Faculty Bibliography

To study the local anesthetic properties of yohimbine in more detail, its effect was examined in vitro on the scorpion toxin-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate [( 3H]BTX-B) to the gating complex in sodium channel preparations from mouse brain cortex. Both equilibrium and kinetic experiments were carried out. Yohimbine inhibited the specific binding of [3H]BTX-B in the vesicular preparation with an IC50 value of 2.2 X 10(-5) M. This is about one order of magnitude higher than the concentration required for antagonism via the alpha 2-adrenoceptors; however, yohimbine is 7-fold more potent in inhibiting [3H]BTX-B binding than lidocaine. In a concentration-dependent manner, yohimbine increased the dissociation constant (Kd) of high-affinity [3H]BTX-B binding without changing the maximal binding capacity (Bmax). The dissociation rate constant was not affected by yohimbine, suggesting competitive inhibition as opposed to the action of local anesthetics involving an allosteric action via receptor sites distinct from the BTX site. Alpha 2-adrenoceptors are apparently not involved because clonidine and alpha-methyl-noradrenaline had no appreciable effect on [3H]BTX-B binding and did not antagonize the inhibitory effect of yohimbine. The present findings indicate a mechanism of local anesthetic action of yohimbine that differs from that of other local anesthetics such as tetracaine and lidocaine involving direct binding to the BTX site, thereby stabilizing a non-permeable form of the sodium channel

Since ethanol has local anesthetic activity its effect was examined in vitro on the scorpion toxin-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) to the gating complex in sodium channel preparations from mouse brain cortex by equilibrium and kinetic experiments. Ethanol inhibited the specific binding of [3H]BTX-B in a vesicular preparation with an IC50 value of 310 mM and a Hill number of 1.0. Ethanol increased the equilibrium dissociation constant of batrachotoxin in a concentration dependent manner without changing the maximal binding capacity and decreased the half-time of the aconitine-induced dissociation of [3H]BTX-B. Thus, ethanol acts as an apparent competitive inhibitor, allosterically affecting the [3H]BTX-B binding, like other local anesthetics. Results of competition experiments in the presence of different concentrations of ethanol and fixed concentration of tetracaine (and vice versa) are consonant with an interaction of ethanol and tetracaine with the same binding sites. Experiment carried out at 32, 37 and 42 degrees C indicated that the effect of ethanol is not mimicked by increasing the temperature

Alcohol has been shown to inhibit veratridine- and batrachotoxin-stimulated sodium flux in rodent brain synaptosomes. In this study, binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) and uptake of [14C]guanidine in cerebrocortical synaptosomes were measured in naive rats belonging to lines with high or low acute sensitivity to ethanol (ANT, Alcohol Non-Tolerant, vs. AT, Alcohol Tolerant, lines) and to lines with high or low voluntary alcohol consumption (AA, Alko Alcohol, vs. ANA, Alko Non-Alcohol, lines). Our rat line pairs did not differ in the equilibrium binding characteristics of [3H]BTX-B nor in the properties of [14C]guanidine uptake, suggesting that the genetic selection has not modulated the genes or the expression of the genes associated with the voltage-sensitive sodium channels

Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine

The present study was an attempt to answer the question whether the local anesthetic [3H]tetracaine labels sodium channels in mouse brain synaptosomes. Binding of [3H]tetracaine had a Kd of 0.19 microM and a Bmax ranging from 3.7 to 5.2 pmol/mg of protein. Local anesthetics other than tetracaine and cocaine-related compounds inhibited [3H]tetracaine binding with Hill numbers between 0.3 and 0.6. Initiation of the dissociation of [3H]tetracaine binding by an excess of unlabeled tetracaine resulted in non-linear curves. These results are consonant with site heterogeneity, negative cooperativity, or complexities arising from the use of synaptosomal preparations instead of broken membranes. There were important differences between the absolute values of the potency of various local anesthetic drugs and cocaine-related compounds in inhibiting binding of [3H]tetracaine and those in inhibiting binding of [3H]batrachotoxinin A 20-alpha-benzoate. Although there were some effects of manipulating the state of the channel with activating toxins on the apparent rate of association and dissociation of [3H]tetracaine binding, the equilibrium binding was not much affected by the toxins. The results indicate that further characterization is necessary before accepting [3H]tetracaine binding as a valid tool for studying sodium channels

Intermittent s.c. and i.p. injections of cocaine (20 mg/kg; total 430 mg/kg for each animal) for 18 days resulted in locomotor stimulation of mice upon challenge with cocaine on the 25th or 26th day, compared with no locomotor stimulation in a saline-pretreated group. In contrast to the sensitization by intermittent cocaine administration, tolerance was found upon challenge after continuous administration of cocaine by minipumps (25 mg/kg/day; total 450 mg/kg for each animal) on a similar schedule. No differences were found between the sensitized and tolerant groups in the levels of cocaine and benzoylecgonine in plasma and brain 12 min after i.p. administration of a challenge dose of cocaine, suggesting that in these chronic experiments the changes in the locomotor response are not accounted for by dispositional effects. In contrast, in animals treated daily for 2 or 3 days i.p. with cocaine and challenged with cocaine 1 day later, there was both a greater locomotor stimulation and a higher level of brain cocaine than in saline-pretreated animals, suggesting a dispositional effect. Among individual animals there was a positive correlation between their locomotor stimulation by the challenge dose and their brain cocaine concentration

Cocaine and its metabolites were measured in plasma and brain from mice injected i.p. with cocaine and monitored for spontaneous locomotor behavior. Cocaine concentrations in the brain reached peak values within 5 min after administration of cocaine. At all time points between 5 and 60 min the concentrations of cocaine in the brain were 7-fold higher, on the average, than those in plasma. The opposite was true for the concentrations of benzoylecgonine; brain to plasma ratios of benzoylecgonine were approximately 0.1 from 5 to 30 min after i.p. cocaine injection. After i.p. injection of either 10 or 25 mg/kg of cocaine, cocaine disappeared from plasma and brain with a half-life of 16 min and benzoylecgonine disappeared from plasma with a half-life of 62 min. There was good correspondence between locomotor stimulation and concentration of cocaine in the brain measured at 12, 22 and 32 min after i.p. administration of 25 mg/kg of cocaine. Among individual animals there was a significant correlation between their locomotor stimulation and their brain cocaine concentration, indicating that differences in cocaine levels in the brain between animals contribute to their different behavioral response; however, the correlation analysis also indicated the role of other factors determining the locomotor response to cocaine

The effect of acute and chronic nicotine on the metabolism of specific brain proteins was examined by measuring incorporation of labeled valine into protein, with densitometric scanning of proteins resolved by gel electrophoresis. Acute and chronic administration of nicotine (0.4 mg/kg per 30 min for 2 hours, s.c., or 0.5 mg/kg per 30 min for 5 days (Alzet mini-pump implanted subcutaneously] reduced incorporation of [14C]valine administered by approximately 6-7%. The results with chronic nicotine administration indicated a lack of tolerance for this effect of nicotine. Mecamylamine, a nicotinic ganglionic antagonist, does not seem to block the inhibition of protein synthesis. Small increases in protein content were observed in a high- and a low-molecular-weight region of SDS-polyacrylamide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective bands. Results are consonant with selective effects of nicotine on the synthesis or degradation of specific brain proteins

Effects of caffeine on monoamine systems in the mouse brain were studied in three lines of experiments. First, concentrations of 10(-7) M to 10(-2) M of caffeine were tested for their potency in inhibiting the carriers involved in the neuronal uptake of dopamine, norepinephrine, and serotonin. The IC50 of caffeine in inhibiting the first two carriers was approximately 10(-2) M, and that in inhibiting the serotonin was 2 x 10(-3) M. Second, concentrations of 10(-5) M to 10(-3) M of caffeine were tested for their potency in affecting the in vitro KC1-induced release of [3H]dopamine from dopamine terminals in the striatum and from norepinephrine terminals in the hypothalamus, and the release of [3H]serotonin from serotonin terminals in the striatum. Little or no effect was observed. Third, caffeine was administered for 3 weeks to mice via their drinking water at 73, 123, and 162 mg/kg per day. No changes were found in their D2-dopaminergic, 5-HT2-serotonergic, or alpha 1-adrenergic receptors in the striatum or cerebral cortex as compared with animals on normal drinking water. All these neurochemical results are consonant with the interpretation of behavioral studies suggesting that caffeine is an only mildly stimulatory drug that should not be grouped with other psycho-stimulant drugs such as amphetamine and cocaine that do affect monoamine systems in the brain