Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1973"

Acetaminophen-induced hepatic necrosis. iv. protective role of glutathione

Abstract: The possibility that glutathione may protect against acetaminophen-induced hepatic necrosis was examined. Pretreatment of mice with diethyl maleate, which depletes hepatic glutathione, potentiated acetaminophen-induced hepatic necrosis, whereas pretreatment with cysteine, a glutathione precursor, prevented hepatic damage. Administration of acetaminophen caused a dose-dependent depletion of hepatic glutathione. Glutathione depletion by acetaminophen was enhanced by treatments that potentiate the hepatic necrosis and covalent binding produced by the toxic metabolite of acetaminophen. Conversely, glutathione depletion was inhibited by treatments that protect against these toxic effects. Moreover, covalent binding of this metabolite to hepatic macromolecules did not occur until the availability of glutathione was exhausted through conjugation with the metabolite. Similarly, alteration of glutathione availability by pretreatment with diethyl maleate or cysteine, respectively, increased or decreased covalent binding of the toxic metabolite. We propose that. a fundamental role of glutathione in the body may be to protect tissues against electrophilic attack by drug metabolites and other alkylating agents.

Acetaminophen-induced hepatic necrosis. i. role of drug metabolism

Abstract: A number of recent reports indicate that massive overdoses of acetaminophen can produce a fulminant hepatic necrosis in man. Acetaminophen-induced hepatic necrosis thereforne was examised in experimental animals. Centrilobular hepatic necrosis similar to that seen in man was shown to be dose dependent in mice and rats. To determine whether acetaminophen-induced hepatotoxicity was mediated throug an active metabolite, acetaminophen concentrations in rat and mouse tissues were compared with the severity of liver damage after alteration of the rate of metabolism of acetaminophen. Phenobarbital pretreatment stimulated the disappearance of acetaminophen from tissues but markedly potentiated the hepatic necrosis. In contrast, piperonyl butoxide pretreatment inhibited the metabolism and disappearance of acetaminophen from tissues yet dramatically protected against hepatic necrosis. Additional studies demonstrated that pretreatment with 3-methylcholanthrene also potentiated acetaminophen-induced hepatic necrosis whereas cobaltous chloride, which inhibits synthesis of cytochrome P-450, prevented the hepatic damage. We propose that acetaminophen-induced hepatic necrosis is mediated by a toxic metabolite of acetaminophen.

Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity

Abstract: The effects of amphetamine, various phenothiazines and haloperidol on dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat midbrain were studied in anesthetized and gallamine-paralyzed animals using a single unit recording technique. d -Amphetamine administered intravenously markedly decreased the spontaneous activity of dopaminergic neurons in the substantia nigra and ventral tegmental area. Antipsychotic phenothiazines and haloperidol increased the firing rate of these cells and reversed the d -amphetamine depression. Promethazin. a phenothiazine lacking antipsychotic efficacy, had no effect. In an experiment designed to correlate changes in firing rate with dopamine metabolism, neostriatal 3,4-dihydroxyphenylacetic acid concentrations were determined before and after administration of chlorpromazine (1.25 mg/kg), amphetamine (1.25 mg/kg) and promethazine (10 mg/kg). Neostriatal 3,4-dihydroxyphenylacetic acid was inceased by chlorpromazine,decreaed by amphetamine and unchanged by promethazine, thus paralleling the effects of these drugs on dopaminergic unit activity. These findings, together with our single unit recording results, are compatible with the neuronal feedback hypothesis orignally suggested as a mechanism by which these drugs might alter dopamine metabolism.

Acetaminophen-induced hepatic necrosis. ii. role of covalent binding in vivo

Abstract: Doses of 3H-acetaminophen (300-750 mg/kg) that cause necrosis in mice were shown to result in large amounts of radiolabeled material covalently bound to mouse liver protein in vivo (2 nmol/mg of protein or approximately one molecule bound per two molecules of protein in microsomes and cytoplasm). Both covalent binding and hepatic necrosis were dose dependent, and the peak level of binding preceded the development of recognizable necrosis by at least one to two hours. Pretreatment of mice with phenobarbital, piperonyl butoxide or cobaltous chloride, which changed the rate of metabolism of 3H-acetaminophen and altered the severity of hepatic necrosis, similarly affected the extent of hepatic binding of radiolabeled metabolite. Furthermore, the degree of binding in individual mice was always directly proportional to the severity of hepatic necrosis regardless of the biologic variation among various animals. Accordingly, we propose that acetaminophen-induced hepatic necrosis may be caused by the covalent binding of a chemically reactive metabolite to vital hepatsic macromolecules.

Mechanism of tolerance development to organic nitrates

Abstract: Tolerance to the vasodilator activity of organic nitrates appears to involve an alteration in a receptor in vascular smooth muscle. An in vitro model of nitrate tolerance was developed by incubation of rabbit thoracic aorta with organic nitrate at alkaline pH, producing a time- and concentration-dependent decrease in sensitivity to glyceryl trinitrate. Cross-tolerance is observed to all organic nitrates studied but not to nonnitrate vasodilators. When aortic strips are incubated at alkaline pH, nitrite formation is enhanced and tissue SH levels decrease. It s proposed that organic nitrate tolerance involves the oxidation of a critical sulfhydryl in the glyceryl trinitrate "receptor." This hypothesis is supported by the reversal of both in vitro - and in vivo -induced glyceryl trinitrate tolerance by the disulfide reducing agent, dithiothreitol.

Acetaminophen-induced hepatic necrosis. 3. Cytochrome P-450-mediated covalent binding in vitro.

Abstract: The binding of 3 H-acetaminophen to hepatic microsomes was studied in vitro . Binding of 3 H-acetaminophen to rat and mouse microsomal protein was linear with time and with protein concentration. Binding occurred by covalent linkage to amino acids of protein. Reduced nicotinamide adenine dinueleotide phosphate and oxygen were necessary for the binding while carbon monoxide or cobaltous chloride pretreatment inhibited it, demonstrating that a cytochrome P-450-dependent. mixed function oxidase mediated the binding. The extent of in vitro binding correlated with treatments that alter hepatic necrosis and in vivo binding, indicating that in vitro binding was a valid index of acetaminophen-induced hepatotoxicity. Analogous studies with 2-acetylaminofluorene showed that its binding to microsomal protein also was dependent on cytochrome P-450. Since the toxicity of 2-acetylaminofluorene results from its conversion to an N-hydroxy derivative, the collective data are consistent with the hypothesis that the hepatotoxic metabolite of acetaminmophen may be an N-hydroxy derivative.

Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin

Abstract: In anesthetized dogs, infusions of angiotensin II into the renal artery caused dosedependent decreases in renal blood flow and release of a prostaglandin E2-like substance into the renal venous blood. Inhibitors of prostaglandin synthesis, indomethacin (0.1-2 mg/kg i.v.) or meclofenamate (1 mg/kg iv.) reduced renal blood flow and urine output but did not substantially affect blood pressure or hindlimb blood flow. After indomethacin or meclofenamate, angiotensin II no longer caused release of prostaglandin E2-like material from kidney and the renal vasoconstrictor action of angiotensin II was augmented. Enhancement of the renal vascular effects of angiotensin II was greatest with low infusion rates of the octapeptide (3-20 ng/kg/min i.a.). Reductions in hindlimb blood flow produced by i.a. infusions of angiotensin II were not accompanied by prostaglandin release and the responses were not augmented by indomethacin. Indomethacin enhanced the renal (but not hindlimb) vasoconstrictor effects of norepinephrine. The results support the hypothesis that intrarenal prostaglandin E2 generation in response to angiotensin II attenuates the increase in renal vascular resistance produced by angiotensin.

Beta-phenylethylamine: a specific substrate for type B monoamine oxidase of brain.

Abstract: β-Phenylethylamine and serotonin were oxidatively deaminated by separate forms of monoamine oxidase of rat brain wimen studied in vitro . These forms, designated as type A and type B enzyme, have different characteristics. For example, type A enzyme deaminated serotonin, was sensitive to the drug clorgyline, was resistant to the drug deprenyl and was heat-stable. In contrast, type B enzyme deaminated β-phenylethylamine, was resistant to clorgyline, was sensitive to deprenyl and was heat-labile. Moreover, type A and type B enzyme activities could be partially separated by centrifugation on a continuous sucrose gradient. β-Phenylethylamine and serotonin are present in mammalian brain. We conclude from our studies that the amines of brain may be cataboblized by specific types of monoamine oxidase in vivo .

Role of brain norepinephrine in audiogenic seizure in the rat

Abstract: Treatments which caused severe reduction of brain concentrations of norepinephrine (NE) and dopamine (DA) by interfering with storage or by simultaneously inhibiting synthesis and promoting release of these biogenic amines markedly intensified audiogenic seizure (AGS). These results imply that depletion of brain NE and/or DA is associated with increased severity of AGS. Several observations provide evidence that NE is more important than DA in the modulation of AGS. For example, the benzoquinolizine, Ro 4-1284, markedly intensified AGS and depleted NE and DA one hour after administration; however, four hours later AGS enhancement was no longer apparent and brain NE concentration showed recovery, while DA remained severely reduced. The pronounecd effects of Ro 4-1284 on AGS and brain catecholammes were prolonged by concurrent treatment with l -α-methyl- p -tyrosine and were still evident 12.75 hours later. In comparison to the combination treatment, l -α-methyl- p -tyrosine caused an equally severe reduction of brain DA, but it produced significantly less reduction of NE and no enhancement of AGS. Intracerebroventricular injection of reserpine intensified AGS and markedly reduced brain NE, but only moderately lowered DA. Imipramine inhibited AGS and effectively antagonized the NE-depleting and AGS-enhancing effects of Ro 4-1284 without reversing DA depletion. It is proposed that NE exerts all inhibitory effect in the brain which tends to limit spread of seizure discharge. Consequently, depletion of brain NE disrupts inhibitory function, allows more efficient spread of seizure activity in the brain and increases severity of AGS.

Activation of the pathway from locus coeruleus to rat cerebellar Purkinje neurons: pharmacological evidence of noradrenergic central inhibition.

Abstract: A direct pathway of norepinephrine-containing axons can be demonstrated by fluorescence histochemistry to arise in the pontine nucleus locus coeruleus and project to the Purkinje cells of the rat cerebellar cortex. To determine the mechanisms underlying the physiological effects of activating this pathway, a pharmacological analysis has been performed by microelectrode techniques. Discrete stimulation of the locus coeruleus inhibits the discharge of cerebellar Purkinje cells. This inhition derives from a hypenpolarization of the Purkinje cell membrane accompanied by no increase in membrane ionic conductance, as shown in previous experiments with iontophoretic administration of norepinephrine and cyclic adenosine monophosphate. The magnitude of the hyperpolarizing response is not related to the initial resting membrane potential. Destruction of the central norepinephrine fiber systems by pretreatment with 6-hydroxydopamine or acute depletion of norepinephrine content by reserpine and α-methyltyrosine blocks the effects of stimulating locus coeruleus. Inhibitions of Purkinje cells produced by stimulation of locus coeruleus are also blocked by iontophoretic administration of prostaglandins E1 and E2 and potentiated by iontophoretic administration of papaverine. Purkinje cell inhibitions produced by activaton of non-noradrenergic Pathways are not similarly affected by any of these treatments. These data provide evidence that activation of the locus coeruleus inhibits the Purkinje cells by a norepinephrine-mediated mechanism.

A specific and sensitive enzymatic-isotopic microassay for serotonin in tissues

Abstract: A sensitive and specific enzymatic-isotopic micromethod for the determination of serotonin in animal tissues is described. The assay can measure as little as 50 pg of serotonin. Organ extracts are incubated with acetyl coenzyme A and rat liver N-acetyltransferase (ES 2.3.1.5) to acetylate seratonin and then incubated with 3 H-methyl-S-adenosyl-1-methionine and pineal hydroxyindole-O-methyltransferase (ES 2.1.1.4), an enzyme that specifically O-methybates N-acetylserotonin. Under the conditions of the assay, 3 H-melationin is the only radioactive product formed in detectable amounts from substrates extracted from tissue. Melatonin is produced in direct proportion to the serotonin added to the incubation mixture. With this assay, the endogenous serotonin content of small rat brain regions and nuclei can be measured.

Sulfhydryl requirement for relaxation of vascular smooth muscle

Abstract: Incubation of aortic strips with ethacrynic acid, at pH 7.4, markedly decreases tissue sulfhydryl (SH) groups and decreases aorta sensitivity to all tested vasodilators. This desensitization to vasodilators could be achieved under conditions which did not alter norepinephrine- or KCl-induced contraction of rabbit aortic strips. There appears to be two distinct vasodilation responses, one activated by direct interaction with a specific receptor site and not necessarily requiring SH oxidation as a primary event, and a second group of nonspecific vasodilators in which vasodilation is correlated with ability to react with SH groups. Besides " specific receptors," a hypothetical model is presented which contends that all vasodilators act through a common intermediate reaction involving critical SH groups witimin the tissue.

Quantitative aspects of precipitated abstinence in morphine-dependent rats

Abstract: The dose-response relationships of naloxone to selected signs of the precipitated abstinence syndrome were studied in male rats rendered physically dependent on morphine by subcutaneous implantation of two morphine pellets. Abstinence behavior was precipitated by i.p. injection of naloxone hydrochloride 72 hours after the first pellet implant. The median effective dose of naloxone was determined for the quantal abstinence responses of diarrhea, abnormal posturing, ear blanching, ptosis, teeth chattering, swallowing movements, escape attempts and wet shakes. Other signs of precipitated abstinence such as the incidence of seminal emissions and chromodacryorrhea as well as the average number of wet shakes and escape attempts per responding animal were found to exhibit a poor dose-response relationship with increasing naloxone dosage in the tested range of 0.04 to 10 mg/kg. Loss of body weight, measured three hours after naloxone administrations, was correlated to the log dose of naloxone. The relative merits of different criteria for quantifying the morphine abstinence syndrome are discussed.

Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons

Abstract: Eleven alkaloids obtained from Veratrum have been compared for their effects on the membrane potential and conductances of squid and crayfish giant axons. They can be classed in three groups. 1) Veratridine, cevadine and protoveratrines A and B cause the membrane to depolarize. The potency decreases in that order, and the concentrations of veratridine and cevadine required for 50% maximum depolarization are estimated to be 3.3 x 10 -5 M and 3.7 x 10 -3 M, respectively. The depolarization by veratridine is due primarily to a selective increase in resting sodium permeability of time membrane and is antagonized by tetrodotoxin. All of them are effective in augmenting and prolonging the negative (depolarizing) afterpotential. 2) Veratramine, isorubijervine, muldaimine (5-veratranine-3β, 11α-diol-11-acetate) and 5-veratranine-3β, 1lα-diol (1 x 10 -4 M) are capable of blocking the action potential with little or no depolarization. 5-Veratranine-3β, 1lα-diol blocks both sodium and potassium conductance increases. 3) Cyclopamine, jervine, rubijervine and veratrosine have no effect on the resting and action potentials. Possible structure-activity relationships for these effects are discussed.

Involvement of nigro-striatal neurons in the in vivo release of dopamine by amphetamine, amantadine and tyramine.

Abstract: After labeling the caudate nucleus of spinal-sectioned cats with 3H-dopamine and perfusing the cerebroventricular system, d - or l -amphetamine, amantadine or tyramine was added to the perfusion inflow. All four drugs caused a concentration-related efflux of 3H-dopamine into the ventricular effluent. When the experiment was repeated using cats with chronic lesions of the nigro-striatal dopaminergic fibers, the drug-evoked efflux was greatly reduced. When similar lesions were made during perfusate collection. a significant drop in 3H-dopamine efflux occurred. The ability of amphetamine and amantadine to increase 3H-dopamine efflux was markedly decreased by this acute lesion; the efflux indueed by tyramine was unaffected. Low concentrations of amphetamine or amantadine but not tyramine, potentiated the efflux of 3H-dopamine elicited by low frequency nigro-striatal pathway stimulation. Thus, the efflux of 3H-dopamine evoked from central dopaminergic synapses by amphetamine and amantadine is primarily dependent upon the impulse activity of neurons in the nigro-striatal pathway; the release by tyramine, although arising from the same terminals, is not dependent upon ongoing impulse activity.

Alcohol withdrawal reactions in mice: effects of drugs that modify neurotransmission

Abstract: Mice were made physically dependent on ethanol by a three-day period of alcohol inhalation with small daily injections of pyrazole to stabilize the blood alcohol levels. The severity of the withdrawal reaction was measured by repeatedly scoring the characteristic convulsions that could be elicited by handling. The effects of neuropharmacological agents on the withdrawal seizures were observed. Drugs that affect cholinergic systems (atropine, dihydro-β-erythroidine or physostigmine) had little or no effect. Similarly, attempts to modify serotonergic neurons with p -chlorophenylalanine, tryptophan or 5-hydroxytryptophan did not affect the withdrawal reaction. However, agents directed at γ-aminobutyric acid neurons modified the seizures; picrotoxin facilitated and aminooxyacetic acid suppressed the convulsions. Drugs that interfere with catecholamine pathways (reserpine, α-methyltyrosine, phentolamine or propranolol) aggravated the withdrawal seizures. Reserpine had the strongest and most prolonged effect. Apparently both γ-aminobutyric acid and catecholamine pathways tend to suppress the hyperexcitability of the alcohol withdrawal state.

DOPAMINERGIC NEURONS: SIMILAR BIOCHEMICAL AND HISTOCHEMICAL EFFECTS OF γ-HYDROXYBUTYRATE AND ACUTE LESIONS OF THE NIGRONEOSTRIATAL PATHWAY

Abstract: γ-Hydroxybutyrate (GHB) and its precursor, γ-butyrolactone (GBL) administered in anesthetic doses have been shown to cause a rapid increase in neostriatal dopamine (DA) levels without haveing similar effects on other brain monoamines. The time course of this effect on DA has been correlated with the behavioral effects and brain levels of the drug. This study reports that high frequency electrothermic lesions placed in the nigro-neostriatal DA pathway of the rat caused a similar increase in neostriatal DA; an 80% increase was seen within 30 minutes. Histochemical fluorescence observations indicated that the site of increase in DA fluorescence observed on the side of the brain with the lesion was similar to that observed after GBL treatment. Biochemical studies with αmethyl- p -tyrosine indicated that after both GBL administration and axotomy, the increased DA levels seem protected from release and/or metabolism. Extracellular recording experiments indicated that GHB and GBL administered parenterally inhibit the firing of the units localized to the DA-containing cells of the substantia nigra. These results suggest that the increase in DA in the neostriatum caused by both GHB and electrothermic lesioning of the DA pathway results from an inhibition of impulse flow in the nigro-neostriatal pathway.

COMPARABLE BEHAVIOR MAINTAINED UNDER FIXED-RATIO AND SECOND-ORDER SCHEDULES OF FOOD PRESENTATION, COCAINE INJECTION OR d-AMPHETAMINE INJECTION IN THE SQUIRREL MONKEY

Abstract: Under a fixed-ratio (FR) schedule, every 10th or every 30th key-press response of squirrel monkeys resulted in either presentation of food or intravenous injection of drug. With optimal doses of cocaine or d -amphetamine and with optimal amounts of food, mean response rate was over one per second. Decreasing the cocaine or d -amphetamine dose resulted in irregular responding at reduced rates. Discontinuing food presentation had the same effect. Increasing the cocaine or d -amphetamine dose resulted in a high response rate at the beginning of each session, followed by decreasing response rates as the session progressed; increasing the amount of food had the same effect. Monkeys were then studied under a second-order fixed-interval schedule of FR components. Each FR component completed during a fixed interval of time (five minutes) produced only a brief light. The first FR component completed after the five-minute interval ended produced a brief light and either cocaine injection or food presentation. Mean response rates of about one per second were maintained consistently as the dose of cocaine injected or the amount of food presented was systematically varied over a wide range. Thus, striking parallels between drug-maintained responding and food-maintained responding occurred over a wide range of parameter values under both FR and second-order schedules.

Role of detoxifying enzymes in bromobenzene-induced liver necrosis

Abstract: The mechanism of 3-methylcholanthrene (3-MC)-induced protection from bromobenzene's hepatotoxicity has been investigated. Determination of the metabolic half-life of bromobenzene indicated that 3-MC induction did not alter the rate of metabolism of bromobenzene in vivo . In contrast, phenobarbital and SKF 525A treatments, which enhance and block, respectively, bromobenzene-induced liver necrosis, enhanced and blocked bromobenzene metabolism. Measurement of bromobenzene metabolism in vitro indicated that 3-MC caused a modest induction of metabolism. Thus 3-MC-induced protection, unlike the SKF 525A effect, is not due to an inhibition of bromobenzene metabolism. Determination of the urinary metabolites of bromobenzene indicated that 3-MC induction caused an increased excretion of bromophenyldihydrodiol and bromocatechol. These metabolites arise from epoxide hydrase-catalyzed hydration of bromobenzene epoxide, suggesting that 3-MC protection is due to an increased capacity to detoxify the chemically highly reactive epoxide. In addition, 2-bromophenol was found to be a major urinary metabolite of hromobenzene in 3-MC-induced rats, suggesting that 3-MC induction may also divert bromobenzene metabolism into a comparatively nontoxic pathway.

Studies of the presynaptic effect of -bungarotoxin on neuromuscular transmission.

Abstract: The blocking effect of β-hungarotoxin (β-BuTX) isolated from the venom of Bungarus multicinctus on neuromuscular transmission in the rat isolated diaphragm was studied. No postsynaptic action on the membrane potential, action potential or the sensitivity to acetylcholine of the motor end-plate was observed. Since conduction in the phrenic nerve and its terminal also was unaffected, the action of β-BuTX seems to be confined exclusively to the presynaptic site. Although the binding of the toxin to action sites was almost complete within 20 minutes, it took more than 60 minutes for the inhibitory effect to appear. Shortly after application of the toxin, an increase in both frequency of miniature end-plate potentials and quantal content of end-plate potentials was observed. The onset of neuromuscular blockade was dependent on the stimulus frequency, being shorter at higher frequencies. Both Ca++-deficient and high Mg++ media antagonized the binding as well as the change after biniding which leads to the inhibitory effect. β-BuTX did not reduce the store of acetylcholine in the diaphragm even after complete neuromuscular blockade when the quantal content of end-plate potentials was markedly reduced. After neuromuscular blockade bursts of miniature end-plate potentials could be evoked by hypertonicity or high K+. It is concluded that the depression of the release of neurotransmitter by β-BuTX is not due to depletion of the store as previously postulated but is due to an inhibition of the mechanism for releasing neurotransmitter. The possibility of a causal link between an initial facilitation and the subsequent inhibition of transmitter release is discussed.

Growth and behavioral changes in developing rats treated intracisternally with 6-hydroxydopamine: evidence for involvement of brain dopamine.

Abstract: Intracisternal administration of 6-hydroxydopamine to immature rats produced marked reductions of norepinephrine, dopamine and tyrosine hydroxylase activity in brain. Accompanying these reductions were alterations in growth and behavior clearly demonstrable when adult. The 6-hydroxydopamine-treated rats showed decrements in eating and drinking, in their intake of a sucrose solution and in the ability to improve performance durimg acquisition of a shuttle-box avoidance response. Since choline acetylase activity and tryptophan hydroxylase activity were not altered in brain after this treatment, it seemed unlikely that these neuronal systems were responsible for the observed deficits. To evaluate the roles of norepinephrine or dopamine in the deficits, methods were developed to reduce these amines preferentially in the brains of immature rats. Animals in which dopamine was preferentially reduced showed similar deficiencies to those described above whereas norepinephrine-depleted rats did not show these alterations. Thus, the data suggest that the decrements in the 6-hydroxydopamine-treated developing rat are due to reduction in brain dopamine and support the view that dopaminerigc neurons are involved in the maintenance of consummatory behavior as well as conditioned avoidance responding.

The disposition of propranolol. 3. Decreased half-life and volume of distribution as a result of plasma binding in man, monkey, dog and rat.

Abstract: The binding of propranolol to plasma has been determined at therapeutic concentrations by equilibrium dialysis in man (93.2%), monkey (99.2%), dog (96.6%) and rat (92.2%) and correlated with its kinetics of disposition after intravenous administration. In nine human subjects the clearance of propranolol from the blood was high and relatively constant at 1.05 liters/min. The apparent volume of distribution (V dβ) which varied 2-fold was proportional to the disposition rate constant, β. In all the species examined Vdβ increased as free drug concentration increased in the order monkey:dog:man:rat, such that the volume of distribution of free drug was constant. This occurred despite large variations in drug clearance among the species (14-92 ml/kg/min), which resulted from differences in hepatic extraction ratio, relative magnitude of liver blood flow and extrahepatic metabolism. When the influence of a variable drug clearance was taken into account, increased drug binding was associated with a decrease in drug half-life. Such differences in binding account in part for species differences in half-life and are entirely responsible for interindividual variation in half-life in man after intravenous administration. These data are consistent with the hypothesis that plasma binding decreases the half-life of propranolol by increasing the rate of drug delivery to its site of elimination. This results because only volume of distribution is dependent on the free drug in the circulation, whereas more than the free fraction of the drug in the circulation is available for clearance by the organs of elimination.

Characterization of the adrenergic receptors mediating a rise in cyclic 3',5'-adenosine monophosphate in rat cerebral cortex

Abstract: Norepinephrine (NE) causes a 4- to 6-fold rise in the cyclic 39, 59-adenosine monophosphate content of chopped rat cerebral cortex. The effect is half-maximal at 6 µM and essentially maximal at 30 µM. The effect of 30 µM NE is only partially blocked by maximally effective concentrations (30 µM) of either propranolol or phentolamine. However, the effect is completely blocked when these inhibitors are added together even at lower concentrations (10 µM each). Isoproterenol (ISO) causes only a 2- to 4-fold rise in cyclic 39, 59-adenosine monophosphate in the same preparation. The effect is half-maximal at 0.4 µM and essentially maximal at 3 µM. The effect of 30 µM ISO is completely blocked by 30 µM propranolol, but phentolamine is without effect. Maximally effective concentrations of NE and ISO together produce an effect similar to that of NE added alone. The results suggest that NE interacts with at least two types of receptors (analogous to alpha and beta ) whereas ISO appears to react with a single type (analagous to beta ). Analysis of the difference in the time courses of the effects of NE and ISO suggests that a slow component of the NE effect may be indirect.

Biogenic amines and narcotic effects. i. modification of morphine-induced analgesia and motor activity after alteration of cerebral amine levels

Abstract: The effect of modification of biogenic amine levels on morphine analgesia and motor activity was studied in the rat. Depletion of brain serotonin by p -chlorophenylalanine (300 mg/kg) or by lesioning of the raphe nuclei clearly did not antagonize morphine analgesia as measured by the hot-plate or tail-flick test. α-Methyltryosine (78 mg/kg) produced a slight potentiation and prolongation of morphine analgesia. Reserpine (5 mg/kg, 24 hours) did not antagonize but instead appeared to produce a slight prolongation of morphine analgesia. However, alterations of amine levels produced rather selective modifications of the hyper- or hypoactivity induced by morphine. Morphine (4 mg/kg) produced an initial excitation lasting two hours followed by a return to normal motor activity. A dose of 16 mg/kg of morphine, however, produced an initial two-hour period of hypoactivity followed by a two-hour period of hyperactivity. p -Chlorophenylalanine pretreatment did not alter the hyperactivity observed after 4 mg/kg of morphine. However, p -chlorophenylalanine pretreatment and lesions in the raphe nuclei not only antagonized but reversed the hypoactivity caused by the higher dose of morphine. α-Methyltyrosine antagonized the immediate and delayed increases in activity produced by 4 and 16 mg/kg of morphine respectively. Moreover, α-methyltyrosine antagonized the hyperactivity observed after chronic (16 mg/kg t.i.d., 5-7 days) morphine administration. Reserpine antagonized both the hypoactivity and the hyperactivity. These data suggest that the motor activity observed after various morphine treatments in rats is dependent, in part, upon a balance between a catecholaminergic system which acts to increase motor activity and a serotonergic system which acts to decrease motor activity. The effects of morphine on these two systems appear to be both dose- and time-dependent.

Self-administration of optical isomers of amphetamine and methylamphetamine by rats

Abstract: Rats intravenously self-administer both optical isomers of amphetamine and methylamphetamine. Responding was maintained under a schedule where every lever-pressing response produced an intravenous drug injections. When self-administration was limited to 6 hour/day sessions, increases in dose produced decreases in response rate and relatively slight inereases in hourly drug intake. Responding was maintained by lower doses of the d -than of the l -isomer of both amphetamine and methylamphetamine. Responding resulted in 2.38 to 2.98 times more l -than d -amphetamine and 4.31 times more l -than d -methylamphetamine being self-administered per hour. At similar molar doses, 1.75 times more l -methylamphetamine than l -amphetamine was self-administered but no difference between d -amphetamine and d -methylamphetamine was found over the range of doses studied. When rats were allowed to self-administer drugs 24 hours/day at doses that produced equal response rates, qualitative effects were similar with both isomers of each drug: alternating periods of responding and no responding, weight loss, body irritation and typically death within two weeks. With onset of self-administration, responding maintained by food and water decreased while activity increased.

Adrenergic-adenosine 3',5'-monophosphate regulation of serotonin N-acetyltransferase activity and the temporal relationship of serotonin N-acetyltransferase activity synthesis of 3H-N-acetylserotonin and 3H-melatonin in the cultured rat pineal gland.

Abstract: The regulation of serotonin N-acetyltransferase actiyity has been studied in cultured pineal glands. Addition of l-norepinephrine (NE) to cultures produces a 10- to 30-fold increase in enzyme activity after six hours of treatment. There is a plateau in enzyme activity for another six hours followed by a spontaneous decrease in enzyme activity to base-line values. NE can stimulate the enzyme over a range of 10-4 to 10-9 M concentratioin. Exposure to NE does not have to be constant for a long-term response. Exposure for as little as 15 minutes to NE produces about a 50% maximal response at six hours. The initial increase in N-acetyltransferase activity is blocked by cycloheximide. Cycloheximide treatment after a gland has been stimulated with NE does not cause a precipitous fall in enzyme activity but does prevent a further increase. Aliphatic amines. indoleamines. an imidazolamine and tyramine are ineffective in stimulating the enzyme. The relative potency of the compounds that stimulate enzyme activity is: l-NE DL-isoproterenol > l-epinephrine > DL-octopamine > d-norepinephrine > 3,4-dihydroxyphenylamine > l-3,4-dihydroxyphenylalanine. The stimulation of enzyme activity is blocked by propranolol and is enhanced by phentolamine. This indicates that the receptor involved is a beta adrenergic receptor and that the response to beta adrenergic stimulation can be influenced by an alpha adrenergic mechanism. The effects of NE on N-acetyltransferase activity are mimicked by dibutyryl cyclic adenosine monophosphate (AMP), which is more effective than theophylline. Cyclic AMP is only slightly effective. The effects of dibutyryl cyclic AMP are blocked by cycloheximide but not by cyclic AMP. The stimulation of N-acetvltransferase by either NE or dibutyryl cyclic AMP is coincident in time and magnitude with the stimulation of the total production of 3H-N-acetylserotonin and 3H-melatonin by glands incubated with 3H-tryptophan. This study describes a striking number of similarities between the factors regulating pineal adenyl cyclase, cyclic AMP, radiolabeled melatonin production from radiolabeled tryptophan, and serotonin N-acetyl-transferase activity in cultured pineal glands. The findings add support to the hypothesis that melatonin production is regulated by the amount of N-acetylserotonin made available for O-methylation and that N-acetylserotonin production by serotonin N-acetyl-transferase is regulated by an adrenergic-cyclic AMP mechanism.

Influence of acetylcholine on contractile force and cyclic nucleotide levels in the isolated perfused rat heart

Abstract: The effects of acetylcholine chloride (ACh) on cardiac contractile force and on myocardial levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP) were studied in spontaneously beating and electrically driven isolated perfused rat hearts. Perfusion of spontaneously beating hearts with Tyrode's solution containing ACh (7.4 x 10-8 M) produced significant decreases in contractile force and heart rate as well as a significant elevation in myocardial cGMP levels. Excellent correlations were obtained between the changes in the concentrations of cGMP and the effects of ACh on heart rate and force of contraction. Myocardial levels of cAMP were decreased by ACh, but this change was not correlated well with either changes in heart rate on contractile force produced by this agent. In electrically driven hearts cGMP levels were increased by ACh infusion (7.4 x 10-8 M), as in the case of the spontaneously beating hearts, and this change was well correlated with the decrease in contractile force. In electrically driven hearts, as in the spontaneously beating preparation, the correlation between reduced cAMP levels and contractile force was not as good as that between elevated cGMP levels and reduced contractile force. The results of the present study suggest that increased intracellular levels of cGMP produced by ACh may be involved in the mediation of the negative inotropic effect of this agent in the isolated perfused rat heart. Furthermore, such increases in cGMP concentrations may be more important than decreases in concentrations of cardiac cAMP with respect to the mediation of this negative inotropic effect.

Disposition of naloxone-7,8-3h in normal and narcotic-dependent men

Abstract: Naloxone-7,8-3H was administered intravenously and orally on separate occasions to the same normal male subject and its disposition was examined. The fate of intravenous naloxone-7,8-3H was also studied in an opiate-dependent subject both while on heroin maintenance and after withdrawal. In all cases the urinary excretioon was rapid but incomplete, nerve exceeding 70% of the dose over 72 hours. Initial plasma concentrations of naloxone were low with a rapid rate of disappearance. Oral naloxone entered plasma quickly but in metabolized form. The volume of distribution, plasma half-life and metabolic clearance rate of naloxone as calculated from the intravenous studies were about 200 liters, 90 minutes and 2500 liters/day, respectively.

Effects of 6-hydroxydopamine treatments on active avoidance responding: evidence for involvement of brain dopamine.

Abstract: Rats treated with pargyline prior to intracisternal injection of 6-hydroxydopamine (6-OHDA) failed to display acquisition of either a shuttle-box avoidance response or a one-way active avoidance response, even when tested as long as 72 days after injection. This treatment had no effect, however, on acquisition of a passive avoidance response. 6-OHDA also caused a significant decrement in performance by rats previously trained in the shuttle-box. When methods of 6-OHDA treatment were employed in which norepinephrine (NE) or dopamine (DA) was preferentially reduced in brain, animals depleted of NE displayed facilitated acquisition of the shuttle-box response, whereas depletion of DA by a single 6-OHDA treatment had little effect. Administration of α-methyltyrosine or reserpine to these preferentially depleted rats performing in the shuttle-box caused a marked decrement in performance by the DA-depleted rats but had little effect on rats depleted of NE. Two injections of 6-OHDA after desipramine treatment, which reduced DA by 85% and NE by 24%, suppressed acquisition of the avoidance response. The data, therefore, suggest that the deficit in avoidance behavior observed in 6-OHDA-treated rats is due mainly to a reduction in brain dopamine and support the view that brain catecholamines are important for the maintenance of conditioned avoidance responding.

Morphine pellet implantation in rats: quantitative assessment of tolerance and dependence

Abstract: The development of tolerance to and physical dependence on morphine was quantitatively examined in rats implanted s. c. with 75-mg morphine pellets. The animals were maintained for up to seven days after pellet implantation and tolerance and physical dependence were assessed at daily intervals. Both tolerance and dependence peaked after three days of pellet implantation and then declined gradually after this time. The withdrawal reaction precipitated by naloxone in morphine-dependent rats was characterized by lacrimation, salivation, an ejaculate-like discharge, diarrhea, hyperactivity and most notably by wet-dog shakes. Since the observed withdrawal synsdrome included virtually every response which had previously been associated with morphine abstinence in the rat, it appears that morphine pellet implantation produces a degree of physical dependence which is at least as great as that which can be obtained with more commonly used techniques. On the basis of these data, it was concluded that the technique of pellet implantation can be used in the rat as a rapid and reliable means of producing a high degree of toleranuce and physical dependence on morphine which are readily quantifiable.