Metyrapone Research Articles

The glucose paradox of cerebral ischemia – a possible resolution

The glucose paradox of cerebral ischemia, the aggravation of delayed neuronal damage by preischemic hyperglycemia, is a well‐documented phenomenon. Recently, we demonstrated that corticosterone (CT) plasma levels were significantly elevated within 30 min after glucose administration (i.p.) only to return to baseline levels by 120 min. In this study we examined the postulate that preischemic glucose loading aggravates the postischemic outcome via the induction of a short‐lived, massive elevation in CT plasma levels. Hence, we tested the ability of the CT synthesis inhibitor metyrapone (MT) and the glucocorticoid receptor antagonist, mifepristone (RU486), to attenuate the aggravated delayed neuronal damage in a rat model of cardiac‐arrest‐induced transient global cerebral ischemia (TGI). Seven different groups of 24‐h‐fasted rats were used and were administered several treatments prior to TGI that were aimed at either inhibiting CT synthesis (MT, 100 mg/kg, i.p.) or at blocking the brain glucocorticoid receptors (RU486, 40 mg/kg, i.p.). Plasma glucose and CT levels were measured 2 min pre‐TGI. Neuronal damage was assessed 7 days post‐TGI in hippocampal slices using electrophysiological means. Plasma glucose levels were not reduced in MT‐ or RU486‐treated rats. Both MT and RU 486 prevented preischemic hyperglycemia‐aggravated ischemic damage, the former presumably via inhibition of CT synthesis, the latter by glucocorticoid receptor antagonism. The present data strongly suggest that preischemic hyperglycemia‐aggravated neuronal damage, i.e. the glucose paradox, results from glucose‐induced CT release, rather than intensification of lactic acidosis in the ischemic brain.

Small changes in whole-body corticosterone content affect larval Rana pipiens fitness components

In amphibians, large changes in tissue corticosterone content (caused by treatment with large doses of hormone) alter tadpole growth and development, but the effects of smaller changes on growth, development, behavior, and morphology are unknown. In the current study, we exposed pre-metamorphic Rana pipiens tadpoles to moderate doses (62 and 125 nM) of exogenous corticosterone by adding it to the rearing water. We then analyzed effects on growth, development, behavior, morphology, and the endogenous corticosterone response to exogenous adrenocorticotropic releasing hormone (ACTH). A 50% elevation in whole-body corticosterone content was associated with slowed growth and development, increased tail muscle depth, and a diminished corticosterone response to ACTH. Behavior was unaffected by corticosterone administration. Treatment with the corticoid synthesis inhibitor metyrapone (MTP) reduced whole-body corticosterone content by 50% and was associated with increased size at metamorphosis but no change in time to metamorphosis. Our findings support the hypothesis that corticoids can mediate growth, developmental, and morphological responses of tadpoles to changing environmental conditions. Our results also demonstrate that even small changes in corticosterone content can have important implications for amphibian fitness.

Role for corticoids in mediating the response of Rana pipiens tadpoles to intraspecific competition.

Competition is known to decrease growth and development rate in tadpoles, but the physiological basis for this phenomenon is poorly understood. We hypothesized that competition results in increased production of stress hormones and that these hormones are responsible for the suppression of growth and development. To test this hypothesis, we measured whole-body corticosterone content in premetamorphic Leopard frog (Rana pipiens) tadpoles raised at two different population densities and three different food levels. Whole body corticosterone content was elevated in tadpoles subjected to either limited food (at low density) or high density. Within the low and intermediate food treatments, high density reduced tadpole growth and slowed development. Limited food slowed growth and development at all densities. Blocking corticoid synthesis by treating tadpoles with metyrapone (MTP) reversed the growth suppression caused by high density (tested in the intermediate food level treatment) but did not alter the effect of density on development rate. MTP treatment did not alter the depressive effect of limited resources on growth or development. Our results suggest that elevated corticoid biosynthesis mediates the negative effect of increased population density (i.e., increased intraspecific competition) on tadpole growth.

Cold-induced increment in rat adrenal gland type II deiodinase is corticosterone dependent.

In this study we analyzed whether corticosterone synthesis is involved in the regulation of adrenal gland type II deiodinase (AG-D2) activity during acute cold exposure. Two well-known inhibitors of steroidogenesis, aminoglutethimide (AGT) and metyrapone (MTP), were administered to male Wistar rats maintained either at room temperature or acutely exposed to cold (1 h at 4 degrees C). AG-D2 activity was measured by the radioiodide release method, and corticosterone circulating levels were measured by competitive protein binding assay. Results show that resting corticosterone levels and AG-D2 activity were lower in both AGT- and MTP-treated rats. Furthermore, the phasic increase normally exhibited by AG-D2 activity in response to acute cold stress was blunted in AGT- and MTP-treated animals. Therefore, we conclude that corticosterone synthesis is necessary in preserving the physiologic response of AG-D2 activity to cold exposure.

New mechanisms of adrenostatic compounds in a human adrenocortical cancer cell line.

Adrenostatic compounds are frequently used in the treatment of patients with Cushing's syndrome and act via direct inhibition of steroidogenic enzymes. However, additional mechanisms may be involved in the blockade of adrenal steroid secretion. We therefore investigated the effects of aminoglutethimide (AG), metyrapone (MTP) and etomidate (ETO) in the human NCI-h295 adrenocortical carcinoma cell line. Cells were incubated with increasing doses of the adrenostatic compounds. Steroid hormone secretion (cortisol, 17-OH-progesterone, DHEA-S) and cAMP synthesis were determined and Northern blot analysis and cell proliferation experiments were performed. ETO was the most potent adrenostatic compound inhibiting P450c11 hydroxylase at low concentrations (IC50 15 nM), and also blocking P450 side-chain cleavage (scc) enzyme (IC50 400 nM) at higher concentrations. The pattern of enzyme inhibition was similar for MTP with an IC50 of 3-5 microM for P450c11 and 17 microM for P450scc, while AG blocked P450scc with an IC50 of 10 microM. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 microM AG: 5% +/- 1%; 30 microM AG: 64% +/- 1%; 3 microM AG: 108% +/- 19% compared with control cells: 100% +/- 11%) but increased glucocorticoid receptor mRNA. The reduced ACTH-R mRNA was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of ACTH-R protein. The simultaneous incubation of hydrocortisone together with AG reversed the inhibitory effect of AG on the ACTH-R expression. AG and ETO inhibited cell proliferation in the NCI-h295 cells, but ETO was much more potent and showed antiproliferative effects at concentrations of 6 microM. The growth inhibition was not reversed by administration of hydrocortisone. Our data demonstrate that adrenostatic compounds not only act by suppression of steroidogenic enzymes but can also influence both ACTH-R expression and cell proliferation in adrenal cells. As these effects occur in vitro at concentrations that are reached during treatment with these drugs in patients, they are probably also of clinical relevance. Particularly the antiproliferative activity of ETO may be useful in Cushing's syndrome due to adrenocortical cancer. The interaction of steroidogenesis, ACTH-R and glucocorticoid receptor expression as well as cell proliferation provides a new concept of the intra-adrenal response to stress in humans.

Reduced cortisol potentiates the exercise-induced increase in corticotropin to a greater extent in trained compared with untrained men

We examined the effect of acute exercise and reduced cortisol on pituitary and adrenal responsiveness and the impact of reduced plasma cortisol on maximal oxygen consumption (V̇ o 2max) in eight trained (T) and eight untrained (UT) males. Subjects completed two graded maximal exercise tests (GXT), each preceded by either overnight metyrapone (MET) or placebo (PLA) administration. Blood samples were collected before and after GXT. With PLA, resting corticotropin (ACTH) levels were higher in T versus UT men; however, cortisol and 11-deoxycortisol were similar between groups. Following GXT on PLA, cortisol was unchanged but 11-deoxycortisol increased in both groups; however, ACTH increased only in UT men. For both groups, cortisol, 11-deoxycortisol, and ACTH were different post-GXT with MET versus PLA. Furthermore, following GXT with MET, the ACTH response was greater in T versus UT subjects. V̇ o 2max was not altered by MET in either group. We conclude that (1) at rest, only ACTH levels differed between T and UT men; (2) individually, the GXT and MET provide a similar ACTH response in UT but not in T subjects; (3) when GXT and MET are superimposed, they provide a stronger stimulus to pituitary and adrenal reserve than either test alone; (4) the combination of MET and GXT elicits a greater ACTH response in T compared with UT men; and (5) an acute reduction in plasma cortisol does not alter V̇ o 2max.

Metabolic Fate of Azasetron Hydrochloride. (IV). In Vitro Metabolism of Azasetron Hydrochloride by Rat and Dog Liver Microsomes.

塩酸アザセトロンの代謝物の排泄率における性差および種差の原因を明らかにする目的で，雌雄ラットおよび雄イヌの肝ミクロソームを用いてin vitro代謝試験を行い，各代謝反応に関与している酵素を明らかにするとともに，酵素活性を雌雄ラットと雄イヌで比較した．各代謝反応の進行にはNADPHが必要であった．イヌにおける窒素原子の酸化活性は，反応液のpHを7.4から8.5にすると著しく増加したが，加熱した酵素液を用いると著しく減少した．また，酸化活性を，メチマゾール(MTZ)が阻害したが，メチラポンはほとんど阻害しなかった．これらの結果は，イヌでは窒素原子の酸化反応をフラビン含有モノオキシゲナーゼ(FMO)が触媒していることを示している．ラットにおける窒素原子の酸化活性は，pHを7.4から8.5にすると著しく増加したが，加熱した酵素液を用いても40%以上の酸化活性が残存した．また，MTZ，メチラポンおよびSKF-525Aは，酸化活性を中程度に阻害した．これらの結果は，ラットでは，FMOとP-450が窒素原子の酸化反応に同程度関与していることを示している．N-脱メチル化活性およびM1の水酸化活性は，加熱した酵素液を用いてもほとんど減少しなかった．さらにこれらの活性をSKF-525Aおよびメチラポンは強く阻害した．したがって，P-450が，N-脱メチル化反応およびM1の水酸化反応を触媒している可能性が高い．続いて，アザセトロソの各代謝反応の肝臓での固有クリアラソス(CLint)を比較した。アザセトロンのN-脱メチル化反応のCLintは雄ラットと雄イヌで大きな差異がないので，この反応が種差の原因ではない．雄イヌでは，雌雄ラットと比べて窒素原子の酸化反応およびベンゼン環の水酸化反応のCLintが大きいので，これが排泄率における種差の原因である可能性が高い．また，雄ラットでは，雌ラットと比べ，アザセトロンのN-脱メチル化反応のCLintが大きいので，これが排泄率における性差の原因である可能性が高い．

Comparative induction of CYP3A and CYP2B in rat liver by 3-benzoylpyridine and metyrapone

3-Benzoylpyridine (3BP) is a major metabolite of HGG-12, an oxime that has been synthesized as a potential antidote to the toxic effects of soman and other anticholinesterases. Structural similarities exist between 3BP, the cytochrome P450 (CYP)-inducer metyrapone (MET) and other 3-substituted pyridines that interact with CYPs. The present study evaluated the regulatory effects of 3BP on CYP expression in rat liver. Both 3BP and MET (100 mg/kg) increased total hepatic microsomal holo-CYP content significantly 24 h after administration to male rats. Pronounced increases in activities mediated by CYP2B (androstenedione 16 β-hydroxylation and 7-pentylresorufin O-depentylation) were produced by 3BP and MET, which correlated with respective 9- and 14-fold increases in CYP2B immunoreactive protein. In addition, both agents slightly increased rates of microsomal CYP3A-dependent steroid 6 β-hydroxylation, troleandomycin metabolite complex formation and total CYP3A immunoreactive protein. Induction of the dexamethasone-inducible CYP3A23 mRNA to 4.5- and 2.5-fold of control was detected in liver of MET- and 3BP-induced rats; CYP3A2 mRNA levels were unchanged. Analogous in vitro studies revealed that MET was a preferential inhibitor of CYP3A-mediated steroid 6 β-hydroxylation activity, but 3BP was inactive against constitutive steroid hydroxylase CYPs. These findings indicate that the structurally related 3BP and MET elicit similar induction effects on CYPs 2B and 3A23 in rat liver after in vivo administration, but differential inhibitory effects of the chemicals on CYP activity in vitro. Recent reports have implicated a microsomal binding site in the induction of CYP3A1/3A23 in rat liver. In light of the present findings, substituted pyridines like 3BP may be useful tools in structure–activity studies to evaluate the physicochemical requirements for binding to this protein.

PITUITARY AND ADRENAL RESERVE: EFFECTS OF TRAINING AND ACUTE EXERCISE.

1564 We examined the effect of training status and acute exercise on pituitary and adrenal reserve and the impact of reduced plasma cortisol on VO2max in 8 trained (T) and 8 untrained (UT) males. Subjects completed 2 graded maximal exercise tests (GXT) each preceded by either an overnight metyrapone(MET) test or placebo (PLA) administration. Blood samples were collected before (PRE) and after (POST) GXT. PRE-PLA plasma adrenocorticotropin (ACTH) was higher in T compared to UT subjects, whereas POST-PLA plasma ACTH increased only in UT (P< 0.05). Additionally, T subjects had higher PRE-MET ACTH levels compared with POST-PLA values. PRE-MET plasma cortisol was attenuated ≈ 70% in both groups compared to PRE-PLA, whereas plasma 11-deoxycortisol and ACTH increased (P<0.05). POST-MET 11-deoxycortisol and ACTH increased compared to PRE-MET in both groups ( P <0.05), whereas plasma cortisol remained unchanged. Further, the POST-MET ACTH response was greater in T than in UT subjects. VO2max was not altered by MET in either group. We conclude that: 1) at rest training does not alter pituitary or adrenal reserve; 2) individually the GXT and MET provide a similar ACTH response in UT but not in T subjects; 3) when these two tests are superimposed, greater ACTH and glucocorticoid responses result compared to either test alone; and 4) an acute reduction in plasma cortisol does not alter VO2max.

The role of endogenous corticosterone in the late-phase response to allergen challenge in the brown Norway rat.

The aim of this study was to assess the role of endogenous corticosterone (CCST) concentrations on the late airway response (LAR) to ovalbumin (OA). Thirty-two Brown Norway (BN) rats were sensitized to OA on Day 0, then divided into three groups. Group 1 (n = 11) received saline (1 ml, subcutaneously) at time (T) = -24, -12, and 0 h prior to a 5% OA challenge on Day 14. Group 2 (n = 11) received metyrapone (MTP), an 11 beta-hydroxylase inhibitor, (10 mg/100 g in 1 ml saline, subcutaneously) at time (T) = -24, -12, and 0 h. Group 3 (n = 10) received MTP on the same schedule as Group 2, plus CCST in the drinking water (16 micrograms/ml) from T = -24 to T = 0 h. Pulmonary resistance (RL) was measured for 8 h following OA challenge to determine early (EAR) and LAR. Blood samples were taken at T = 0 and T = 8 h to determine serum CCST levels. Bronchoalveolar lavage (BAL) fluid was collected at T = 8 h. Serum CCST levels were significantly reduced in the MTP group (235 ng/ml +/- 14 SEM) compared with control (564 +/- 38, p < 0.0001) and MTP+CCST animals (349 +/- 19, p < 0.0005). There were no differences in either baseline RL or EAR between the groups. However, the MTP group had a smaller LAR (6 ml/cm H2O/s*min +/- 2 SEM) than the control group (19 +/- 5, p < 0.02). The effect of MTP on LAR was reversed by treatment with CCST (21 +/- 3, p < 0.005). Total cell counts (p < 0.05) and eosinophils (p < 0.01) were increased in the BAL fluid of MTP rats versus control and MTP+CCST animals. We conclude that depletion of endogenous CCST in the BN rat diminishes the LAR to allergen challenge. These results indicate that physiologic levels of CCST are not necessary for development of the EAR, but they play a permissive role in the LAR to inhaled allergen.

Enhancement of thiazopyr bioefficacy by inhibitors of monooxygenases

AbstractThe preceding paper described inhibition of thiazopyr metabolism in plant seedlings by inhibitors of cytochrome P450 monooxygenases, and the lack of inhibition by esterase inhibitors. We now describe greenhouse evaluation of the effects of these metabolic inhibitors on the bioefficacy of thiazopyr. Inhibitors of cytochrome P450 monooxygenases, piperonyl butoxide (PBO), 1‐aminobenzotriazole (ABT), metyrapone (MET) and tetcyclacis (TET), all enhanced the bioefficacy of thiazopyr against pigweed and other plant species. In contrast, inhibitors of esterases, tributyl phosphate (TBP) and triphenyl phosphate (TPP), produced only slight enhancement of thiazopyr activity. The effect of PBO was dose‐dependent and was demonstrated against barnyardgrass, grain sorghum, redroot pigweed, seedling johnsongrass, and giant foxtail. PBO demonstrated no enhancement of thiazopyr activity in velvetleaf, tall morningglory, cotton, or soybeans. Bioefficacy was most enhanced via exposure of seedling shoots to PBO and thiazopyr. The combination of results from the present and the preceding papers suggests that PBO enhances thiazopyr bioefficacy by effectively inhibiting thiazopyr metabolism in plants.

Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3α-hydroxysteroid dehydrogenase from Pseudomonas testosteroni

3α-Hydroxysteroid dehydrogenase (3α-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3α-HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co-substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3α-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3α-HSD. Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3α-HSD seem to be members of the short-chain alcohol dehydrogenase family.

Enalapril cytotoxicity in primary cultures of rat hepatocytes. I. Effects of cytochrome P450 inducers and inhibitors

Enalapril maleate (EN) incubated with primary cultures of rat hepatocytes was cytotoxic in concentrations of 0.5 mM or greater. Toxicity was measured by release of lactate dehydrogenase (LDH) into the culture medium at 24 h. SKF525A, α-naphthoflavone (αNF) and metyrapone (MTP) reduced the toxicity of EN. In vivo pretreatment with phenobarbital (PB) and β-naphthoflavone (βNF) had minor protective effects on the responses of hepatocytes to EN exposure. However, in vivo pretreatment with pregnenolonel6α-carbonitrile (PCN) substantially potentiated EN cytotoxicity. These results suggest that the cytocidal hepatotoxicity of EN in primary culture depends to some degree on metabolic activation by a cytochrome P450 species.

Cytochrome P-450-dependent metabolism in alveolar type II epithelial cells: modulation by platelet-activating factor

Platelet-activating factor (PAF), an ether lipid mediator released from activated pulmonary pahagocytes, weas evaluated for its ability to affect cytochrome P-450-dependent activities in isolated rat alveolar type II cells. The data indicate that at non-toxic doses, PAF caused an increase in β-naphthoflavone (BNF) inducible/α-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. A high concentrations of PAF, inhibition of both EtOPx'ase and metyrapone (MP) sensitive benzyloxyphenoxazone debenzylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAF analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF, PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in both intact cell and sonicated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type II cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.

The occurance of carbonyl reduction in continuous cell lines emphasizes the essentiality of this metabolic pathway

Using the ketone compound metyrapone (MPON) as a substrate for carbonyl reduction it has been verified for the first time that various permanent cell lines in culture express carbonyl reducing activity. This is even true for the dedifferentiated and fibroblasloid cell line V79, emphasizing the essentiality of this metabolic pathway. MPON reducing enzyme activities are located in the endoplasmic reticulum as well as in the cytoplasm of the cells. Compared to MPON-reductase in rat liver microsomes, no immunological homology to microsomal C2REV7 rat liver hepatoma cell MPON-reductase could be detected, indicating differences in antigenic determinants between the enzymes of the solid organ and respective cells in continuous culture.

Corticosterone, foraging behavior, and metabolism in dark-eyed juncos, Junco hyemalis

Male, dark-eyed juncos, Junco hyemalis, were held in captivity under conditions simulating winter temperature and photoperiod. Birds were administered corticosterone (B), metapyrone (MET), or control (empty) implants in silastic tubing. B implants resulted in higher plasma levels of B, especially on the first day following implantation. After 4 days, B-implanted birds had significant atrophy of the flight muscles and increased storage of fat in the furcular and abdominal adipose depots. Despite the increased fat deposition, no differences in adipose tissue lipoprotein lipase (LPL) activity or plasma levels of glycerol or free fatty acid were observed after 4 days of treatment. There were no differences in muscle LPL activity when enzyme activity was expressed per tissue. However, when enzyme activity was expressed per gram of tissue, muscle LPL was significantly greater in the B-treated birds. Therefore, despite the atrophy of muscle tissue, the LPL enzyme had been preserved. No differences were observed in either foraging behavior or food consumption, even in light of the dramatic changes in fat and muscle masses in B-treated birds. These data are discussed in light of the chronic effects of B which may be applicable to the advanced stage of stress in birds.

In vivo and in vitro Effects of Glucocorticoids on Lymphocyte Proliferation in Man: Relationship to Glucocorticoid Receptors

Interrelations between the hypothalamic-pituitary-adrenal system (HPA) and the immune system represent a well-documented biological phenomenon. While in vitro administration of glucocorticoids may inhibit concanavalin A (Con A)- and phytohemagglutinin (PHA)-induced T-cell proliferation, pokeweed mitogen (PWM)-driven B-cell mitogenesis is relatively resistant to glucocorticoids. To further explore the link between the HPA and the immune system in relation to glucocorticoid receptor function, dose-response curves were obtained for Con A- and PHA-induced T-cell mitogenesis, PWM-generated B-cell mitogenesis and spontaneous lymphocyte proliferation in 13 healthy controls. Glucocorticoid effects were assessed in vivo by depletion of endogenous glucocorticoids after oral administration of 1.5 g metyrapone (MET) and subsequent glucocorticoid replacement, and in vitro by incubation of the cells with different doses of dexamethasone (DEX). There was a significant decrease in PWM-induced B-cell mitogenesis and a more pronounced effect of DEX administered in vitro on spontaneous lymphocyte proliferation after MET treatment when compared with the DEX plus MET pretreated condition in vivo. These data suggest that the inhibition of spontaneous lymphocyte proliferation by glucocorticoids in vitro is related to glucocorticoid receptor function. The decrease in PWM-generated B-cell proliferation following cortisol depletion by MET may be seen in connection with impaired glucocorticoid-mediated induction of interleukin-1 receptor synthesis.

Lung cytochrome P450-dependent benzyloxyphenoxazone debenzylase and ethoxyphenoxazone deethylase activities in total microsomal and isolated alveolar type II cells: Responses to changes in assay conditions with special reference to non-linear dependence at low enzyme concentration

1. 1. Responses of cytochrome P450-dependent ethoxyphenoxazone deethylase (EtOPx'ase) and benzyloxyphenoxazone debenzylase (BzOPx'ase) activities to changes in assay conditions were measured in total microsomal and isolated alveolar type II (t 11) cells from rats pretreated with β-naphthoflavone. 2. 2. Whereas microsomal EtOPx'ase activity was unaffected by storage at −80°C for up to 4 months, BzOPx'ase activity began to decline after only 1 month. 3. 3. The microsomal and type II activities were unaffected by changes in pH (7.2–8.0) or salt content. 4. 4. The type II activities increased after sonication 2.3–2.7-fold or in the presence of 10 μM dicumarol 1.7–1.9-fold. 5. 5. Type II BzOPx'ase was sensitive to metyrapone (MP) whereas EtOPx'ase was sensitive to α-naphthoflavone (ANF). I 50, values for the tu activities were calculated as: 0.63 μM—MP (BzOPx'ase), 80 μM—MP (EtOPx'ase), 0.024 μM—ANF (EtOPx'ase). At the highest concentration of ANF (10 μM), 50% inhibition t 11, BzOPx'ase was not observed. The results were similar to those obtained with the total lung microsomal fraction. 6. 6. Microsomal and t 11 BzOPx'ase activities exhibited non-linear dependence at low enzyme concentration. Linearity was restored by 0.5 mM dimyristoylphosphatidylcholine.

The influence of adrenalectomy on α-adrenoceptor responses in rat cerebral cortex slices

Experiments were undertaken to examine the effect of depletion of adrenal steroids on neurotransmitter-stimulated cAMP accumulation in rat cerebral cortex slices. Long-term depletion due to bilateral adrenalectomy (ADX) and short term depletion due to the steroid synthesis inhibitor metopirone (MET) caused a significant increase in α1-adrenoceptor-mediated potentiation of β-adrenoceptor-stimulated cAMP accumulation. The effect of MET was prevented by corticosterone treatment. This effect of ADX was selective in that there was no change in α1-adrenoceptor-mediated inositol phosphate accumulation. In addition, ADX had no effect on potentiation of isoprenaline-stimulated cAMP by either the GABAB agonist baclofen or the phorbol ester, 4β-phorbol dibutyrate. However, 2-chloroadenosine-stimulated cAMP accumulation was significantly reduced in ADX rats.

Nature of endothelium-derived relaxing factor: are there two relaxing mediators?

The role of arachidonic acid in forming endothelium-derived relaxing factor remains controversial. This controversy may be explained if more than one factor exists. To test this hypothesis, the effects of various inhibitors of arachidonic acid metabolism were studied. The perfusate from canine femoral arteries with endothelium was bioassayed with coronary artery rings without endothelium. Treatment of the perfused segment (but not the bioassay ring) with inhibitors of phospholipase A2 (quinacrine) or cytochrome P450 (metyrapone) had no effect on the basal relaxing activity of the effluent; treatment with the inhibitor of lipoxygenase, nordihydroguaiaretic acid, significantly depressed it. With increasing concentrations of acetylcholine, a biphasic concentration-relaxation curve was obtained; the enzyme inhibitors depressed or prevented the first phase but did not affect the second phase. Infusion of arachidonic acid or soybean lipoxidase directly on the bioassay ring did not cause relaxation; together, they evoked concentration-dependent relaxations. These data suggest that acetylcholine can trigger the release of two chemically different relaxing mediators from the endothelium of the canine femoral artery. One factor may be a product of lipoxygenase (or epoxigenase). The second factor is not a metabolite of arachidonic acid and may be released under basal conditions. The existence of two (or more) chemically different endothelium-derived mediators may help to explain the controversial data regarding the nature of the factor(s).