Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1966"

p-CHLOROPHENYLALANINE: A SPECIFIC DEPLETOR OF BRAIN SEROTONIN

Abstract: p -Chlorophenylalanine has been found to be a potent and selective depletor of brain serotonin (5HT) in mice, rats and dogs. Brain 5-hydroxy-3-indolylacetic acid (5HIAA) content was also depleted by the drug, but catecholamine concentrations were only slightly decreased. Peripheral stores of 5HT were also lowered. In rats, p -chlorophenylalanine reduced the normal increase in brain 5-hydroxyl-3-indolyl compounds following L-tryptophan loading (without apparently affecting tryptophan uptake into brain), completely prevented the increase in brain 5HT accompanying inhibition of monoamine oxidase by pargyline and blocked the increase in brain 5HIAA usually observed after reserpine treatment. p -Chlorophenylalanine slightly diminished the usual increase in brain 5HT in rats following 5-hydroxytryptophan (5HTP) administration, but decreased the rate of disappearance of excess 5HT and antagonized the increase in brain 5HIAA. p -Chlorophenylalanine did not inhibit monoamine oxidase or 5HTP-decarboxylase in vitro and exerted no effect on monoamine oxidase or 5HTP decarboxylase activity of rat tissues in vivo. In contrast, p -chlorophenylalanine inhibited liver tryptophan hydroxylase in vitro and strongly suppressed the tryptophan- and phenylalanine-hydroxylating capabilities of livers of rats treated with it. These results suggest that p -chlorophenylalanine may effect 5HT depletion by inhibiting the biosynthesis of this monoamine, possibly by blocking tryptophan hydroxylation. A blockade of uptake of amino acid precursor might also contribute to the effect of decreasing 5HT biosynthesis. The slow depletion (2-3 days) of brain 5HT induced by p -chlorophenylalanine suggests that an active metabolite might be formed. p -Chlorophenylpyruvic acid exerted essentially the same pharmacologic effects as the amino acid, but it cannot be ascertained at present whether it is the active metabolite because of the interconversion of α-amino acids and α-keto acids in vivo. p -Chlorophenethylamine may be excluded as the metabolite responsible for the action of p -chlorophenylalanine because of the brief duration of the amine in brain and the short lasting, nonselective decrease of both 5HT and norepinephrine produced by the amine. A study of structural variation in the phenylalanine series indicated a specific requirement of a single chlorine substituent in the para position for potent in vivo activity. Rats treated with p -chlorophenylalanine displayed few apparent signs, and certainly not sedation. p -Chlorophenylalanine did not block characteristic signs elicited by reserpine or tetrabenazine in rats. Accordingly, the central actions of reserpine and reserpine-like drugs may possibly be dissociated from both 5HT concentrations and the formation of new 5HT in brain.

Application of steady state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines

Abstract: A method of measuring the rate of synthesis of catecholamines in various tissues takes advantage of the steady state relationship in which the rate of catecholamine formation is equal to the rate of efflux. After tyrosine hydroxylase is blocked with α-methyltyrosine, the brain levels of norepinephrine (NE) and dopamine and the NE levels in various peripheral tissues of rats and rabbits decline exponentially. The rate of synthesis of the catecholamines is calculated from the product of the rate constant of amine decline and the normal catecholamine level. The value for heart NE yielded by this method is almost identical with that obtained from the decline in radioactivity after labeling with dl -H3-NE. The application of this method to studies in the rat shows that the turnover time for NE in heart, salivary gland and iris is about 12 hr, compared to 6 hr for brain NE, 3 hr for brain dopamine and 2 hr for NE in the cervical sympathetic ganglia. In the rabbit hypothalamus, NE is formed five times more rapidly than in the midbrain, though the turnover times are almost identical. This suggests that the rate of synthesis might be similar in each adrenergic unit and that variations in rates of synthesis in different brain areas are a function of the number of neurons per gram of tissue.

Antiamphetamine effects following inhibition of tyrosine hydroxylase

Abstract: (α-MT), a potent inhibitor of tyrosine hydroxylase, antagonizes the hyperactivity, the sniffing-licking-gnawing syndrome, the anorexia and the nondiscriminated avoidance rate-stimulating effects of amphetamine, as well as the syndrome produced by 2 amphetamine-like drugs. Theseeffects of α-MT appear to be correlated with in vivo inhibition of tyrosine hydroxylase, since other inhibitors, notably m -iodo-L-α-methyltyrosine and m -bromo-L-α-methyltyrosine, also antagonize amphetamine. The gross norepinephrine depletion produced by α-MT may be related to its antiamphetamine effect, but gross norepinephrine depletion is not itself sufficient for amphetamine antagonism since amine depletors such as DL-α-methyl- m -tyrosine (α-M- m -T) do not antagonize amphetamine. The behavioralprofile of α-MT appears unique: α-MT differs from chlorpromazine in not profoundly disrupting avoidance behavior and in not altering symptoms produced by intravenous norepinephrine, tryptamine and apomorphine or by a monoamine oxidase (MAO) inhibitor + either 3,4-dihydroxyphenylalanine (dopa) or α-M- m -T. It differs from phenobarbital in that α-MT exertsno anticonvulsant effect and is not an effective sedative. Its antiamphetamine action clearly distinguishes α-MT from norepinephrine depletors which act by a release mechanism. The antiamphetamine effects of α-MT and other tyrosine hydroxylase inhibitors suggest that a critical level of norepinephrine at the receptor is required for amphetamine to exert its customary effects. It is speculated that this level derives from a functional pool of norepinephrine in thecentral nervous system, highly susceptible to blockade of norepinephrine biosynthesis at the tyrosine hydroxylase step.

Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold.

Abstract: When rats were exercised or exposed to cold, the levels of norepinephrine in heart, spleen and brain showed little change. Appreciable decreases were observed in adrenal epinephrine following severe exercise. However, when the tyrosine hydroxylase inhibitor, α-methyl-tyrosine, was administered prior to the exercise or exposure to cold, norepinephrine and epinephrine levels fell markedly in most tissues. Furthermore, incorporation of radioactivity from administered tyrosine-C14 into norepinephrine and epinephrine was increased 2-to 3-fold as a result of exercise. These findings indicate that the increased sympathetic stimulation, presumably associated with such stressful conditions, induces increased synthesis of norepinephrine and epinephrine. The regulatory mechanism most likely operates at the tyrosine hydroxylase step, which is ratelimiting.

Studies on the absorption, distribution and excretion of indomethacin in various species

Abstract: Indomethacin is promptly and completely absorbed after oral administration. The half-life of the drug in plasma varies among species, ranging from 20 min to 4 hr. Most of the dose is eliminated from the body in 24 hr, the route of excretion varying among species. The drug is extensively excreted in the bile, largely reabsorbed and excreted in the urine in most species except the dog, in which reabsorption of biliary material does not result in eventual urinary excretion of the drug. Only minimal amounts of free drug are found in the urine. No evidence was obtained for accumulation of the drug in man. Little passage into cerebrospinal fluid takes place. Indomethacin is concentrated in various tissues of the guinea pig, but not of the rat. The drug is highly bound to plasma protein. No evidence for accumulation of the drug in the intestinal wall of either rats or guinea pigs was seen. The dog was shown to convert indomethacin to indomethacin glucuronide, which is excreted in the bile.

Application of steady state kinetics to the synthesis rate and turnover time of serotonin in the brain of normal and reserpine-treated rats

Abstract: A new method of measuring the rate of synthesis of brain serotonin (5-HT) takes advantage of the steady state relationship in which the molar rates of 5-HT synthesis and 5-hydroxyindoleacetic acid (5-HIAA) efflux are equal. The method is based on the dynamic steady state of 5-HT and its metabohte 5-HIAA, with rates of formation equal to rates of metalbolism and elimination. After monoamine oxidase was blocked with a number of inhibitors, including pargyline and tranylcypromine, the brain levels of 5-HIAA decline exponentially. The rate of synthesis of 5-HT is calculated from the product of the rate constant of 5-HIAA decline and the normal 5-HIAA level. After depletion of 5-HT stores by reserpine, the 5-HT synthesis rate is not decreased; in fact, the rate possibly may be increased. In the brain stem, 5-HT is formed twice as rapidly as in the rest of the brain, although the rate constants of 5-HIAA efflux are almost identical. This suggests that the rate of synthesis might be similar in each serotonergic unit, and that variations in rates of synthesis in different brain areas are a function of the number of neurons per gram of tissue.

Regional studies of catecholamines in the rat brain. iv. effects of drugs on the disposition and metabolism of h3-norepinephrine and h3-dopamine

Abstract: The effects of reserpine, a monoamine oxidase inhibitor, Catron (pheniprazine), amphetamine and desmethylimipramine (DMI) on the metabolism of dl -H 3 -norepinephrine were examined in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, hippocampus and cortex of the rat brain. Reserpine reduced the accumulation of H 8 -norepinephrine to varying extents in the different brain regions. In reserpine-pretreated animals, there was also an elevation in the levels of H 3 -deaminated metabolites and a fall in H 3 -normetanephrine in all regions. After Catron, there was an increased accumulation of H 3 -norepinephrine in all areas of the brain except in the striatum; deaminated metabolites were severely reduced, while H 3 -normetanephrine levels were strikingly elevated in all areas of the brain, including the striatum. Both amphetamine and DMI reduced the accumulation of H 3 - norepinephrine in the cerebellum, medulla oblongata and hypothalamus, but produced only small changes in other areas. Amphetamine caused a striking elevation in the levels of H 3 -normetanephrine throughout the brain, while DMI produced only small elevations in the medulla oblongata and cortex. O-methylated deaminated metabolites were unaffected by either drug, though catechol deaminated metabolites were reduced, particularly after amphetamine. H 3 -amphetamine was evenly distributed in the brain at a concentration of about 8 µg/g (5 x 10 -3 M) after an intraperitoneal injection of 15 mg/kg. Amphetamine at this concentration almost completely inhibited the enzymatic deamination of H 3 -norepinephrine in vitro. The levels of H 3 -norepinephrine were reduced in the medulla oblongata, cerebellum and hypothalamus after the intraventricular injection of H 3 -dopamine in d - amphetamine- or DMI-pretreated animals. H 3 -dopamine levels in these regions and in the striatum were unaffected or increased after amphetamine or DMI.

Effect of ethanol on plasma corticosterone levels

Abstract: Following interperitoneal administration fo ethanom to rats, the effect of this alcohol on pituitary-adrenal function has been studied by measuring changes in plasma corticosterone concentration. Doses of ethanol ranging from 0.5 to 4.0 g/kg increased plasma steroid levels in a roughly parallel relationship to the size of the dose. After 2 g/kg of ethanol, the duration of the adrenocortical response was approximately 6 hr, which corresponded to the period of detectable blood alcohol concentration. When ethanol was given to hypophysectomized animals. no elevation of corticosterone concentration was observed. Pretreatment of intact animals with pentobarbital and morphine produced a complete blockade of the steroid response to ethanol. Pentobarbital alone reduced the response to about 50% of the usual ethanol effect. Since the intraperitoneal injection of a local anesthetic prior to ethanol administration failed to alter the action of ethanol, it is assumed that peritoneal irritation could not have been a part of the involved mechanism of action. Repeated daily injections of ethanol for 1 wk did not result in pituitary-adrenal adaptation and decreased steroid response to a subsequent injection as has been demonstrated for certain other centrally acting depressant drugs. These observations suggest that ethanol induces hypersecretion of corticotropin by acting through an unexplained central nervous system mechanism.

Field stimulation as a means of effecting the graded release of autonomic transmitters in isolated heart muscle

Abstract: A method is described for the use of field stimulation to produce pronounced and reproducibly graded sympathomimetic and parasympathomimetic effects in preparations of isolated heart muscle without disturbing the rate or rhythm of contraction. In addition to causing the release of autonomic neurotransmitters, field stimuli can alter the strength of contraction of heart muscle by at least three other mechanisms. If they are recognized, the latter need not interfere greatly with the method. The parasympathomimetic component of the response to field stimulation is abolished by atropine. There is a definite cholinergic response in the cat papillary muscle, though it is much less prominent than in atrial muscle. Maximally effective amounts of norepinephrine are readily liberated by field stimulation; the effects of submaximal stimulation are enhanced by cocaine. The adrenergic component of the response to field stimulation is markedly reduced by propranolol, and is absent in the chronically denervated heart. It is reduced after the administration of reserpine, and in cats (but not guinea pigs) it may be eliminated entirely by pretreatment with reserpine. In guinea-pig atria, reserpine pretreatment reduces the sympathomimetic response to field stimulation and the inotropic action of tyramine to about the same extent. There are important differences between the effects of reserpine pretreatment on the inotropic and chronotropic actions of tyramine. With proper precautions, it is possible to drive preparations of isolated heart muscle electrically without producing appreciable sympathomimetic or parasympathomimetic effects. However, many methods of stimulation in common use are likely to cause the release of substantial amounts of the autonomic neurotransmitters.

Comparison of the effects of dopamine, isoproterenol, norepinephrine and bradykinin on canine renal and femoral blood flow.

Abstract: The effects of arterial injections of dopamine on femoral and renal blood flow were compared with those of isoproterenol, norepinephrine and bradykinin after POB pretreatment. Dopamine was found to have a vasodilator action on the hind limb, which was antagonized by pronethalol and DCI. The potency of dopamine as a femoral vasodilating agent was very low compared to that of isoproterenol. In contrast, dopamine caused renal vasodilation by a qualitatively different mechanism, as indicated by a higher relative potency within the series of adrenergic agonists and the lack of antagonism by selective beta adrenergic blockade. This property cannot be attributed to conversion of dopamine to tetrahydropapaveroline which causes renal vasodilation by a beta adrenergic mechanism. Also, the metabolism of dopamine to norepinephrine,3,4-dihydroxyphenylacetic acid or 3-methoxy-4-hydroxyphenylethylamine is not the basis for this property. Furthermore, the renal vasodilation was not antagonized by atropine or eliminated by reserpine pretreatment. The ability of dopamine to dilate the renal vascular bed without producing a qualitatively similar direct effect on the femoral vascular bed sets it apart from agents such as papaverine and the nitrites. The physiologic significance of the renal vascular effects of dopamine remains to be determined.

ALTERATIONS IN BEHAVIOR AND BRAIN CATECHOLAMINE LEVELS IN RATS TREATED WITH α-METHYLTYROSINE

Abstract: α-Methyltyrosine (α-MT) has been proposed to cause central nervous depression by blocking synthesis of brain catecholamines. However, interpretation of these reports is complicated by the toxicity of this drug. In the present study, the effects of α-MT on behavior and brain catecholamine content of rats were examined with doses and routes of administration that did not cause toxicity. Behavioral tests consisted of conditioned avoidance responding, rotarod performance and spontaneous locomotor activity. Single intraperitoneal injections of α-MT caused marked loss of avoidance responding only in doses that were toxic. Subcutaneous injections were only slightly effective in depressing behavior because of the poor absorption of α-MT. Oral (200 mg/kg) or multiple intraperitoneal injections (3 x 50 mg/kg) of α-MT impaired avoidance responding, rotarod performance and spontaneous locomotor activity without producing obvious toxic effects. The content of α-MT in the brain and depletion of brain norepinephrine and dopamine bothexhibited a time course similar to that of the behavioral depression.

A sensitive and specific enzymatic isotopic assay for tissue histamine.

Abstract: A sensitive, specific and relatively simple and rapid enzymatic-isotopic method for the estimation of histamine in biologic materials is described. Tissues are incubated with tracer amounts of H3-histamine and C14-S-adenosylmethionine in the presence of the enzyme, histamine methyl transferase, which specifically methylates histamine. H3-C14-methylhistamine istamine is the only radioactive product extracted. The ratio of C14/H3 is directly proportional to the amount of unlabeled tissue histamine present in the incubation mixture. This assay can be utilized to detect as little as 2 ng of tissue histamine.

An experimental pain method sensitive to morphine in man: the submaximum effort tourniquet technique.

Abstract: There is great practical need for a dependable method of assessing effects of analgesics on experimental pain in man. Past use of threshold pain has not provided such a method. A technique, called here the submaximum effort tourniquet technique, was developed to produce experimental pain in man which simulates the duration and severity of pathologic pain more closely than earlier experimental methods. Pain was produced by having the subject squeeze a hand exerciser 20 times after a tourniquet had been inflated around his upper arm. Under appropriately controlled drug and placebo conditions, 45 male subjects (experiments I, II, III combined) were studied to determine the amount of time required for pain to build up to levels designated as slight, moderately distressing, very distressing and unbearable. Intravenous morphine, 10 mg/70 kg b.wt., was found to allay significantly the development of moderately distressing, very distressing and unbearable pain. There were indications that the 10-mg dosage of morphine might also be capable of allaying development of slight pain and that the 5-mg dosage might be capable of allaying development of unbearable pain, but these effects were not conclusive. Although the effect of 10 mg of morphine was more dependable ( i.e. , more statistically significant) at the higher pain levels than at the lower levels, the magnitude of the morphine effect was approximately a fixed multiple of the placebo score. Indeed, the largest morphine effect (expressed as percent of the placebo effect) was on the lowest pain level. When averaged for the four pain levels, the 10-mg dosage of morphine prolonged pain tolerance about 70%. It was concluded that appropriate use of the submaximum tourniquet technique produces a type of experimental pain which is responsive to the analgesic effects of 10 mg of intravenous morphine.

Storage and synthesis of norepinephrine in the reserpine-treated rat brain

Abstract: The retention of H3-norepinephrine in the rat brain after the injection of H3-norepinephrine into the lateral ventricle of the brain was severely reduced 6 hr after a single injection of reserpine. The ability to retain small amounts of H3-norepinephrine in the brain recovered rapidly between 24 and 48 hr after reserpine at a time when the animals were recovering from the most obvious behavioral effects of the drug. During this period the endogenous norepinephrine content of the brain was markedly depressed and rose only slowly to 40% of normal levels after 8 days. In reserpine-treated rat brains the accumulation of H3-norepinephrine could be inhibited by pretreatment with a second dose of reserpine or increased by treatment with an inhibitor of monoamine oxidase. Treatment of reserpinized animals with amphetamine or DMI produced changes in the metabolism of H3-norepinephrifle which indicate that these drugs are still able to inhibit norepinephrine uptake in such animals. These results are consistent with other studies which suggest that the primary action of reserpine is to impair the intracellular storage of norepinephrine rather than to inhibit the uptake of norepinephrine into adrenergic neurons. The small amount of H3-norepinephrine retained in the brains of 24-hr reserpinized animals was resistant to release by amphetamine or by the administration of an additional dose of reserpine. After the administration of C14-tyrosine or H3-dopamine into the lateral ventricle of normal or reserpinized rat brains it was possible to demonstrate that all the enzymatic steps involved in the biosynthesis of norepinephrine can occur in reserpinized animals.

Inhibition of dopamine-beta-hydroxylase by disulfiram in vivo.

Abstract: Disulfiram is a potent inhibitor of dopamine-β-oxidase which lowers norepinephrine content of the tissues. It prevents conversion of dopamine-H3, tyramine-H3 and other phenylethylamines to their β-hydroxylated derivatives, and presumably interferes with endogenous norepinephrine synthesis. The findings reported support the view that the diminished norepinephrine level in tissues following disulfiram treatment is a consequence of inhibition of dopamine-β-hydroxylase. The results suggest that β-hydroxylation can be made ratelimiting in catecholamine synthesis, even though it may not be the rate-limiting step under physiologic conditions.

Metabolism of drugs and carcinogens by human liver enzymes

Abstract: Human liver contains enzyme systems which catalyze the ring hydroxylation of 3,4-benzpyrene, the side chain oxidation of pentobarbital, the O-dealkylation of acetophenetidin and the N-dealkylation of 3-methyl-4-monomethylaminoazobenzene. The rate of metabolism of these substrates by hepatic enzymes was compared in man and rat. The enzyme system which hydroxylates 3,4-benzpyrene was studied in detail. This enzyme system in man is similar to that found in the rat; both are localized in the microsomal fraction and require reduced triphosphopyridine nucleotide (NADPH) for activity. Methods are described for measuring oxidative microsomal enzyme activity in small samples of human liver.

The role of brain serotonin in the mechanism of the central action of reserpine

Abstract: Reserpine sedation is associated with changes in brain serotonin (5-HT) rather than with catecholamines. Thus, after blockade of catecholamine synthesis, catecholamines can be reduced by 80% without producing sedation ; in contrast, reserpine elicits sedation in doses that reduce both 5-HT and catecholamines by only 55%. In addition, there is a time correlation between the effects of reserpine on behavior and impairment of the process that accumulates exogenous 5-HT in brain tissue. This process is recovered in 48 hr when rabbits have recovered from sedation, even though 5-HT stores are still largely depleted. After reserpine, the rate constant of 5-HT efflux is dose-dependent, and is maximal after a dose (5 mg/kg iv.) which depletes 5-HT at a half-life of about 7 min. Intensity and duration of sedation are correlated with initial rates of 5-HT release rather than the final extent of depletion. After doses of reserpine that deplete the monoamines at a maximal rate, the steadystate levels calculated from the rate of synthesis and the rate constant of efflux are 12% of normal for 5-HT and 3% of normal for norcpinephrine (NE). Studies showing that 5-hydroxytryptophan (5-HTP) in doses that elicit excitation also causes the release of brain NE may remove a key argument against the view that reserpine acts through free 5-HT.

Influence of drugs on behavior controlled by internal and external stimuli

Abstract: Three pigeons were trained to peck an illuminated disk for a food reinforcer which was delivered according to a fixed interval (FI) schedule of reinforcement. Under one set of conditions, the stimulus projected on the disk remained constant during the 5-min FI. Under a second set of conditions, the symbol projected on the disk changed once per minute during the interval, thus providing the birds with a "clock." These two conditions were chosen to represent behavior controlled by internal and external stimuli respectively, and were presented to the bird in an irregular order. Greater changes in response distribution were produced by amphetamine, scopolamine and pentobarbital when no "clock" was available; with the added clock, the virds responded mostly near the end of the interval, and drug-induced changes in response rate were for the most part confined to the end of the interval. This confirms for these drugs the hypothesis that behavior largely controlled by externally based discriminative stimuli is more resistant to drugs than behavior controlled by internal stimuli. Chlorpromazine and promazine, however, did not display such a pronounced differential effect on response distribution.

Pharmacologic studies on analgesics—vii. significance of the calcium ion in morphine analgesia

Abstract: In order to determine the significance of calcium in the physiologic function of the central nervous system with reference to the analgesic effect of opiates, the effect of calcium ion on the analgesic responses to morphine, meperidine and ohton was studied, using the methods of electrical stimulation and tail flick in mice. The intracisternal administration of calcium markedly suppressed the analgesic effects of morphine, meperidine and ohton, which were given either subcutaneously or intracisternally. Calcium also antagomzed the established analgesic effects of these drugs. In contrast, decalcifying agents such as ethylenediaminetetraacetic acid·2Na, cyclohexanediaminetetraacetic acid.2Na, sodium citrate and sodium oxalate injected intracistennally enhanced the analgesic effects of morphine, meperidine and ohton, and antagonized the antagonistic effect of calcium on the analgesic response. The calcium complexes of these chelating agents and other cations such as magnesium, barium, strontium, zinc, ferrous, aluminum, potassium and sodium had no analgesic effect themselves, and affected neither the analgesic response to morphine nor the antagonistic action of calcium on the analgesia produced by morphine. These results suggest that calcium in the central nervous system is involved in the mechanism of analgesia produced by opiates.

The physiologic disposition and metabolism of norepinephrine in immunosympathectomized animals

Abstract: The uptake and metabolism of H3-norepinephrine was examined in immunosympathectomized (IS) rats and mice. IS affected the uptake of H 3 -norepinephrine in different tissues to markedly different extents. In the rat heart and spleen, H 3 -norepinephrine uptake was reduced to less than 10% of normal; H 3 -norepinephrine uptake was 30 to 60% of normal in salivary glands, liver and intestine, and was unaffected in uterus, urinary bladder, brain and seminal vesicles. A significant increase in H 3 -norepinephrine uptake was found its IS vas deferens. In general, the reduction in the accumulation of H 3 -norepinephrine in various IS tissues paralleled the reduction in the endogenous catecholamine content of these tissues. A significant increase in the serotonin content of IS mouse intestine was found. After intravenous injection in the mouse, H 3 -norepinephrine was metabolized more rapidly in IS animals than in normal. In all tissues of IS rats there was a relative increase in the accumulation of H 3 -metabolites of norepinephrine, mainly accounted for by an increased content of the O-methylated metabolite, normetanephrine. However, there were no changes in the activity of catechol-O-methyl transferase in IS mouse tissues. A decreased monoamine oxidase activity was found in IS salivary glands, but no differences in the activity of this enzyme were observed in other IS tissues. Aromatic L-amino acid decarboxylase activity was decreased in IS heart, salivary glands and intestine, but not in liver or brain. The β-hydroxylation of H3-α-methyltyramine was impaired in IS tissues, suggesting a decreased dopamine-β-oxidase activity. The rate of turnover of adrenal medullary catecholamines was more than twice as fast in IS mice as in normals. Repeated injections of antiserum during the first week after birth failed to abolish H 3 -norepinephrine uptake completely in any rat tissue. The decreases in norepinephrine uptake in the rat heart produced by various amounts of antiserum can be used as a sensitive bioassay procedure for the estimation of nerve growth factor antiserum.

Potentiation by hydrocortisone of responses to catecholamines in vascular smooth muscle

Abstract: Potentiation by hydrocortisone (HC) of responses to catecholamines has been studied in the dog and in the aortic strip preparation. The site of the potentiating action of HC is at the adrenergic receptor level. This conclusion is supported by the observations that potentiation was obtained for both the alpha and beta adrenergic effects of catecholamines in vivo and in vitro . The failure to demonstrate potentiation by HC of the aortic strip responses produced by agents other than catecholamines (with one exception, synephrine) indicates that there is considerable specificity in the action of HC. The observation that potentiation was obtained in reserpine-treated tissues both with and without cocaine indicates that the action of HC is not dependent upon the integrity of the endogenous catecholamine stores. Evidence was not found for a direct relationship between the potentiating action of HC and membrane depolarization or the availability of extracellular calcium for the muscle response produced by a stimulating agent. It is suggested that HC increases the affinity of the catecholamine or possibly its p-hydroxy analog for the adrenergic receptor by modifying the interaction of the p -hydroxyl group in the molecule with the receptor.

Anticonvulsant properties of some benzodiazepines.

Abstract: The anticonvulsant potencies (ED50) of chlordiazepoxide (Librium), diazepam (Valium) and 7 experimental benzodiazepines were determined in mice by the following 3 tests: maximal electroshock seizure pattern (MES) test, low-frequency electroshock seizure threshold (l.f. EST) test and pentylenetetrazol (Metrazol) seizure threshold (s.c. Met) test. In addition, the dose of eachdrug which produced overt evidence of neurotoxicity in 50% of animals (TD50) was determined andprotective indices (PI = TD50/ED50) were calculated. Three of the more potent agents (chlordiazepoxide; compound 5, 7-nitro-5-(2-chlorophenyl)-3H-1,4-benzodiazepine-2(1H)-one; and compound 7, 7-nitro-5-(2-fluoromethylphenyl)-3H-1,4-benzodiazepine-2(1H)-one) were compared with trimethadione for ability to abolish the tonic extensor component of maximal pentylenetetrazol seizures(MMS test). Intravenously administered chlordiazepoxide, compound 5 and trimethadione also werecompared for ability to elevate by 100% the intravenous pentylenetetrazol seizure threshold. Finally, the effects of chlordiazepoxide and compound 5 on synaptic transmission were investigated in unanesthetized spinal cats. On the basis of the results obtained, the following conclusions appear justified: 1) Except for compounds 5 and 7 by the MES test, all 9 benzodiazepines tested are effective by all 3 tests; however, compound 6 (7-nitro-1-methyl-5-(2-fluorphenyl)-3H-1,4-benzodiazepine-2(1H)-one) was effective by the l.f. EST test only in a dose greatly in exces of the TD50. Compounds 5 and 7 were ineffective by the MES test, but were remarkably active by the l.f. EST, s.c. Met and MMS tests. 2) Compound 1 (7-chloro-4,5-dihydro-5-phenyl-3H-1,4-benzodiazepine-2(1H)-one) has the highest PI by the MES test (8.4) and compound 5 has the highest PI by the l.f. EST (3081) and s.c. Met (7983) tests. 3) Compound 5, administered intravenously, was determined to be 4750 times more potent than trimethadione in elevating by 100% the threshold for seizures induced by the intravenous administration of pentylenetetrazol. 4) Although chlordiazepoxide and compound 5 had no significant effect on transmission of isolated impulses in monosynaptic and polysynaptic pathways or on posttetanic potentiation in unanesthetized spinal cats, both drugs decreased the response to repetitive stimulation. This depressant effect was abolished with appropriate doses of pentylenetetrazol. In view of the favorable profile of anticonvulsant action of compound 1 and the unique antipentylenetetrazol activity of compound 5, these, and possibly other henzodiazepines included in this study, appear worthy of cautious clinical trial in epilepsy.

The actions of the catecholamines on transmission in the superior cervical ganglion of the cat

Abstract: An elecrotphysiologic study was made of the effects of norepinephrine (NE), epinephrine (E) and isoproterenol (ISO) on transmission in the superior cervical ganglion of the cat. NE produced ganglionic hyperpolarization and blocked ganglionic transmission. These actions were blocked by dihydroergotamine (DHET), and enhanced by dichloroiso-proterenol (DCI) and pronethalol. After the administration of DHET, NE produced ganglionic depolarization and enhanced transmission. These actions were blocked by DCI and pronethalol. Although the actions of E on ganglionic transmission were similar to those of NE, differences were noted between the ganglionic potentials evoked by three amines. For example, although both agents evoked ganglionic hyperpolarization, the E-induced hyper-polarization was followed in many experiments by a phase of depolarization. This depolarization evoked by E was blocked by DCI and pronethalol. ISO produced a depolarization of the ganglion and enhanced transmission. These actions of ISO were blocked by the beta adrenergic blocking agents, but were unaffected by DHET. The above findings indicate that sympathetic ganglia contain two pharmacologically distinctive adrenoceptive sites. It is considered that one site, which was blocked by alpha adrenergic blocking agents, mediated catecholamine-induced inhibition and ganglionic hyperpolarization and that the other site, which was blocked by beta adrenergic blocking agents, mediated catecholamine-evoked facilitation and ganglionic depolarization.

The adrenergic blocking activity of N-tert.-butylmethoxamine (butoxamine).

Abstract: The effects of butoxamine upon a number of adrenergic responses were determined in the anesthetized dog and the isolated rat uterus. Studies in the dog included a determination of the intrinsic effects of butoxamine on mean arterial pressure, heart rate, intestinal motility, cardiac contractile force, retractor penis muscle contractions and femoral vascular flow. These studies in the dog also included the effects of pretreatment with butoxamine on the responses to epinephrine, isoproterenol, ethylnorepinephrine and phenylephrine on these same parameters. Studies with the isolated, spontaneously contracting rat uterus consisted of a determination of both the intrinsic action of butoxamine and its ability to modify the uterine inhibitory responses to epinephrine, isoproterenol, norepinephrine, phenylephrine and papaverine. Butoxamine reduced the vasodilator response to isoproterenol and reversed the vasodilator response to ethylnorepinephrine. Butoxamine also produced isoproterenol and ethylnorepinephrine blood pressure "reversal" (depressor to pressor). None of the other parameters measured were reduced to any significant extent in the dog. In the isolated rat uterus, butoxamine produced a blockade of the uterine inhibitory responses to the catecholamines that was similar to the blockade produced by N-isopropylmethoxamine, dichloroisoproterenol and pronethalol. Butoxamine appears to be capable of producing a selective blockade of some, but not all, beta adrenergic receptors.

The metabolism of tritiated digoxin in renal insufficiency in dogs and man.

Abstract: The metabolism of tritiated digoxin was studied in dogs with acute renal insufficiency induced by bilateral nephrectomy as well as bilateral ureter ligations. Eight-day metabolic studies were obtained in patients with chronic renal disease after a single intravenous injection of tritiated digoxin. In animals with renal insufficiency, the heart as well as other organs participated in the retention of activity. Tissues such as heart, liver, bile and small intestines which had a high level of activity in the control animals showed the greatest gain in radioactivity. More than 80% of activity in heart, liver and kidney tissue, obtained from sham-operated as well as nephrectomized animals, was eluted from an alumina column with 20% ethanol in chloroform. Filter paper chromatography of this fraction of heart and liver showed that the predominant peak of activity migrated with digoxin; lesser peaks of activity correspond to digoxigenin-mono- and bis-digitoxoside. Only one peak of activity which migrated with digoxin was found in kidney tissue. Two patients with renal disease had an impaired ability to eliminate a single intravenously administered dose of digoxin. The total urinary activity was one-half that excreted by control subjects. Fecal excretion of digoxin and metabolites was increased, but not sufficiently to compensate for the diminished renal excretion. The difference in biotransformation of digoxin in renal patients as compared to control subjects was detailed. The findings of a diminished urinary excretion of digoxin-H3 as well as renal retention of digoxin-H3 in tissues provides a metabolic basis for the observed sensitivity to digoxin in renal failure.

Comparative effect of antirheumatic drugs on adjuvant-induced polyarthritis in rats

Abstract: The effect of several clinically useful antiarthritic drugs on adjuvant-induced polyarthritis in rats is compared with that of indomethacin. The drugs were given daily, either orally or by intraperitoneal injection, beginning on the day of adjuvant injection. The doses used were comparable to the commonly used human doses, given in milligrams per kilogram. Adjuvant containing 6.0 mg of Mycobacterium butyricum per milliliter of heavy mineral oil was given by intradermal injection into a hind foot pad. Swelling occurred at the adjuvant depot site within 24 hr, and disseminated arthritis appeared in 10 to 14 days. Each joint and the injection site were scored daily for swelling and redness. Indomethacin, prednisolone and phenylbutazone were shown to decrease the swelling at the injection site and to suppress the generalized arthritis. Aspirin was only effective in suppressing the inflammation at the injection site. Chloroquine and hydroxychloroquine were without demonstrable activity. Because the doses of all drugs were within the range used for treating patients, adjuvant arthritis of rats is established as an experimental model for testing antiartliritic drugs. It is suggested that this model would be more useful for drug evaluation with improved techniques for measuring joint swelling and tenderness.

THE EFFECTS OF 5-HYDROXYTRYPTOPHAN AND l-TRYPTOPHAN ON SPINAL SYNAPTIC ACTIVITY

Abstract: The potentials evoked by supramaximal stimulation of an L7 dorsal root or peripheral nerve and recorded from the L7 ipsilateral ventral moot and a dorsal rootlet were studied after the injection of 5-hydroxytryptophan (5-HTP) and l -tryptphan. The injection of 5-HTP produced a marked increase in the monosynaptic spike height and depression of the polysynaptic and dorsal root reflexes. This was accompanied by spontaneous motoneuronal discharge and an average decrease of 0.2 msec in the stimulus-response latency. An increase in the monosynaptic spike height averaging 310% ± 30 (S.E.) but the polysynaptic and dorsal root reflexes exhibited only a This action was slow in onset, reaching a maximum about 90 min after injection. A the time of maximal effect, cord levels of 5-hydroxytryptamine (5-HT) Were markedly elevated. l -Tryptophan in doses of 100 mg/kg produced an average increase in the monosynaptic spike height of 172% ± 31 (S.E.), but the polysynaptic and dorsal root reflexes exihibited only a moderate increase. It is postulated that the increase in 5-HT produced by 5-HTP and l-tryptophan occurs largely in descending fibers in the cord, and that this excess 5-HT induces, either directly or indirectly via interneurons, an increase in motoneuronal excitability.

Failure to anesthetize human subjects by intravenous administration of magnesium sulfate.

Abstract: Two human subjects were given MgSO4 by slow intravenous infusion. The concentration of Mg rose to 15.3 and 14.6 mEq/liter of plasma, from a control value of 1.4 mEq/liter. Profound paralysis of skeletal muscles was produced, with the exception of the diaphragm, the adductors of the vocal cords and some of the facial muscles. The electrocardiogram showed slowing of atrioventricular conduction and alterations of the ventricular action potential. Both subjects remained aware of the surrounding, and in rational contact with the observers. Pain was keenly felt; vision and hearing were normal. Eye centering and conjugate eye movements did not fail. Nystagmus could be provoked in one subject. The electroencephalogram showed diminished α rhythm and decreased voltage, but none of the common signs of sleep, coma or anesthesia. One of the subjects had amnesia over a short period of time coincident with the occurrence of cyanosis. The other subjects, whose ventilation remained adequate, could remember every detail of the experiment. It is concluded that general anesthesia, or analgesia, cannot be produced by parenteral administration of magnesium. It is doubtful whether in these two experiments any depression of the central nervous system was produced at all by the drug.

Centrally mediated cardiovascular effects of angiotensin II.

Abstract: The intraventricular administration of angiotensin in chloralose-anesthetizedcats produced centrally mediated cardiovascular effects which consisted of hypertension and tachycardia and, in more than 50% of the experiments, contraction of the nictitating membrane. Section of the spinal cord at the C1 level essentially abolished the response to intraventricularly administered angiotensin. The data presented suggest that physiologic levels of brain norepinephrine are necessary for the production of the angiotensin-induced pressor response following intraventricular administration and that either a reduction orelevation of brain norepinephrine significantly decreases the response. Additional data have been presented providing evidence that angiotensin acts directly or indirectly on centralsympathetic structures, thereby inducing a prolonged increase in peripheral blood pressure.The intraventricular administration of norepinephrine produced a centrally mediated hypotensive effect and bradycardia which could be reversed by "central depletion" of norepinephrine by reserpine or metaraminol.

Responses of spinal cord interneurons to acetylcholine, norepinephrine and serotonin administered by microelectrophoresis.

Abstract: Biochemical, histochemical and pharmacologic evidence suggest a functional role for acetylcholine (ACh), norepinephrine (NE) and serotonin (5-HT) in the spinal cord, possibly as synaptic transmitters. However, except for ACh sensitivity of Renshaw cells, Curtis, Phillis and Watkins (1961) did not find other neurons in the spinal cord responsive to ACh, NE or 5-HT. We reinvestigated this problem in the lumbosacral segments of decerebrated and ether-anesthetized cats using 5-barrel glass micropipette electrodes to administer ACh, NE and 5-HT electrophoretically to single neurons at the site of extracellular action potential recording. We found that a number of interneurons in the spinal cordresponded to the electrophoretic administration of ACh, NE and 5-HT with either facilitation or depression of firing. Some neurons responded to one, others to two and some to all three compounds with no correlation in the direction of their responses. The finding of neurons in the spinal cord responsive to the electrophoretic administration of ACh, NE and 5-HT providesa basis for studying the possible involvement of these substances in synaptic transmission.