James A. Ruth

An analysis of cocaine effects on locomotor activities and heart rate in four inbred mouse strains.

The effects of cocaine on Y-maze activity and heart rate have been examined in four inbred strains of mouse (BALB, C57BL, C3H and DBA). In addition, brain [3H]-cocaine concentrations were measured at the time of maximal response to cocaine. Cocaine produced a dose-related increase in Y-maze cross activity in C3H, DBA and C57BL, with C3H mice being considerably more sensitive than DBA or C57BL. Cocaine was without effect on Y-maze cross activity in BALB mice. Cocaine produced a biphasic effect on rearing activity in C3H mice, a dose related depression in BALB mice, and was without effect on C57BL and DBA mice. At the highest dose studied (15 mg/kg), cocaine produced a small decrease in heart rate in C3H mice. Strain differences in behavior were maximal 15 minutes after a dose of 5 mg/kg, IP. At this dose and time interval, brain [3H]-cocaine concentrations were not significantly different among the four strains of mice. The results suggest a genetically-determined difference in CNS sensitivity to cocaine.

Uptake of L-[3H] glutamic acid by crude and purified synaptic vesicles from rat brain.

Abstract Rat brain synaptic vesicles suspended in a medium comprised of potassium tartrate displayed saturable accumulation of L-[ 3 H] glutamic acid at 37° (Km 2.0 × 10 −4 M; 311±13 pmol/mg protein), which was stable for periods up to 60 min. The accumulation was temperature sensitive and partially ATP-dependent, uptake levels being reduced to 18.7±0.8 pmol/mg protein at 4°, and to 141±4 pmol/mg protein in the absence of ATP. Fractionation of a crude vesicle preparation on a discontinuous sucrose gradient demonstrated the accumulation to be specifically associated with the synaptic vesicle fraction.

Enkephalins modulate the responsiveness of rat atria in vitro to norepinephrine.

Potential interactions between opiate peptides and catecholamines in mammalian heart were examined using isolated spontaneously beating rat atria as a test system. Methionine-enkephalin (ME), leucine-enkephalin (LE), phe-met-arg-phe amide (FMRFamide), D-ala2, N-methyl-phe4, met (O)5-ol-enkephalin (FK 33-834), methionine-enkephalin arg6 arg7 (ME arg6 arg7) and beta-endorphin had no effect on basal beating rate of isolated atria at all concentrations up to 10(-5) M. The positive chronotropic effect of norepinephrine (NE) on atrial rate is, however, significantly attenuated by enkephalin peptides. Thus, the maximal chronotropic effect of NE (an increase from 317 +/- 7.0 to 473 +/- 7.3 beats per minute (bpm) in 250 gm rats at a dose of 10(-5)M NE) is decreased by 42% in the presence of 10(-7)m ME. The action of ME is completely blocked by addition of 10(-7)M naloxone, which by itself has no effect on NE-induced positive chronotropy or basal beating rate. The dose-effect curve for ME attenuation of NE-induced positive chronotropy is bell-shaped, i.e., both 10(-8) M and 10(-5) M ME have no significant effect on NE positive chronotropy. Other enkephalin peptides acted in a similar manner to ME; LE (10(-7) M) and FK 33-834 (10(-8) M) decreased maximal NE-induced positive chronotropy 42 and 27%, respectively. The molluscan cardioexcitatory peptide FMRFamide (10(-7) M) also decreased maximal NE positive chronotropy, about 30%. In contrast, beta-endorphin did not significantly affect NE stimulation of atrial rate. We conclude that enkephalins can modulate the noradrenergic responsiveness of rat atria in vitro. The possible physiological relevance of this interaction is discussed.

NMDA agonists and antagonists alter the hypnotic response to ethanol in LS and SS mice

Involvement of glutamate neurotransmission in the differential response of long-sleep (LS) and short-sleep (SS) mice to acute ethanol was examined by measuring the effect of centrally administered glutamate receptor agonists and antagonists on blood ethanol concentration (BEC) at loss of righting response following intragastric administration of ethanol. NMDA coinjected with glycine, and quinolinic acid (QA), decreased sensitivity to ethanol in both lines of mice. SS mice were more sensitive to QA than were LS. The NMDA antagonists 2-amino-5-phosphonovaleric acid (APV), MK-801 and an inhibitor of glutamate synthesis, methionine sulfoximine, increased sensitivity to ethanol in both lines of mice. MK-801 effects were line dependent with SS being more sensitive. In addition, coinjection of APV, Mg++ or Zn++ with QA blocked the decrease in sensitivity seen with QA alone. These results demonstrate that NMDA agonists and antagonists alter the acute hypnotic response to ethanol in both LS and SS mice, and support the hypothesis that ethanol exerts its effects in part by altering glutamatergic neurotransmission.

Leucine-enkephalin modulation of catecholamine positive chronotropy in rat atria is receptor-specific and calcium-dependent.

Leucine-enkephalin (LE) at 10(-8) M reduces the maximum chronotropic response of isolated spontaneously beating rat atria to exogenously added (-)-norepinephrine (NE) by approximately 27%, with no effect on the NE ED50 (1.5 X 10(-7) M) for positive chronotropy. This modulatory effect of LE is completely blocked by addition of 10(-7) M naloxone, and seems to be catecholamine-receptor specific, since the positive chronotropic response to forskolin is unaltered in the presence of LE. Isoproterenol (ISO)-induced positive chronotropy is also attenuated by LE. This effect is markedly dependent on the extracellular calcium concentration: LE actually causes a greater than two-fold enhancement of the positive chronotropic effect of ISO at low (0.5 mM) extracellular calcium concentration. A possible role for enkephalins to modulate catecholamine action on the heart via an alteration of catecholamine-induced inward calcium flux is discussed.

Metabolism of trans, trans-muconaldehyde by aldehyde and alcohol dehydrogenases: Identification of a novel metabolite

The metabolism of trans,trans-muconaldehyde (MA), a highly reactive alpha,beta-unsaturated dialdehyde, was examined in vitro using purified yeast alcohol and aldehyde dehydrogenases (ADH and ALDH, respectively). In the presence of NAD(+)-fortified ALDH, the mono-oxidation product (acid/aldehyde) was the primary metabolite formed with trace amounts of the dioxidation product (trans,trans-muconic acid). In NADH-fortified reactions with ADH, both the mono- and direduction products (hydroxy/aldehyde and dihydroxy, respectively) were readily detected. Oxidation and reduction products of MA were formed in incubates containing both dehydrogenases together with either NAD+ or NADH. Unexpectedly, an additional metabolite was detected, which was a major product in both NAD(+)- and NADH-fortified systems containing ALDH and ADH in combination and whose formation could be inhibited by pyrazole (an ADH inhibitor). ALDH-mediated oxidation of a synthetic standard of the hydroxy/aldehyde derivative of MA resulted in formation of this new metabolite, which was also a major product formed by rat hepatocytes incubated with MA. Using HPLC/photodiode array detection, the new metabolite was found to cochromatograph and have a uv spectrum identical to that of a synthetic standard of the hydroxy/acid derivative of MA. The metabolite was confirmed as the hydroxy/acid derivative of MA after preparative HPLC, TMS derivatization, and GC/MS analysis. The hydroxy/acid metabolite was not formed during ADH-mediated reduction of the mono-oxidation product of MA, suggesting that this metabolite was formed by yeast dehydrogenases via a primary reduction of MA and subsequent oxidation of the hydroxy/aldehyde to the hydroxy/acid. These data show that the hydroxy/acid derivative is a novel metabolite of MA, which arises from the interaction of both oxidative and reductive routes of metabolism.

Differential inhibition of synaptosomal accumulation of [3H]-monoamines by cocaine, tropacocaine and amphetamine in four inbred strains of mice

The relative ability of cocaine, tropacocaine and amphetamine to inhibit the uptake of [3H]norepinephrine (NE), [3H]dopamine (DA) and [3H]5-hydroxytryptamine (5HT) was examined in whole brain synaptosomes from BALB, C3H, C57BL and DBA inbred mouse strains. With inhibition of [3H]NE uptake, synaptosomes from BALB and C57 mice were substantially more sensitive to cocaine inhibition than those from DBA or C3H. Moreover, with BALB and C57 tissue, amphetamine was as potent as cocaine, whereas with C3H and DBA, amphetamine and tropacocaine were much less potent inhibitors of [3H]NE uptake. With respect to [3H]DA accumulation, synaptosomes from BALB, C57 and DBA were equally sensitive to cocaine inhibition, while C3H synaptosomes were significantly less sensitive. In each of the four strains, amphetamine was more potent than cocaine, and tropacocaine far less potent. The relative potencies of the three drugs varied significantly among the four strains. With [3H]5HT accumulation, synaptosomes from DBA were exquisitely sensitive to cocaine inhibition, followed by BALB and lastly by C57 and C3H. In each of these strains, amphetamine and tropacocaine were equipotent at [3H]5HT inhibition, and less potent than cocaine. The results suggest that there are pronounced genetic differences in sensitivity to monoamine uptake inhibition by cocaine, which may arise from genetic differences in either carrier topology or other site of cocaine interaction. The results further suggest that genetic behavioral differences to cocaine and amphetamine may involve complex neurotransmitter interactions.

Leucine enkephalin antagonizes norepinephrine-induced 45Ca++ accumulation in rat atria

Exposure of rat atrial slices to 10(-5) M norepinephrine (NE) for 10 minutes increases 45Ca++ accumulation from 1.64 +/- 0.10 to 2.23 +/- 0.06 nmol/mg tissue. In the presence of leucine enkephalin (10(-8) M), NE-stimulated 45Ca++ uptake is reduced to 1.44 +/- 0.10 nmol/mg tissue. The effect of leu-enkephalin is reversed in the presence of 10(-7) M naloxone, NE-stimulated 45Ca++ uptake being increased to 2.17 +/- 0.15 nmol/mg tissue. The results support a direct interaction of leu enkephalin with beta-agonist-stimulated Ca++ flux in rat atria, and correlate with the previously reported enkephalin antagonism of NE-induced positive chronotropy in the same tissue.

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

Abstract The MgATP-stimulated accumulation of (-)- 3 H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K + concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)- 3 H-NE (Km 1 × 10 −6 M) in this system (37°) was examined under saturating (10 −5 M) and non-saturating (2 × 10 −7 M) concentrations of NE. At 10 −5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated in vivo ) and 6.0±0.79 (without MgATP). At 2×10 −7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2 x 10 −7 M).

Leucine-enkephalin increases norepinephrine-stimulated chronotropy and 45Ca++ uptake in guinea-pig atria

Norepinephrine (NE) (10(-5) M) increases the beating rate of isolated, spontaneously beating guinea-pig atria 78 +/- 3 beats per minute. Leucine-enkephalin (10(-7) M) increases the maximal chronotropic response to NE by 38%, i.e., by 30 beats per minute. In the presence of 10(-7) M leucine-enkephalin and 10(-7) M naloxone, the chronotropic response to NE is reduced to 38 +/- 3 beats per minute, a value observed in the presence of naloxone alone. Neither naloxone nor leucine enkephalin significantly altered the inotropic response to NE, or significantly altered the basal beating rate. Parallel effects were observed when 45Ca++ uptake by atrial tissue was examined. Incubation of atrial slices with 10(-5) M NE for 10 minutes minimally stimulated 45Ca++ uptake from 1.27 +/- 0.04 to 1.45 +/- 0.17 nmol/mg tissue. In the presence of 10(-7) M leucine-enkephalin, 45Ca++ uptake was increased to 1.95 +/- 0.14 nmol/mg tissue. 45Ca++ uptake was reduced to control values (1.19 +/- 0.09 nmol/mg tissue) in the presence of NE, leucine-enkephalin and naloxone (10(-7) M). The data are consistent with a leucine-enkephalin augmentation of NE-induced chronotropy in guinea-pig atria due to an enhancement of NE-dependent Ca++ accumulation. This effect of leucine-enkephalin is opposite that previously reported for rat atria, in which leucine-enkephalin inhibits both NE-induced positive chronotropy and Ca++ influx.