts its antifertility effect at nonspermicidal concentrations and that low levels of gossypol should be tested for their contraceptive action when placed vaginally.     

3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney: possible involvement in 11-deoxycorticosterone formation in situ

3 beta-Hydroxysteroid isomerase dehydrogenase, capable of acting on C21- and C19-3 beta-hydroxy-5-ene-steroids has been found in guinea-pig kidney at equivalent levels to those in guinea pig testes. Of the 3 beta-hydroxy-5-ene-steroids present in guinea pig serum, 21-hydroxypregnenolone occurs in highest concentration (17 nM) followed by pregnenolone (10 nM), whereas 17 alpha-hydroxy-pregnenolone and dehydroepiandrosterone occur in very low concentrations (less than 0.5 nM). Furthermore, the concentration of 21-hydroxypregnenolone relative to 11-deoxycorticosterone (the mineralocorticoid of the guinea pig), is 10:1 (Nishikawa and Strott, Steroids 41 (1983) 105-120). The apparent Km value for 21-hydroxypregnenolone, for the reaction yielding 11-deoxycorticosterone as catalysed by guinea pig kidney microsomes, was 85 nM and the Vmax 33 pmol/min per mg protein. Pregnenolone was a competitive inhibitor (apparent Ki = 5 microM) in the above reaction. A sex difference in the level of the enzyme in the kidney was found (activity in the female was one-third of that in the male) which may indicate that the enzyme is under partial androgen control. 3 beta-Hydroxysteroid isomerase dehydrogenase activity was also detected in guinea pig liver and again it was lower in the female. Whilst the exact role of 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney remains uncertain, the data suggest that it may utilise blood-borne 21-hydroxypregnenolone, the later then playing the role of a prohormone.     

棉酚对鼠类生精细胞LDH-X活性的影响

本文测定了连续饲喂棉酚达6周的大鼠和小鼠的生精细胞的LDH-X活性。结果表明,棉酚能够明显地抑制大鼠成熟精子的LDH-X活性;而对睾丸LDH-X活性的抑制,与对照相比,无显著性差异。在小鼠中,未发现棉酚对成熟精子及睾丸生精细胞中的LDH-X活性产生具统计学意义的抑制作用。本文结合精子发生过程及LDH-X的特殊功能,对棉酚抗生育作用的可能机理进行了讨论。     

Acute hypokalemic effect of gossypol

Clinical trials had demonstrated that chronic ingestion of gossypol induced infertility in males. Hypokalemia of various severities were reported in volunteers taking gossypol. The purpose of this study is to investigate the effects of acute gossypol infusion into alkalotic rats. Plasma potassium concentration decreased from 4.08 to 2.87 mM after gossypol infusion with minimal change in urinary potassium excretion. The hypokalemic effect of gossypol was also observed in nephretomized rats. Some of the factors that are known to affect potassium distribution between the extracellular and intracellular fluid compartment have been investigated in these rats. Plasma glucose concentration was not significantly altered. Gossypol induced hypokalemia was not blocked in rats treated with propranolol. It is therefore believed that the acute hypokalemic effect of gossypol is a direct shifting of potassium into cells.     

Influence of ketamine and propranolol on plasma renin activity (PRA) in female rabbits and guinea pigs.

1. Plasma renin activity was measured in non pregnant rabbits and guinea pigs under Ketamine-induced general anesthesia after pretreatment either with Propranolol or with a Placebo. Study was performed using a radio-immunoassay for angiotensin I. 2. Twenty minutes after the beginning of the observation period, renin activity in rabbits who had received Placebo alone (11.47 +/- 2.35 ng/ml/h) or associated with Ketamine (11.36 +/- 2.54 ng/ml/h) was similar. However, enzyme activity was significantly lower (P less than 0.001) when Propranolol was associated with Ketamine (3.97 +/- 0.58 ng/ml/h) or with Placebo (4.10 +/- 0.55 ng/ml/h). 3. In the same way, renin activity was significantly higher (P less than 0.001) in guinea pigs without Propranolol than in those who had received this drug. 4. These findings indicate that stress induced by general anesthesia with Ketamine or by simple manipulation of animals (Placebo) was accompanied by an excessive increase in plasma renin activity. Propranolol maintained the level of this enzyme activity within normal limits.     

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.     

Administration of isolated chicken L-gulonolactone oxidase to guinea pigs evokes ascorbic acid synthetic capacity

l-Gulonolactone oxidase was purified from chicken kidney microsomes in order to test whether this enzyme had potential advantages in our enzyme therapy studies. Chicken was selected because it has an enzyme that is structurally distinct from the enzyme in mammals and has high enzyme activity. An essentially homogeneous preparation of chicken l-gulonolactone oxidase is obtained by a seven-step procedure. Certain characteristics of this enzyme are presented. The enzyme was found to be quite unstable. However, immunoprecipitates of the enzyme are greatly stabilized. Therefore, this form was administered to young ascorbic acid-deficient guinea pigs that had been supplemented with l-gulonolactone. These animals showed a marked increase in plasma ascorbic acid concentrations.     

The effects of starvation and experimental diabetes on phosphoenol-pyruvate carboxykinase in the guinea pig.

1. Approx. 85% of liver phosphoenolpyruvate carboxykinase is associated with the mitochondrial fraction in the fed guinea pig. Enzyme activity is unchanged in diabetes, but doubles during starvation. In contrast with earlier reports, both cytoplasmic and mitochondrial activities were found to be increased. 2. In kidney cortex, total enzyme activity is increased in both starved and diabetic animals. These changes are associated with increases in the cytoplasmic activity alone. 3. In diabetic animals the mean blood-glucose concentration was 23.1 mM. Other blood metabolites were lower than those in the rat, and the animals did not show significant ketosis. 4. Changes in the rates of gluconeogenesis from lactate and propionate paralleled those in phosphoenolpyruvate carboxykinase activity.     

Differential binding of (+) and (-) gossypol to plasma protein and their entry into rat testis.

Concentrations of (+) and (-) gossypol were measured by high performance liquid chromatography after they were incubated with plasma proteins in vitro. The concentration of (-) gossypol decreased more than the concentration of (+) gossypol. A similar decrease in free gossypol concentrations in the blood plasma of rats was observed after intravenous infusion of gossypol enantiomers. The concentration of (-) gossypol was also found to be lower than the concentration of (+) gossypol at the blood-testis barrier. The biological effect of (-) gossypol probably results from its stereospecific binding to extra- and intracellular proteins in vivo and inhibition of the biological activity of some proteins.     

In situ structural analysis of microsomal UDP-glucuronyltransferases by radiation inactivation

The structure of the UDP-glucuronyltransferases in microsomes from guinea pig and rat liver was examined in situ by radiation inactivation analysis. The p-nitrophenol conjugating activity of guinea pig microsomes increased at lower doses of radiation; at higher doses (greater than or equal to 36 megarads), activity showed a first order decline yielding a target size of 71 +/- 9 kDa. Treating microsomes with Triton X-100 eliminated the activation seen at lower doses of radiation and yielded a simple exponential decrease in activity which gave a larger target size (95 +/- 18 kDa). A monoexponential decrease in activity was seen in sonicated microsomes, at greater than or equal to 36 megarads. The same response was obtained when the reaction was assayed in the reverse direction. The estrone conjugating activity of guinea pig microsomes was similarly activated at lower doses of radiation and declined at higher doses (greater than or equal to 36 megarads), with a target size of 57 +/- 11 kDa. Allosteric activation of the enzyme by UDP-N-acetylglucosamine was eliminated by lower doses of radiation. Thus, activation of the enzyme by radiation, detergent, sonication, and UDP-N-acetylglucosamine appear to be interdependent. These activations are postulated to be due to the existence of the enzyme in an oligomeric form which can be dissociated into monomers with higher activity. The same biphasic activation-inactivation curves were obtained for p-nitrophenol conjugation in rat liver microsomes. The target sizes were 54 +/- 8 kDa (p-nitrophenol in the forward direction) and 66 +/- 10 kDa (p-nitrophenol in the reverse direction). Thus, the enzyme appears to be smaller in rat liver as compared with guinea pig liver. Lithocholate glucuronidating activity in rat liver microsomes (at greater than 36 megarads) gave a target size of 74 +/- 1 kDa.     

Effect of Gossypol on Some Oxidative Respiratory Enzymes

Gossypol was examined in relation to its effect on certain enzymes and enzyme complexes associated with the tricarboxylic acid cycle and the electron transport system. Succinic dehydrogenase and cytochrome oxidase activity from sweet potato was completely inhibited by gossypol at 7.5 x 10(-3)m and 2.0 x 10(-3)m, respectively. Succinoxidase activity of the same preparations was fully inhibited at a lower concentration, 2.5 x 10(-4)m. This concentration did not affect either succinic dehydrogenase or cytochrome oxidase, the primary and terminal enzymes of the succinoxidase complex. The nature of the intermediate step or steps inhibited at this concentration is not yet known. Gossypol was further shown to inhibit phosphorylation at concentrations having no appreciable effect on oxidation. Inhibition in general was not reduced by increased substrate concentrations in the enzyme systems examined, with the exception of cytochrome c for cytochrome oxidase. Bovine serum albumin was partially effective in reducing gossypol inhibition, provided that it was present before enzyme exposure to gossypol.     

Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.     

Increased sensitivity of the enzymatic isotopic assay of histamine: measurement of histamine in plasma and serum.

The use of rat kidney instead of guinea pig brain as the source of histamine-N-methyltransferase for the enzymatic assay of histamine was found to improve the sensitivity of the assay. A partially purified preparation (ammonium sulfate fractionation) of the kidney enzyme was 20- to 50-fold more active than the guinea pig preparation, and sufficient enzyme for 14,000 assays could be prepared from six rats. The kidney enzyme, unlike the guinea pig brain enzyme, was free of interfering enzyme activities and gave low values for assay blanks. The two enzymes otherwise had similar properties. The low blank values permitted direct measurement of histamine in normal plasma without the need to isolate and concentrate histamine from the sample. Plasma histamine levels in normal individuals ranged from 0.2-1.4 (mean 0.6, n = 19) ng/ml.     

Aerosolized neutral endopeptidase reverses ozone-induced airway hyperreactivity to substance P.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on airway neutral endopeptidase (NEP) activity and bronchial reactivity to substance P in guinea pigs. Reactivity after ozone or air exposure was determined by measuring specific airway resistance in intact unanesthetized spontaneously breathing animals in response to increasing doses of intravenous substance P boluses. The effective dose of substance P (in micrograms) that produced a doubling of baseline specific airway resistance (ED200SP) was determined by interpolation of cumulative substance P dose-response curves. NEP activity was measured in tracheal homogenates made from each animal of other groups exposed to either ozone or room air. By reverse-phase high-pressure liquid chromatography, this activity was characterized by the phosphoramidon-inhibitable cleavage of alanine-p-nitroaniline from succinyl-(Ala)3-p-nitroaniline in the presence of 100 microM amastatin. Mean values of the changes in log ED200SP were 0.27 +/- 0.07 (SE) for the ozone-exposed group and 0.08 +/- 0.04 for the air-exposed group. We found that phosphoramidon significantly increased substance P reactivity in the air-exposed animals (P less than 0.01), but it had no effect in the ozone-exposed group. This finding was associated with a significant reduction in tracheal homogenate NEP activity of ozone-exposed animals compared with controls: mean values were 18.1 +/- 1.9 nmol.min-1.mg protein-1 for the ozone-exposed group and 25.1 +/- 2.4 nmol.min-1.mg protein-1 for air-exposed animals (P less than 0.05). Inhalation of an aerosolized NEP preparation, partially purified from guinea pig kidney, reversed the substance P hyperreactivity produced by ozone exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

棉酚对大鼠肾脏γ-谷氨酰转肽酶动力学的影响

本文应用动力学分析观察了棉酚对大鼠肾脏γ-谷氨酰转肽酶(γ-GT)的抑制作用。实验结果证实了棉酚在体外是大鼠肾脏γ-GT的抑制剂,而且抑制常数远小于r-GT的天然抑制剂——马尿酸。在不同浓度的棉酚作用下,改变双底物浓度,测定其活力并应用Lineweaver-Burk双倒数作图法,测得棉酚在两种底物情况下,对γ-GT的抑制作用均呈非竞争性抑制。     

Changes in phospholipase A2 in myometrium of the guinea pig uterus during pregnancy.

Phospholipase A2 activity has been measured in membrane and cytosolic fractions from non-pregnant and pregnant guinea pig myometrium has been studied. Enzyme activity was measured with 1-stearoyl-2- [3H]arachidonoyl-phosphatidylcholine exhibiting Michaelis-Menton kinetics with Km of 83.8 +/- 21.6 and 53.2 +/- 14.1 for membrane and cytosolic enzymes respectively. Fractionation of the myometrium from non-pregnant guinea pigs suggested that 35% of the activity was membrane associated compared with 20% (P < 0.01) in tissue from pregnant animals. In the presence of 1 mM calcium total activity rose from 3.03 +/- 0.41 to 1737 +/- 368 nmol/h per uterus between non-pregnant and late pregnancy. Calcium activated the membrane enzyme, but the effect was greater late in pregnancy with almost a 6-fold increase in activity at 1 mM calcium compared with a doubling in membrane from non-pregnant guinea pigs. The K0.5 for calcium activation was about 150 microM. Immunoblotting with anti-human-110 KDa phospholipase A2 showed in guinea pig uterus a 34 KDa form of the enzyme that, consistent with changes in activity, showed a fifteen-fold increase in quantity between non-pregnant and late pregnancy. The data are consistent with dramatic increases in the capacity for arachidonic acid release and prostaglandin production in the guinea pig myometrium late in pregnancy.     

Gossypol-induced inhibition of guinea pig sperm capacitation in vitro

The effect of gossypol acetate at various concentrations (10(-6) to 10(-4) M) on guinea pig sperm forward progressive movement, capacitation, and the acrosome reaction was explored in vitro. We found that 10(-4) M gossypol completely abolished the forward progressive motility of the sperm, and that this inhibition of motility was proportional to the concentration of gossypol used. Also, a dose-dependent decrease in acrosome reactions occurred with concentrations of the agent as low as 5.0 X 10(-6) M. However, we observed that such prevention of the acrosome reaction apparently happens at the capacitation stage rather than during the acrosome reaction itself. Inhibition of capacitation by gossypol was reversible--once the spermatozoa were capacitated in gossypol-free medium, the compound did not block the reaction.     

Gossypol prevents activation of purified proenzyme of human seminal plasma acidic proteinase

Gossypol inhibits the potential activity of the proenzyme form of human seminal plasma acidic proteinase, but has no effect on the active enzyme under the conditions tested. Inhibition of proenzyme is rapid and pH-dependent: 50% inhibition can be observed at gossypol concentrations of approx. 1.5 X 10(-5) M. SDS-polyacrylamide gel electrophoresis indicates that treatment of proenzyme with gossypol prevents the formation of active enzyme that normally occurs on acidification. Determination of free amino groups with 1-fluoro-2,4-dinitrobenzene suggests that gossypol reacts with 7.8 out of the 11.0 lysine residues in proenzyme: such a reaction could interfere with the activation process.     

Studies on a peptidase acting on a synthetic collagenase substrate Developmental pattern in rat granuloma tissue and distribution of enzyme in the tissues of various animals

The developmental pattern in experimental rat granuloma tissue and the distribution in the tissues of a few animals (monkey, rabbit, guinea pig anrat) of a peptidase acting on a synthetic collagenase substrate, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) has been studied. Maximum enzyme activity was found in 4-month-old rats and on the fourth day of implantation of the cotton wick. Pz-peptidase appears to have a ubiquitous distribution in animal tissues; the highest enzyme activity was generally found in liver, intestine and kidney of the animals. The total activity in other organs (spleen, heart, lungs and brain) was much less compared to that of liver, intestine or kidney.