Differential effects of cyclic nucleotides and their analogs and various agents on cyclic GMP-specific and cyclic AMP-specific phosphodiesterases purified from guinea pig lung.

The differential effects of various compounds on the activities of cyclic GMP-specific phosphodiesterase (cyclic GMP-PDE) and cyclic AMP-specific phosphodiesterase (cyclic AMP-PDE) purified from the guinea pig lung were examined. Cyclic IMP, 2'-deoxy cyclic GMP and 2'-O-monobutyryl cyclic GMP were found to be the most potent inhibitors of the cyclic GMP-PDE, with I₅₀ (concentrations of the compounds inhibiting 50 per cent of the activity) values of 1.5, 4.5 and 35 μM respectively. These compounds, however, were at least 30–600 times less potent in inhibiting the cyclic AMP-PDE. In contrast, 2'-O-monobutyrul cyclic AMP and 2'-deoxy cyclic AMP were the most potent inhibitors of cyclic AMP-PDE, with I₅₀ values of 10 and 45 μM respectively; these compounds, however, were at least 30–100 times less potent in inhibiting cyclic GMP-PDE. Ethanol (13%, v/v) stimulated cyclic GMP-PDE 80 per cent while conversely inhibiting cyclic AMP-PDE 75 per cent. Most of the phosphodiesterase inhibitors studied were found to be more selective for cyclic AMP-PDE, with the possible exception of 1-methyl-3-isobutylxanthine, which was more specific for inhibiting cyclic GMP-PDE. The differential inhibition of the two classes of phosphodiesterases by a wide variety of compounds shown in the present study suggests a possibility of selective regulation of the tissue levels of respective cyclic nucleotides through specific or preferential inhibition of their enzyme activities.     

Covalent binding of prostaglandins G2 and H2 to components of ram seminal vesicle microsomal fraction.

Following incubation of 1-¹⁴C arachidonic acid with a microsomal preparation of ram seminal vesicles, 90 per cent of the products formed were classical prostaglandins and hydroxyfatty acid. In addition, approximately 8 per cent of the products formed possessed radioactivity which was covalently bound to the microsomal pellet after acid precipitation and solvent extraction. The amount of covalently bound material derived from arachidonate was related to the concentration of free prostaglandin (PG) endoperoxides in the incubation mixture, indicating that these reactive intermediates were probably responsible for the generation of the covalently bound material. Protein-associated radioactivity was also observed following incubation of ¦ 1-¹⁴C ¦PGG₂ or ¦ 1-¹⁴C ¦PGH₂ with a heat-denatured preparation of ram seminal vesicle microsomes. Such preparations do not convert PGG₂ to PGH₂; thus, the binding of PGG₂ was not due to its prior conversion to PGH₂. The rate constant for the covalent binding of PGG₂ (0.19 min^?1) was approximately twice that for PGH₂ (0.11 min^?1), suggesting that both 9, 11-endoperoxy and 15-hydroperoxy groups participate in the binding reaction. Moreover, the rate constant of binding derived from arachidonate (0.12 min^?1) approximated those of the purified endoperoxides. In an active microsomal preparation, glutathione reduced covalent binding and increased PGE₂ formation without greatly affecting oxygen consumption and, therefore, without affecting total product formation from arachidonic acid. Glutathione, therefore, probably inhibited covalent binding by lowering free endoperoxide concentrations in the incubation mixture. Diethyldithiocarbamate and phenylbutazone, but not para-aminophenol or butylated hydroxyanisole, inhibited the covalent binding of PGG₂ to heat-denatured tissue protein. These results suggest that the covalent binding reaction may proceed via the formation of free radicals.     

2-Acetylaminofluorene metabolism in rat liver microsomes: formation of 9-hydroxy-2-acetylaminofluorene and effect of hepatic enzyme modifiers.

9-Hydroxy-2-acetylaminofluorene [N-(9-hydroxy-9H-fluoren-2-yl)acetamide; 9-OH-2-AAF] was isolated and identified as a metabolite of 2-acetylaminofluorene (2-AAF) in rat liver microsomes. Evidence for this metabolite was provided by thin-layer chromatography (tlc), inverse isotope dilution analysis, pH partition, and nmr spectroscopic studies. 9-Oxo-2-acetylaminofluorene [N-(9-oxo-9H-fluoren-2-yl)acetamide; 9-OXO-2-AAF] was also detected and identified (by tlc) as a microsomal metabolite of 2-AAF. Microsomal conversion of 2-AAF to 9-OH-2-AAF and 9-OXO-2-AAF was dependent upon the presence of NADPH and was inhibited by a carbon monoxide atmosphere. Increased formation of 9-OH-2-AAF was noted in microsomes obtained from rats treated with the hepatic enzyme modifiers, phenobarbital and pregnenolone-16α-carbonitrile and inhibited in the presence of metyrapone. In contrast, treatment with 3-methylcholanthrene or chlordane did not modify 9-OH-2-AAF or 9-OXO-2-AAF formation. Also, rates of 9-OXO-2-AAF formation were increased only by prior administration of phenobarbital and were unaffected by metyrapone. These data provide evidence for the involvement of specific types of cytochrome P-450 in the 9-carbon hydroxylation reaction of 2-AAF.     

Direct extraction radioassay for catechol-Q-methyl-transferase activity

An improved radiochemical assay for catechol-O-methyl-transferase is presented that is particularly suitable for use with large numbers of samples. 3,4-Dihydroxyphenylacetic acid is converted to radioactively-labelled homovanillic acid in the presence of s-adenosyl-l-[methyl-³H]methionine. This product is extracted into an organic solvent-scintillant mixture and scintillation counting is performed without further manipulation.     

Examination of the metabolism in vitro of parathion (diethyl p-nitrophenyl phosphorothionate) by rat lung and brain.

These studies have indicated there is present in rat lung microsomes a mixed-function oxidase enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. In addition, analogous to previous results using rat liver microsomes, the sulfur atom released in the metabolism of parathion to paraoxon was found to covalently bind to lung microsomes. In contrast to liver, the metabolism of parathion by rat lung microsomes is not inducible by pretreatment of the animals with phenobarbital or 3-methylcholanthrene. The metabolism of parathion by lung microsomes is stimulated by NADPH and oxygen and is inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. There is also present in a particulate fraction of rat brain equivalent to microsomes an enzyme or enzyme system capable of metabolizing parathion to paraoxon and to diethyl phosphorothioic acid. The metabolism of parathion to paraoxon by rat brain microsomes is also accompanied by the release and covalent binding of the sulfur atom of parathion. The activity in rat brain microsomes is stimulated by oxygen and NADPH and inhibited by carbon monoxide, anaerobic conditions, SKF-525A and piperonyl butoxide. These data suggest that cytochrome P-450-containing mixed-function oxidase enzyme systems are responsible for the NADPH-stimulaled catalytic activity toward parathion found in rat lung and rat brain microsomes.     

Modulation of fluoropyrimidine metabolism in L1210 cells by L-alanosine

L-Alanosine, an analogue of aspartic acid which inhibits the conversion of inosine monophosphate to adenosine monophosphate (AMP), was evaluated in L1210 cells as a modulator of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism. L-Alanosine resulted in increased intra-cellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), enhanced FUra metabolism to ribonucleotide derivatives, and resulted in more FUra residues incorporated into RNA. Sequential addition of L-alanosine and FUra also resulted in synergistic cytotoxicity as determined by soft agar cloning. Adenine antagonized these biochemical and biological effects of L-alanosine. L-Alanosine also augmented the rate at which FUrd was metabolized and was also associated with a greater incorporation of the FUra residues into RNA. Cytotoxicity after sequential L-alanosine and FUrd was also synergistic. The mechanism by which L-alanosine altered the metabolism of FUrd, however, was different from the way in which it enhanced FUra metabolism in that aspartic acid and not adenine was able to reverse the effects of L-alanosine on FUrd metabolism and cytotoxicity. L-Alanosine appeared to augment the RNA-directed activity of FUra and FUrd in that there was no correlation between the enhanced metabolism and cytotoxicity of these two fluoropyrimidines and either levels of fluorodeoxyuridylate (FdUMP) which inhibits thymidylate (TMP) synthetase or inhibition of the ability of cells to incorporate deoxyuridylate into acid-precipitable material.     

Inhibition of dog brain synaptosomal Na+ -K+ ATPase and K+-stimulated phosphatase activities by long chain n-alkyl-amine and -piperidine, and N'-alkylnicotinamide derivatives

The effects of piperidine, primary amine, and nicotinamide aliphatic derivatives on dog brain snyaptosomal Na⁺-K⁺ ATPase were investigated. These derivatives inhibited the enzyme activity in a manner dependent on alkyl chain length. Kinetic studies revealed that inhibition of Na⁺-K⁺ ATPase activity by long chain alkyl derivatives (C₁₂-C₁₈) was biphasic and non-competitive with respect to the inhibitor and substrate (ATP) concentrations respectively. These long alkyl derivatives caused changes in the Hill coefficient that suggest the occurrence of a possible conformational change in the enzyme molecule. Dual-inhibitor experiments showed that both saturated and unsaturated pentadecylpiperidine (C₁₅-pip) derivatives inhibited Na⁺-K⁺ ATPase by the same mechanism. Oleylamine (C_18:1-NH₂), and N-dodecylnicotinamide (C₁₂-NA⁺Cl^?) derivatives apparently inhibited the enzyme activity by a mechanism different from that of piperidine derivatives. Low concentrations of C_15:1-pip, C_18:1-NH₂, and C₁₂-NA⁺Cl^? inhibited dog brain Na⁺-K⁺ ATPase activity only, but at higher inhibitor concentrations K⁺-stimulated phosphatase activity was inhibited as much as Na⁺-K⁺ ATPase. It is concluded that long chain n-alkyl derivatives of piperidines, amines, and nicotinamide have a cationic detergent-like action on Na⁺-K⁺ ATPase, possibly by the disruption of protein-phospholipid interactions. The mechanism by which these compounds inhibit the overall Na⁺-K⁺ ATPase may involve two different inhibitory sites: one, a high affinity inhibitory site, binding to which inhibits the Na⁺-stimulated phosphorylation reaction, and the other, a low affinity inhibitory site, binding to which inhibits the K⁺-stimulated dephosphorylation reaction. These possible mechanisms may provide an explanation for the observed biphasic inhibition kinetics.     

Stimulation of the glucuronidation of digitoxigenin-monodigitoxoside by liver homogenates from spironolactone-pretreated rats

Pretreatment of rats with spironolactone (SPL) produces an increased formation of the glucuronide conjugates of digitoxigenin-mono-digitoxoside (DMD) present in the bile after administration of digitoxin. For the digitoxin series of cardiac glycosides, DMD is the preferred substrate for in vitro conjugation with glucuronic acid by rat liver homogenates. The present experiments were performed to determine whether SPL pretreatment could produce an inceased glucuronidation of DMD by rat liver homogenates. Three different substrate concentrations (3, 30, and 100 nmol DMD/g of liver homogenate) were incubated under identical conditions with homogenates from control and SPL-pretreated rats. At 3.0-nmol. treatment with SPL produced a slight, but significant, increase in the production of aqueous alcohol-soluble metabolites of DMD by the experimental homogenates. With 30 nmol, treatment with SPL produced a 1.57-fold increase in the formation of aqueous alcohol-soluble metabolites of DMD. At 100 nmol, treatment with SPL produced a 3.44-fold increase in the formation of the aqueous alcohol-soluble metabolites of DMD. Treatment of these aqueous alcohol-soluble fractions (30-nmol substrate incubations only) with β-glucuronidase converted nearly 99% of the activity to a nonconjugate fraction. For both the control and the SPL-treated groups, greater than 92% of this nonconjugate fraction was determined by high-pressure liquid chromatography (HPLC) to be identical to the parent substrate (i.e., DMD). Thus, by selective enzyme treatment of the apparent conjugate and subsequent HPLC identification of the released products, the aqueous alcohol-soluble metabolites were identified as glucuronide conjugates of DMD. Pretreatment with spironolactone increased the rate of glucuronidation of DMD by rat liver homogenates.     

Analysis and differentiation of mechanism of tolerance to methadone in methadone-fed rats.

Rats fed methadone in drinking water (0.5 mg/ml) for 1 or 2 weeks developed tolerance to the analgesic effect of methadone as measured by the hot-plate method. This decreased analgesia was accompanied by an increased methadone N-demethylase activity measured in vitro and an increased methadone metabolism in vivo as judged by increases in the percentages of total ¹⁴C as ¹⁴C-water-soluble metabolites in the liver and urine 3 hr after administration of [¹⁴C]methadone. Two-day oral methadone administration and 4-day phenobarbital pretreatment also increased methadone metabolism both in vitro and in vivo and decreased the analgesic effect of methadone but not of morphine, indicating that increased methadone metabolism contributed to the development of tolerance to methadone analgesia (dispositional tolerance). Methadone-fed rats also developed cross-tolerance to morphine-induced analgesia. Two-week feeding caused tolerance to the analgesic effects of methadone and morphine to a greater degree. However, there was no significant difference between the increases in methadone metabolism produced by 1- and 2-week feedings. These results indicate that dispositional tolerance did not account for all of the tolerance seen after chronic methadone feeding. This suggests that chronic methadone feeding also caused cellular adaptive tolerance. This suggestion is further supported by the evidence that 4-day phenobarbital pretreatment, enhanced methadone metabolism more than 2-week methadone feeding, but the decrease in methadone analgesia caused by phenobarbital pretreatment was not as great as that caused by 2-week methadone feeding. It is concluded that tolerance to methadone analgesia as a result of chronic methadone feeding arises due to both increased metabolic inactivation of methadone and cellular adaptation to the drug in the brain.     

Effect of adrenochrome on calcium accumulation by heart mitochondria

The effects of adrenochrome (1–100 μg/ml or 5.6 × 10^?6 to 5.6 × 10^?4 M) on calcium binding, calcium uptake, and ATPase activities of rat heart mitochondria were investigated. Depression, by adrenochrome, of mitochondrial calcium binding and uptake was observed under in vitro conditions, whereas mitochondrial ATPase activity was not altered appreciably. The inhibitory effect of adrenochrome on calcium uptake activity was independent of the pH of the incubation medium, but it was dependent upon the dose of drug and the time of incubation. High concentrations of calcium in the incubation medium antagonized the adrenochrome-induced depression. The inhibition of mitochondrial calcium uptake by adrenochrome was of a mixed type. Furthermore, the depressed calcium uptake activity was observed after washing adrenochrome-treated mitochondria with buffer. Significant decreases in calcium accumulating activities were also seen in mitochondria isolated from hearts perfused with various concentrations of adrenochrome (5–50 μg/ml) for 10 min, or with 50 μg/ml adrenochrome for various time periods. Contractile force of the perfused rat heart with various concentrations of adrenochrome (5–50, μg/ml) decreased in a dose-dependent manner. It is suggested that the cardiac contractile failure and myocardial cell necrosis induced by adrenochrome may partly be due to its inhibitory effect on the calcium accumulating ability of mitochondria.     

Factors affecting the induction of DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a toxic contaminant in some preparations of chlorinated phenols, is a potent inducer of a number of enzymes including rat liver DT-diaphorase (EC 1.6.99.2). The present study has shown that the induction of DT-diaphorase by TCDD is prevented by prior administration of actinomycin-D. In addition, it has been shown that administration of TCDD brings about an increase in NADPH-diaphorase activity in a number of extrahepatic tissues of the rat. In contrast to the rat, the adult male guinea pig, the species most sensitive to the toxic effects of TCDD, exhibits little or no increase in DT-diaphorase activity in various tissues in response to TCDD administration. The compound 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD) is much less toxic than TCDD. OCDD was also shown to be much less potent as an inducer of rat liver DT-diaphorase than TCDD.     

Effects of hexachlorocyclohexane isomers on the mitogenic response of bovine lymphocytes.

The α-, β-, γ- and δ-isomers of hexachlorocyclohexane (HCH) were evaluated as modifiers of the mitogenic response in cultured bovine lymphocytes. None of the four isomers were mitogenic alone; however, the concurrent administration of αHCH and phytohemagglutinin-P (PHA) stimulated DNA synthesis in lymphocyte cultures 2.2-fold over that obtained with PHA alone. βHCH was inactive in the test system whereas both γ- and δHCH severely inhibited the PHA response. Treatment of lymphocytes with the microsomal enzyme inhibitor SKF-525A blocked the co-mitogenic activity of αHCH without affecting the basal PHA response. The role of hexachlorocyclohexane-sensitive metabolic systems in the regulation of mitogenesis in lymphocytes is discussed.     

Characterization of [3H]muscimol binding to mouse brain membranes.

The binding of [³H]muscimol {[methylene-³H(N)]-3-hydroxy-5-aminoethyl isoxazole} to a membrane fraction from mouse brain was studied as a possible model for the GABA recognition site of the γ-aminobutyric acid (GABA)-anionophore receptor complex. Kinetic studies showed two distinct association and dissociation rate constants, 2.3 × 10^?4 and 1.4 × 10^?5 sec^?1 nM^?1 for association and 1.2 × 10^?2 and 1.0 × 10^?3 sec^?1 for dissociation. Equilibrium analysis (Scatchard plot) of binding data also indicated two types of sites with K_D equal to 9 × 10^?9 and 70 × 10^?9 M. Detergents (0.1% Triton X-100, 0.1% Lubrol PX or 5% Tween 20) had no effect on the binding of [³H]muscimol or other conformationally restricted agonists such as imidazoleacetic acid, isoguvacine, THIP (4,5,6,7-tetrahydroisoxazole [5,4-c] pyridine-2-ol), and (±)-isonipecotic acid. In contrast to this, the detergents potentiated the ability of the less conformationally restricted agonists such as GABA and β-alanine in displacing [³H]muscimol. Binding of bicuculline was reduced by all of the detergent treatments.     

Cyclic AMP-dependent amino acid uptake in intestine--the importance of beta-adrenergic agonists.

Cyclic AMP (cAMP)-dependent stimulation of neutral and dibasic amino acid uptake by intestinal mucosa was investigated by the use of everted intestinal rings, β-adrenergic receptor sites on the mucosal cell itself were implicated, since epinephrine and isoproterenol stimulated uptake in vitro, which was blocked by propranolol. Glucagon, vasopressin and norepinephrine had no effect on tissue cyclic AMP levels or on amino acid uptake but cholera toxin given in vivo increased both parameters tested in vitro. Theophylline and isoproterenol caused an increase in cyclic AMP levels in the villus tip cells, where these drugs also stimulated amino acid uptake. Neither drug increased cAMP in crypt cells. Thus, the cyclic AMP-stimulated uptake of amino acids in vitro is a function of the villus tip cells. Moreover, when metabolic products, such as cAMP, are measured in whole intestinal mucosa, it cannot be assumed that the change is uniform throughout the tissue, but may involve only one type of cell on the intestinal villus.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

Renal functional correlates of methyl mercury intoxication: interaction with acute mercuric chloride toxicity.

To determine the effect of methyl mercury and its possible interaction with mercuric chloride on renal function, male Sprague-Dawley rats were treated with methyl mercuric chloride (1 mg/kg per day × 20 days ip) and/or mercuric chloride (1 mg/kg ip at Day 20) in a 2 × 2 factorial experimental design. Methyl mercury depressed urine osmolality and N-methylnicotinamide (NMN) uptake by renal cortical slices but did not affect the uptake of p-aminohippurate (PAH), blood urea nitrogen concentration (BUN), urine volume, or body weight. Urinary excretion of the lysosomal enzymes, β-galactosidase and acid phosphatase, appeared to be decreased, but excretion of the brush border enzyme, alkaline phosphatase, was not affected. Mercuric chloride treatment increased enzyme excretion, BUN, and uptake of NMN by renal cortical slices, while it decreased PAH uptake and urine osmolality, BUN concentration was further increased by combined treatment, yielding the only significant treatment interaction between methyl mercury and mercuric chloride. Prostaglandin E₂ synthesis and release by renal medullary tissue in vitro was not depressed by methyl mercuric chloride pretreatment nor was renal ammoniagenesis or gluconeogenesis. The effects of methyl mercury upon lysosomal enzyme excretion and NMN accumulation are suggestive of lysosomal and mitochondrial dysfunction. The failure to detect significant interaction between methyl mercury and mercuric chloride indicates that methyl mercury neither potentiates nor protects against acute mercuric chloride toxicity at this time and dose.     

Influence of some divalent cations on heart sarcolemmal bound enzymes and calcium binding.

The actions of Ni²⁺, Co²⁺ and Mn²⁺ on the rabbit heart sarcolemmal ATPases, calcium binding, and adenylate cyclase activities were studied. The ability of sarcolemma to hydrolyze ATP was stimulated by 0.1–4 mM concentrations of Ca²⁺, Mg²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal Ca²⁺ ATPase (22.8 if $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$) and Mg²⁺ ATPase (21.6 $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$ were depressed by 0.25–4 mM-Co²⁺, Ni²⁺ and Mn²⁺, and the order of their potency was Ni²⁺ > Co²⁺ > Mn²⁺. The sarcolemmal Na⁺-K ⁺ ATPase activity (9.4 $\text{μmoles P}{}_{\mathrm{i}}\text{mg}$ of $\text{protein}\text{hr}$) was decreased by 0.10–4 mM concentrations of Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal calcium binding in the presence of 0.1 mM Ca²⁺ (98 $\text{nmoles}\text{mg}$ of $\text{protein}\text{5 min}$) was depressed by 0.25 mM or higher concentrations of Co²⁺, Ni²⁺ and Mn²⁺, whereas that in the presence of 1.25 mM-Ca²⁺ (772 $\text{nmoles}\text{mg}$ of $\text{protein}\text{5 min}$) was decreased by 2–4 mM-Co²⁺, Ni²⁺ and Mn²⁺. The sarcolemmal adenylate cyclase activities in the absence (124 pmoles cyclic $\text{AMP}\text{mg}$ of $\text{protein}\text{min}$) and presence of 2 mM-NaFI517 pmoles cyclic $\text{AMP}\text{mg}$ of $\text{protein}\text{min}$) were decreased by 0.1–4 mM-Co²⁺ or Ni²⁺ and stimulated by 0.1–4 mM-Mn²⁺. The contractile force of the isolated rabbit heart was decreased by varying degrees by 0.1–1 mM of divalent cations (Ni²⁺ > Co²⁺ > Mn²⁺). These results indicate sarcolemma is one of the sites involved in the cardiodepressant actions of Ni²⁺, Co²⁺ and Mn²⁺.     

Actions of phenothiazine analogues on dopamine-sensitive adenylate cyclase in neuronal and glial-enriched fractions from rat brain.

Dopamine (DA) at 10^?4M readily activated adenylate cyclase in homogenates of neuronal and glial-enriched fractions prepared from rat cerebral cortex, thalamus, striatum and the total homogenate from the striatum. Several derivatives of phenothiazines were tested for their ability to modify either the control component or the DA-sensitive receptor moiety of the enzyme. Dihydroxy analogues of chlorpromazine (CPZ), prochlorperazine, perphenazine. promazine and 7.8-dioxo-CPZ exhibited the most potent antagonism of either basal or DA-induced activation of the enzyme. In some cases at lowest concentrations the basal activity of adenylate cyclase was enhanced by these dihydroxy compounds. Parent compounds and corresponding monohydroxy metabolites of CPZ, prochlorperazine, perphenazine and fluphenazine were less potent toward antagonism of control enzyme preparations, but nevertheless exerted rather powerful antagonism at the DA-sensitive receptor site of adenylate cyclase. In this regard, 8-hydroxy derivatives were somewhat more potent than respective 7-hydroxy derivatives. Likewise, prochlorperazine and corresponding analogues were overall the most potent compounds. The 7-methoxy derivative of CPZ, along with thiothixene. thioridazine and haloperidol, was observed to exert a weaker antagonism of the DA-sensitive enzyme. Weakest inhibitory actions on either control or DA-sensitive sites of adenylate cyclase were seen with promazine, 2-OH- and 3-OH-promazine, clozapine, promethazine. CPZ-SO and 7,8-diMeO-CPZ. Phenothiazine, 3-OH-phenothiazine and 2-Cl-7,8-dioxo-phenothiazine were without effect. These findings suggest that molecular actions of pharmacologically active phenothiazines within the central nervous system are not totally reflected by the parent compounds, but may instead be additionally manifested by one or more metabolites.