An orally effective, long-acting dopaminergic prodrug: (-)-10,11-methylenedioxy-N-propylnoraporphine

In rat vas deferens, (±)-170 150 proved to be an α₂-adrenoceptor blocking agent with a high $\text{α}{}_2\text{α}{}_1$ selectivity ratio In rat cerebrocortical membranes, (±-170 150 was seven times more potent than yohimbine for inhibiting specific [³H]clonidine binding. However, (±)-170 150 appeared to be 3.5 times less selective for α₂-adrenoceptors sites than yohimbine. These results indicate that (±)-170 150 is a potent preferential α₂-adrenoceptor blocking agent, as had been shown by in vivo experiments.     

Excretion of THC and its metabolites in ewes' milk

After single iv injections of either 0.02 mg/kg or 1 mg/kg of $\text{[}{}^{14}\text{C}\text{]Δ}{}^9\text{-}\text{tetrahydrocannabinol}$, [¹⁴C]THC, to lactating ewes, radioactivity was detected in the milk at all subsequent time intervals tested (4–96 hr). Radioactivity was found in unchanged THC as well as in various unidentified metabolites. Only about 15% of the administered radioactivity was excreted by the ewes in the first 48 hr; most of this was in the urine and feces. Radioactivity appeared in the feces and urine of a lamb suckling milk from a ewe injected with [¹⁴C]THC, indicating transfer of THC and its metabolites via the milk. These results confirm previous literature reports indicating slow elimination of THC, and show that milk is an additional route of excretion.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Mechanisms of accumulation of tyramine,metaraminol, and isoproterenol in isolated chromaffin granules and ghosts

The effects of the transmembrane pH gradient (ΔpH) and the transmembrane potential gradient (ΔΨ) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. biol. Chem.254, 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [¹⁴C]methylamine distribution and, in the presence of ATP, generate a ΔΨ of 80 mV, positive inside, as measured by [¹⁴C]thiocyanate distribution. When tryamine, metaraminol, and isoproterenol (1–50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5–21 μM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [¹⁴C]serotonin and [³H]isoproterenol was totally inhibited by reserpine, [¹⁴C]tryramine accumulation was inhibited by only 60% and [¹⁴C]metaraminol uptake was unaffected. The ATP-dependent generation of a ΔΨ produced a stimulation of amine uptake in the order: serotonin > isoproterenol > tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient $\text{(Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext><mtext>+</mtext>}}\text{)}$ and the electrochemical gradient for each of the sympathomimetic amines $\text{(Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>A</mtext>}}\text{)}$ was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of ΔpH alone. In the presence of ΔΨ alone, [¹⁴serotonin, [¹⁴C]tyramine, and [³H]isoproterenol were accumulated, but no [³H]metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the ΔpH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologie agents for their in vivo action are discussed.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

Distribution and excretion of 2,3,7,8-tetrachlorodibenzofuran in C57BL/6J and DBA/2J mice

The distribution and excretion of the toxic pollutant, 2,3,7,8-tetrachlorodibenzofuran (TCDF), was studied in male C57BL/6J and DBA/2J mice (22–29 g). [¹⁴C]TCDF was administered iv at a dose of 0.1 μmmol/kg. The liver was the major site of TCDF accumulation, with more TCDF in the livers of C57BL/6J mice compared to DBA/2J mice. TCDF had a half-life of approximately 1.8 days in the livers of both strains. At 7 hr and 1 day, respectively, radioactivity was redistributed to adipose tissue of C57BL/6J mice and DBA/2J mice. The terminal $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of TCDF in adipose tissue of C57BL/6J mice was 1.1 days, whereas it was 6.8 days in DBA/2J mice; the sixfold longer half-life in DBA/2J mice may be related to the approximately 36% greater adipose tissue content of this strain which may sequester more TCDF. More than 80 and 55% of the dose was excreted in the feces of C57BL/6J and DBA/2J mice, respectively, within 10 days as polar metabolites. The whole body half-life of TCDF was 2 days in C57BL/6J and 4 days in DBA/2J mice. Thus, DBA/2J mice sequester more of the TCDF dose in adipose tissue, accounting for a relatively slower rate of clearance and lower concentrations of TCDF at the putative target site(s) for toxic action.     

Effects of GABA receptor agonists on [3H] flunitrazepam binding to rat cerebellar and hippocampal membranes

The effects of GABA agonists on [³H] flunitrazepam binding were examined in membranes from CNS areas proposed to contain different populations of benzodiazepine binding site subtypes ($\text{BZ}{}_1\text{BZ}{}_2$). Since these effects were broadly similar in both cerebellar and hippocampal membranes, it seems unlikely that GABA receptors interacting with the proposed $\text{BZ}{}_1\text{BZ}{}_2$ binding sites haue markedly different properties. Sodium chloride clearly facilitated the potentiating effect of muscimol on [³H] flunitrazepam binding but produced a complex interaction uith the effects of GABA itself and the partial agonist imidazoleacetic acid in the phosphate buffer system used.     

Studies of the metabolism of N-methyl containing anti-tumour agents: 14CO2 breath analysis after administration of 14C-labelled N-methyl drugs, formaldehyde and formate in mice

The ¹⁴CO₂; content of the breath was analysed after administration of the following N-¹⁴ CH₃ labelled drugs to mice: aminopyrine, hexamethylmelamine (HMM), pentamethylmelamine (PMM), procarbazine and caffeine. Except for aminopyrine, the ¹⁴CO₂; exhalation rate plots declined monophasically with half lives of 91 min ([¹⁴C]-HMM), 97 min ([¹⁴C]-PMM), 68 min ([¹⁴C]procarbazine) and 92 min ([¹⁴C]caffeine). The ¹⁴CO₂ exhalation rate peaked rapidly after aminopyrine administration and declined bi-phasically with an initial $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 15 min and a terminal $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 126 min. The ¹⁴CO₂; plots after both [¹⁴C]-HMM and [¹⁴C]aminopyrine were influenced by pre-treatment of mice with proadifen. Pretreatment with phenobarbitone shortened the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the ¹⁴CO₂ appearance rate after [¹⁴C]HMM by 24% but did not change the ¹⁴CO₂ curve after administration of [¹⁴C] aminopyrine. The ¹⁴CO₂ exhalation rate plots after administration of H¹⁴CHO and H¹⁴COOH were virtually identical with that obtained after [¹⁴C]aminopyrine and not influenced by either proadifen or phenobarbitone pretreatment. The ¹⁴CO₂ exhalation rate profile obtained on metabolism of [¹⁴C]aminopyrine in mice thus appears to be determined by the rate of the oxidation of formaldehyde or formate to CO₂. Only 24% of the label injected with the N-methyl moieties of [¹⁴C]HMM and 21% of the label in [¹⁴C]procarbazine were exhaled as ¹⁴CO₂, whereas 49% of the N-¹⁴CH₃ in [¹⁴C]aminopyrine were metabolized to ¹⁴CO₂. It remains to be determined whether this difference and the difference in the shapes of the ¹⁴CO₂ exhalation profiles obtained with the cytotoxic N-¹⁴CH₃ drugs as compared to [¹⁴C]aminopyrine, are related to the biochemical processes mediating their antineoplastic activity.     

Muscarinic receptors in lung and trachea: Autoradiographic localization using [3H]quinuclidinyy benzilate

[³H]Quinuclidinyl benzilate binding to slide-mounted frozen sections of ferret lung was of high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{63 ± 14}\text{pM}\text{,}\text{mean}\text{±}\text{S.E.}\text{,}\text{n}\text{= 4}$), and characteristic of interaction with muscarinic cholinergic receptors. Light microscopic autoradiography showed muscarinic receptors to be localized predominantly to smooth muscle of trachea and intrapulmonary cartilaginous airways, and to submucosal glands. There was much less labelling of bronchiolar smooth muscle, airway epithelium and vascular smooth muscle and no labelling of alveoli. This distribution of receptors parallels that of cholinergic innervation.     

Tissue distribution of [14C]DDT in the lobster after administration via intravascular or oral routes or after exposure from ambient sea water

A pharmacokinetic approach to studying the fate and distribution of [¹⁴C]DDT was employed using the lobster, a species of obvious economic importance. The radioactive pesticide was administered by 3 different routes; intravascular, oral and by exposure from the ambient water. After intravascular administration there was very rapid removal of [¹⁴C]DDT from the plasma accompanied by a strikingly persistent increase in the amount of radioactivity in the hepatopancreas. Most (>90%) of the radioactivity in this organ was shown by TLC to be the parent pesticide. Seven days after injection of [¹⁴C]DDT approximately 90% of the administered radioactivity was found in the hepatopancreas and the concentrations in this organ decreased with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 46 days. One month after treatment with 0.1 mg/kg of [¹⁴C]DDT, the only other organs which contained more than 1% of the administered dose were egg masses and muscle. When the pesticide was administered to the lobster from ambient water or from food, the hepatopancreatic compartment again dominated, with more than 90% of the absorbed dose occurring in this organ 7 days after treatment. Studies conducted of residue levels in untreated lobsters indicated that the egg masses contained the largest concentration of total DDT metabolites (1 ppm). The hepatopancreas contained about 0.4 ppm while the carcass (muscle) contained about 0.1 ppm. These distribution studies suggest that while the lobster may protect itself from DDT toxicity by sequestering the pesticide in the hepatopancreas and in egg masses, bioconcentration in these tissue could be hazardous to species consuming these parts of the lobster.     

Drug-protein conjugates-VII: Disposition of [3H]-Ethinylestradiol-protein conjugates in the rat

[³H]17α-Ethinylestradiol ([³H]EE₂; 5 μg/kg, 98.5 μCi) was administered to a female rat. After 18 hr less than 0.02% of the dose was present per ml plasma. Approximately 60% of radioactivity present in plasma was irreversibly bound to proteins, as determined by exhaustive solvent extraction and by high performance ion exchange chromatography of proteins after removal of unbound metabolites with activated charcoal. After chronic administration of [³H]EE₂ (5 μg/kg; 2 μCi per day) for 22 days, there was a three- to fourfold accumulation of radioactivity in the plasma, together with an accumulation of radioactivity in the lung, liver, kidney, spleen and brain, compared to animals receiving a single dose. The spleen showed the greatest ( >tenfold) significant (P < 0.001) accumulation of radioactivity. There was a greater increase in radioactivity irreversibly bound to the soluble fraction than to the microsomal fraction of the liver. [³H]EE₂ was conjugated to rat serum proteins by incubation with rat microsomes in vitro. Upon administration to female rats, the [³H]EE₂-rat serum protein conjugate had a small volume of distribution (12.5 ± 0.5 ml), and its plasma concentration declined slowly $\text{(}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 450 ± 140}\text{min}\text{)}$. Immunization of male New Zealand White rabbits with a chemically synthesized conjugate of 2-hydroxyethinylestradiol (2-OH-EE₂) and human serum albumin produced antibodies which bound EE₂ and 2-OH-EE₂ but not estrone. These data indicate that although reactive metabolite formation represents a minor biotransformation, drug protein conjugates may accumulate during chronic administration.     

Uptake,distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri)

Uptake, distribution, and effects of CCl₄ were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl₄ in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl₄ residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of ¹⁴C residue(s) from the tissue samples were most rapid in muscle ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 1.7}\text{hr}$) and relatively prolonged for liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 38.9}\text{hr}$). Maximum liver concentrations of ¹⁴C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl₄ concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl₄.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin tissue distribution, excretion, and effects on clinical chemical parameters in guinea pigs

The tissue distribution of ¹⁴C-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult male guines pigs was studied up to 15 days following its ip injection (2.0 μg/kg). On Day 1, the highest levels of radioactivity (% of original dose/g tissue) were located in the adipose tissue, adrenals, liver, spleen, intestine, and skin. All other tissues examined contained less than 0.3%/g tissue. By Day 15, the level of radioactivity in the liver increased to nearly three times its initial value. An increase in radioactivity was also noted in the adrenals, kidneys, and lungs. These increases appeared to be due to the mobilization of fat stores and the subsequent redistribution of radioactivity contained in these stores to other organs. Following a single intraperitoneal dose of 0.5 μg [³H]TCDD/kg the excretion of ³H in the urine and feces appeared to be linear up to 23 days. Assuming the excretion of radioactivity would continue in a linear manner, the time for excretion of half the administered dose by way of the urine and feces was calculated to be 30.2 ± 5.8 days. The effect of TCDD (1.0 μg/kg) upon various clinical chemical parameters was determined periodically up to 14 days and compared to pairfed controls. Statistically significant increases in plasma albumin, total protein, iron, urea nitrogen, cholesterol, and triglycerides were observed in TCDD-treated pigs.     

Studies on the respiratory uptake and excretion and the skin absorption of methyl n-butyl ketone in humans and dogs

Methyl n-butyl ketone (MnBK) has produced peripheral neuropathy in experimental animals and is implicated in an occupationally produced neuropathy. Since occupational exposure to MnBK is by inhalation or skin contact, both the absorption and elimination of MnBK vapor and its absorption through skin were investigated. Studies were carried out first with male beagle dogs and subsequently with human volunteers. Humans exposed for 7.5 hours to 10 or 50 ppm or for 4 hr to 100 ppm of MnBK vapor absorbed between 75 and 92% of the inhaled vapor. Unchanged MnBK was not eliminated extensively in the postexposure breath or in urine. 2,5-Hexanedione, a metabolite of MnBK known to be neurotoxic in rats, was found in the serum of humans exposed to either 50 or 100 ppm of MnBK. The absorption and elimination of MnBK in dogs was similar to that observed in humans. The skin absorption of [1-¹⁴C]MnBK or a $\text{9}\text{1}$ ($\text{v}\text{v}$) mixture of methyl ethyl ketone $\text{(MEK)}\text{[1-}{}^{14}\text{C]MnBK}$ was determined by excretion analysis. Two volunteers exposed by skin contact to [1-¹⁴C]MnBK absorbed 4.8 μg min^?1 cm^?2 and 8.0 μg min^?1 cm^?2, respectively. Skin exposure to $\text{MEK}\text{[1-}{}^{14}\text{C]MnBK}$ resulted in the respective absorption of 4.2 and 5.6 μg min^?1 cm^?2 by two individuals. Two volunteers given an oral dose of [1-¹⁴C]MnBK (2 μCi; 0.1 mg/kg) excreted 49.9 and 29.0% of the dose, respectively, as respiratory ¹⁴CO₂ within 3 to 5 days and 27.6 and 25.0% of the dose, respectively, in urine within 8 days. Both [1-¹⁴C]MnBK and $\text{MEK}\text{[1-}{}^{14}\text{C]MnBK}$ were absorbed through the skin of dogs. These findings show that MnBK is readily absorbed by the lungs, the gastrointestinal tract, and through the skin, is not eliminated extensively unchanged in breath or urine, and is metabolized to CO₂ and 2,5-hexanedione. Radioactivity derived from [1-¹⁴C]MnBK was excreted slowly by man, suggesting that repeated daily exposure to high concentrations of MnBK may lead to a prolonged exposure to neurotoxic metabolites.     

Disposition and metabolism of [3H] cocaine in acutely and chronically treated monkeys

Monkeys were chronically treated with cocaine, 1 mg/kg subcutaneously twice daily for the first week and four times daily for the subsequent 21 weeks, followed by the same dose of [³H] cocaine injected intravenously. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of cocaine in the plasma, cerebrospinal fluid, liver, kidney and heart of acutely and chronically treated monkeys were 1.32, 0.85; 0.91, 0.75; 1.60, 1.29; 0.90, 0.91; and 1.02, 0.91 h, respectively. In areas of the central nervous system (CNS) the values for the acute and chronic group ranged between 0.75 – 0.90 and 0.62 – 0.84 h, respectively. With the exception of the temporal cortex, cerebellum and caudate nucleus, the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ were not significantly different in the two groups. Norcocaine, benzoylnorecgonine, benzoylecgonine, and minor amounts of ecgonine were the metabolites of cocaine in the brain of monkeys. Significant amounts of total radioactivity due to benoylnorecgonine and benzoylecgonine persisted in the CNS of chronically treated monkeys. Norcocaine constituted approximately 3, 21, 8, and 6% of the values of cocaine in the CNS areas in the acutely treated monkeys, and 14, 13, 14, and 16% in the chronically treated ones, 0.25, 0.5, 1 and 2 h after cocaine injection. A biphasic pattern of peak levels in the plasma and bile of chronically treated monkeys indicated enterohepatic circulation of cocaine. Brain to plasma and CSF to plasma ratios of cocaine were higher in the chronic than in the acute group. The amounts of cocaine excreted in urine and feces as a percentage of dose were 0.23 – 5.1 and 0.1 – 0.23 in the acute group, 0.54 – 7.0 and 0.1 – 0.14 in the chronic group. Major excretion of radioactivity occurred in urine within 24 h and the mean values of the total radioactivity in urine and feces (96 h) in the acute and chronic groups were 60.4 and 63.2% of the dose, respectively. Norcocaine, benzoylnorecgonine, benzoylecgonine, ecgonine methyl ester, ecgonine, and unidentified compounds were the urinary metabolites of cocaine in the two groups. The percentage of benzoylecgonine was higher, that of benzoylnorecgonine lower, in the chronic than in the acute group and the values of other metabolites were not markedly different in the two groups. Data support the postulate that chronic treatment of monkeys with cocaine does not produce dispositional tolerance.     

Effects of haloperidol and apomorphine on catecholamine metabolism in brain slices: Reserpine-like effects of haloperidol

The accumulation, release and catabolism of [³H]dopamine (DA) and [³H]norepinephrine (NE) synthesized from [³H]tyrosine were measured in mouse striatal and substantia nigral slices. Apomorphine inhibited both [³H]NE and [³H]DA accumulation $\text{(}\text{ic}{}_{50}\text{< 10}{}^{\mathrm{?6}}\text{M}\text{)}$, presumably by acting on a presynaptic receptor. Haloperidol (10^?8 M) caused a small, but significant increase in [³H]DA accumulation from [³H]tyrosine in the presence of 26 mM K⁺, possibly reflecting blockade of presynaptic receptors activated by released DA. However, at higher concentrations (10^?6 to 10^?5 M), haloperidol inhibited [³H]DA and [³H]NE accumulation. Reserpine also potently inhibited catecholamine synthesis; chlorpromazine had only a weak effect, and fluphenazine was ineffective. Both haloperidol (10^?5 M) and reserpine (10^?7 M), but not chlorpromazine and fluphenazine, markedly increased the formation of labeled dihydroxyphenylacetic acid (DOPAC) and increased the spontaneous release of labeled DA from striatal slices preloaded with [³H]tyrosine or [¹⁴C]DA. These data suggest that haloperidol has some direct effects on DA metabolism that are unrelated to DA-receptor blockade. Because the effects of haloperidol are apparently independent of DA release, haloperidol may elevate cytoplasmic DA by altering its vesicular storage. This would, in turn, increase the spontaneous release of labeled DA by diffusion, the oxidation of DA to DOPAC by monoamine oxidase, and the end-product inhibition of tyrosine hydroxylase.     

Characteristics of [3H]fentanyl binding to the opiate receptor

The present study characterises the binding of the highly lipophilic opiate agonist [³H]fentanyl to homogenates of the rat central nervous system. At 25°C, association of [³H]fentanyl with its binding site was rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.5}\text{min}$). Dissociation from the binding site was biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{'}\text{s}\text{= 4.0}\text{and}\text{100}\text{min}$) suggesting the existence of high and low affinity binding sites. Scatchard plots of saturation isotherms were curvilinear, confirming the presence of high (K_D = 0.46 nM) and low K_D = 4.26 nM) affinity binding sites. Increasing temperature and the concentration of sodium ion decreased the [³H]fentanyl binding. Opiate agonists, antagonists and mixed agonist-antagonists were all potent (IC₅₀'s < 20 nM) in displacing [³H]fentanyl and displacement by levorphanol and dextrorphan indicated that [³H]fentanyl binding was stereospecific. The μ and δ selective peptides, morphiceptin and [D-Ala²,D-Leu⁵]enkephalin, had IC₅₀ values of 87 and 9.2 nM respectively. The regional distribution of [³H]fentanyl binding was in the rank order striatum ? midbrain > hypothalamus > cortex > hippocampus > brainstem > spinal cord > cerebellum. Comparison of [³H]fentanyl, [³H]naloxone and [³H-d-Ala², d-Leu⁵]enkephalin binding in the hypothalamus-thalamus (μ-enriched) compared with the frontal cortex-striatum (δ-enriched) indicated that the pattern of [³H]fentanyl labelling was similar to that obtained with [³H]naloxone, but differed from that obtained with [³H-d-Ala²,d-Leu⁵]enkephalin. These characteristics suggest that [³H]fentanyl binds to the μ-opiate receptor. These findings are discussed in relation to the high lipid solubility of fentanyl as compared with morphine.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.