Alteration of Cx43:Cx40 expression ratio in A7r5 cells.

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.

Gap junction permeability is diminished in proliferating vascular smooth muscle cells.

In atherosclerosis and hypertension, vascular smooth muscle cells (SMCs) are stimulated to proliferate and exhibit enhanced gap junction protein expression. Our goal was to determine whether gap junction function differs in proliferating vs. growth-arrested SMCs. A7r5 cells (embryonic rat aortic SMCs) did not proliferate in media with reduced serum ( approximately 90% of cells in G0/G1 phase after 48-96 h in 1% fetal bovine serum). Dye coupling was less but electrical coupling was comparable in proliferating vs. growth-arrested A7r5 cells, suggesting differences in junctional permselectivity. In growth-arrested cells, junctional conductances measured with potassium glutamate, tetraethylammonium chloride, and KCl were well predicted by the conductivities of these solutions. In contrast, junctional conductances measured with potassium glutamate and tetraethylammonium chloride in proliferating cells were significantly greater than predicted by the conductivities of these solutions. These results suggest that junctions between growth-arrested cells are permeated equally well and simultaneously by anions and cations, whereas junctions between proliferating cells are poorly permeated by large molecules of either charge and equally well but not simultaneously by small anions and cations. The data indicate that A7r5 cells regulate chemical coupling independent of electrical coupling, a capacity that could facilitate growth control while protecting vasomotor responsiveness of vessels.

D1 agonist dihydrexidine releases acetylcholine and improves cognitive performance in rats.

Dihydrexidine is a selective, full-efficacy dopamine D1 receptor agonist that has displayed therapeutic potential in Parkinson's disease by reversing motor deficits of MPTP-treated monkeys. The present study monitored the effects of dihydrexidine on acetylcholine release in rat brain by using in vivo microdialysis. Moderate doses of dihydrexidine [3 and 10 mg/kg, intraperitoneally (I.P.)] elevated extracellular concentrations of acetylcholine by 40-60% in rat striatum; higher doses did not significantly alter acetylcholine release. SCH 23390 blocked the dihydrexidine-induced increase, indicating a D1 receptor-mediated action. A more robust stimulatory effect of dihydrexidine on acetylcholine release was observed in prefrontal cortex (to 300% of basal output) than in striatum. Dihydrexidine was also evaluated in a passive avoidance procedure in rats to determine if its neurochemical effects translated into cognition-enhancing activity; in this assay, dihydrexidine (0.3 mg/kg, I.P.) significantly improved the scopolamine-induced deficits. The results of these studies suggest that the acetylcholine-releasing properties of dihydrexidine and other D1 agonists may underlie their cognition-enhancing activity and thus may have clinical value in the treatment of dementia.

Effects of strychnine-insensitive glycine receptor ligands in rats discriminating dizocilpine or phencyclidine from saline.

Several pharmacologically distinct sites are known to modulate the N-methyl-D-aspartate (NMDA) receptor/ion complex, including a site within the ion channel which binds uncompetitive antagonists like phencyclidine (PCP) or dizocilpine. Glycine acts as a co-agonist for activation of the NMDA receptor complex through a strychnine-insensitive receptor, which is a potential target for novel therapeutic agents (e.g., anticonvulsants, antidepressants). We evaluated the behavioral effects of glycine receptor ligands in rats trained to discriminate either dizocilpine or PCP from saline, to predict whether glycine receptor ligands might induce undesirable PCP-like subjective effects in humans. Dizocilpine ([+]-MK-801), (-)-MK-801 and PCP produced dose-dependent substitution in these rats with potencies in accord with NMDA receptor affinity. Pentobarbital and drugs acting at other sites of the NMDA receptor, including competitive antagonists (NPC 12626 and LY 274614) and the polyamine antagonist, ifenprodil, did not substitute for either dizocilpine or PCP. In contrast to the uncompetitive antagonists like PCP, none of the strychnine-insensitive glycine receptor ligands substituted. Neither the full agonist, glycine; the partial agonists, 1-amino-1-cyclopropanecarboxylic acid, D-cycloserine or (+)-3-amino-1-hydroxypyrrolid-2-one; nor the antagonists, 7-chloro and 5,7-dichlorokynurenic acid, mimicked the discriminative stimulus effects of dizocilpine or PCP. Further, co-administration of 1-amino-1-cyclopropanecarboxylic acid did not significantly enhance the discriminative stimulus effects of dizocilpine. Intracerebroventricular administration of D-serine, a selective agonist of the strychnine-insensitive glycine receptor, neither mimicked nor blocked the discriminative stimulus effects of PCP. These data suggest that functional antagonists of the strychnine-insensitive glycine receptor may be devoid of the subjective side effects characteristic of NMDA channel ligands.

The D1 agonist dihydrexidine releases acetylcholine and improves cognition in rats.

Neurochemical and behavioral studies have elucidated extensive interactions between dopaminergic and cholinergic systems in brain areas associated with movement and cognition. The initial goal of these studies was to evaluate the effect of the anti-Parkinson drug dihydrexidine (DHX), a dopamine D1 full-efficacy agonist, on brain acetylcholine (ACh) release using in vivo microdialysis techniques. Moderate doses of DHX (3 and 10 mg/kg) produced approximately a 50% increase in striatal ACh release that was blocked by the D1 agonist SCH23390 (0.3 mg/kg). A higher dose of DHX (17.5 mg/kg) was less effective in raising striatal ACh, possibly due to D2 receptor activation. In frontal cortex, DHX (10 mg/kg) evoked a more robust increase in ACh release (+200%) that was blocked by SCH23390 (0.3 mg/kg). Since elevations in brain ACh are associated with cognitive improvement, the effectiveness of DHX in a passive avoidance model of learning and memory was also evaluated. These studies revealed a significant improvement in performance by 0.3 mg/kg DHX in scopolamine-induced amnestic rats. These results provided support for the hypothesis that DHX improves cognitive performance as a consequence of ACh release in relevant brain regions. Further, D1 agonists may have novel therapeutic potential in the treatment of dementia.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy"): pharmacology and toxicology in animals and humans.

(+/-)3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy"), a ring-substituted amphetamine derivative first synthesized in 1914, has emerged as a popular recreational drug of abuse over the last decade. Pharmacological studies indicate that MDMA produces a mixture of central stimulant and psychedelic effects, many of which appear to be mediated by brain monoamines, particularly serotonin and dopamine. In addition to its pharmacologic actions, MDMA has been found to possess toxic activity toward brain serotonin neurones. Serotonergic neurotoxicity after MDMA has been demonstrated in a variety of experimental animals (including non-human primates). In monkeys, the neurotoxic dose of MDMA closely approaches that used by humans. While the possibility that MDMA is also neurotoxic in humans is under investigation, other adverse effects of MDMA in humans have been documented, including various systemic complications and a number of untoward neuropsychiatric sequelae. Notably, many of the adverse neuropsychiatric consequences noted after MDMA involve behavioral domains putatively influenced by brain serotonin (e.g., mood, cognition and anxiety). Given the restricted status of MDMA use, retrospective clinical observations from suspecting clinicians will probably continue to be a primary source of information regarding MDMA's effects in humans. As such, this article is intended to familiarize the reader with the behavioral pharmacology and toxicology of MDMA, with the hope that improved recognition of MDMA-related syndromes will provide insight into the function of serotonin in the human brain, in health as well as disease.

Evaluation of the neurotoxicity of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine, PMMA).

These studies assessed the neurotoxic potential of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine; PMMA), an amphetamine analog that has surfaced in the illicit drug market. Repeated subcutaneous injections of PMMA caused lasting, dose-related reductions in regional brain concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and in the density of [3H]paroxetine-labelled 5-HT uptake sites. Comparison of the neurotoxic potential of PMMA to that of para-methoxyamphetamine (PMA) and 3,4-methyl-enedioxymethamphetamine (MDMA) showed that equivalent doses of PMMA and PMA (80 mg/kg) produced comparable depletions of 5-HT, but that these depletions were not as pronounced as those induced by a lower dose of MDMA (20 mg/kg). Striatal DA was not affected on a long-term basis by any of the ring-substituted amphetamines evaluated in this study. These data suggest that PMMA, like PMA and MDMA, produces long-term (possibly neurotoxic) effects on brain serotonin neurons, but that PMMA is less potent than MDMA as a 5-HT neurotoxin. Further, they raise concern over the illicit use of PMMA since humans could be more sensitive than rodents to the 5-HT neurotoxic effects of PMMA and related drugs.

Assessment of the role of alpha-methylepinine in the neurotoxicity of MDMA.

To assess the potential involvement of metabolism of 3,4-methylenedioxymethamphetamine (MDMA) to the catechol alpha-methylepinine in producing serotonergic neurotoxicity, we attempted to correlate aspects of this reaction with the neurotoxicity profile of MDMA. In contrast to the stereoselectivity of S-(+)-MDMA in causing persistent declines in rat brain 5-hydroxyindole levels, no stereochemical component to the metabolic reaction was apparent. Rat liver microsomes generated a significantly greater amount of alpha-methylepinine than did mouse microsomes, but similar amounts of metabolite were produced by brain microsomes from the two species. Formation of alpha-methylepinine by hepatic, but not brain, microsomes was inhibited by SKF 525A and induced by phenobarbital, possibly indicating a tissue specificity in cytochrome P-450-dependent metabolism of MDMA. To directly assess whether alpha-methylepine is a likely mediator of MDMA neurotoxicity, the compound was administered intracerebroventricularly. No persistent declines in biogenic amines or their metabolites were observed one week following treatment. These data suggest that alpha-methylepinine alone is not responsible for the neurotoxic effects of MDMA.

MDMA transiently alters biogenic amines and metabolites in mouse brain and heart.

(+-)-3,4-Methylenedioxymethamphetamine (MDMA) (10, 20, and 40 mg/kg) was administered to male CF-1 mice which were sacrificed 3, 6, or 24 hours posttreatment for analysis of brain and cardiac biogenic amines and metabolites. In contrast to reported effects of MDMA in the rat, the highest dose of MDMA transiently elevated mouse brain 5-hydroxytryptamine (5-HT) 3 hours following drug treatment. Levels of dopamine were not significantly affected. 5-Hydroxyindoleacetic acid and dihydroxyphenylacetic acid were significantly lowered by MDMA at the two early time points. The highest dose of MDMA produced a transient depletion of norepinephrine in mouse brain and heart tissue. Only the effects of MDMA on cardiac norepinephrine were prevented by pretreatment of animals with desipramine. A regimen consisting of four daily doses of 40 mg/kg MDMA only produced significant declines in 5-HIAA, dopamine and homovanillic acid levels one week following the last dose. These data confirm previous reports that mice are resistant to the neurotoxic effects of MDMA suggesting that a species variation in response to MDMA exists.

Nocturnal depletion of hypothalamic dynorphin in anorexic Walker-256 tumor-bearing rats.

Rats implanted with the Walker-256 (W-256) tumor exhibit marked anorexia that is most apparent at night. In this model, the hypothalamic kappa opioid system was examined for deficits that might contribute to this tumor-induced anorexia. In anorexic tumor-bearing rats (TBR), nocturnal levels of ir-DYN-8 were significantly reduced in the hypothalamus, but ir-DYN-17 levels were not. Accumulation of 3H-etorphine or 3H-ethylketocyclazocine, a putative ligand for the kappa receptor subtype, was not increased in the hypothalamus of the TBR, as might have been expected if there were less endogenous dynorphin to occupy the opioid receptors in this region. In vitro binding assays with 3H-ethylketocyclazocine indicated that dynorphin depletion in the TBR was not sufficient to increase the numbers of kappa opioid receptors in the hypothalamus. Also, the sensitivity of kappa opioid receptors involved in feeding was not altered in the TBR as indicated by an intact feeding response to ketocyclazocine. In summary, the marginal deficits of hypothalamic dynorphin in W-256 tumor-bearing rats that coincide with the phase of tumor-induced anorexia may contribute to the reduction in food intake.

Stereochemical effects of 3,4-methylenedioxymethamphetamine (MDMA) and related amphetamine derivatives on inhibition of uptake of [3H]monoamines into synaptosomes from different regions of rat brain.

3,4-Methylenedioxymethamphetamine (MDMA) is a recently popularized recreational drug, although some have advocated its psychotherapeutic potential. Since the pharmacology of MDMA is largely uncharacterized, the stereochemical profiles of MDMA and some of its homologs were derived on inhibition of synaptosomal uptake of [3H]monoamines and compared to those of amphetamine and the hallucinogenic phenylisopropylamine 2,5-dimethoxy-4-methylamphetamine (DOM). In contrast to the 5-fold stereoselectivity observed with amphetamine, only the S-(+) enantiomer of MDMA and 3,4-methylenedioxyamphetamine (MDA) inhibited [3H]dopamine uptake into striatal synaptosomes. Neither stereoisomer of the alpha-ethyl homolog of MDMA, N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), inhibited [3H]dopamine uptake. The two stereoisomers of amphetamine and the MDMA-related compounds were equipotent in inhibiting [3H]norepinephrine uptake into hypothalamic synaptosomes. Both stereoisomers of MDMA, MDA and MBDB were potent inhibitors of [3H]serotonin uptake into hippocampal synaptosomes, but only S-(+)-amphetamine produced an appreciable inhibition of [3H]serotonin uptake. Neither stereoisomer of DOM inhibited synaptosomal uptake of any [3H]monoamine. These results suggest that MDMA and its homologs may be more closely related to amphetamine rather than to DOM in their biochemical mode of action. The pronounced effects of the methylenedioxy-substituted compounds on [3H]serotonin and [3H]norepinephrine uptake implicate these neurotransmitters in the pharmacological effects of these drugs.

Effects of 2% sodium chloride imbibition on various opiate related hyperphagic conditions.

Dynorphin is one of the most potent appetite stimulants among the endogenous opioids. In this study, we describe the anorexic effects of 5 days of forced 2% NaCl drinking in rats, a regimen which depletes vasopressin as well as dynorphin in the neurohypophysis. Feeding induced by direct activation of kappa-opioid receptors with ketocyclazocine was unaffected by the NaCl regimen. However, 2% NaCl imbibition reduced 2-deoxy-D-glucose (2-DG) induced feeding by 65% and spontaneous nocturnal feeding by 38%. Feeding subsequent to 24 hour food deprivation was not decreased. Naloxone-resistant hyperphagia induced by insulin and spontaneous daytime feeding were also not reduced. The combination of naloxone (3.0 mg/kg) and the NaCl regimen produced an additional 50% reduction in 2-DG induced feeding and an extra 40% decrease in nocturnal feeding. Naloxone, given with 2% NaCl to food deprived animals, retained its appetite suppressing activity, indicating that the NaCl regimen did not deplete the endogenous opioid which mediates food deprivation hyperphagia. These results demonstrate that 2% NaCl imbibition suppresses certain opioid mediated hyperphagias. However, the failure of 2% NaCl to affect all of the naloxone-sensitive types of feeding and the independence of naloxone-sensitive and NaCl-sensitive components suggests that NaCl drinking does not deplete dynorphin in the brain areas which mediate opiate-sensitive hyperphagias.