Inhibition of the in vivo biosynthesis and changes of catecholamine levels in rat brain after α-methyl-p-tyrosine; time- and dose-response relationships

Summary Male Sprague-Dawley rats were given 0.407 mmoles/kg of D,l--methyl-p-tyrosine methylester HCl (H44/68; -MT) at eleven time-points between 0–24h, or 8 doses between 0.013–1.628 mmoles/kg of the drug at 1 h before i. v. injection of 160 Ci tyrosine-2,6-³H. The rats were killed 15 min after tyrosine-³H and brain -MT, tyrosine and catecholamines (endogenous and labelled), and plasma -MT and tyrosine (-³H) were chromatographically isolated before being assayed spectrophotofluorimetrically (endogenous) or by liquid scintillation methods (labelled compounds).A delayed penetration of -MT from plasma into brain, different elimination rates of -MT in plasma and brain, and decreasing brain/plasma drug concentration on increasing -MT dosages indicated, that -MT in brain and plasma belong to different pharmacokinetic compartments.The endogenous levels of catecholamines in the time-response experiment, declined to a minimum 4 h after -MT administration, where the dopamine level was 38% and the noradrenaline level 51% of the saline controls. Kinetic data of the catecholamine elimination is given. In the dose-response experiment the decrease in the endogenous catecholamine levels was doserelated up to 0.407 mmoles/kg of -MT, with no further decline on higher doses.The maximal inhibition of brain catecholamine synthesis occurred within 30 min after -MT administration and the inhibition correlated better with the brain than with plasma -MT content. The inhibition was dose-related with a maximal synthesis inhibition of 95% for dopamine and 80% for noradrenaline at the highest dose of -MT. The duration of synthesis inhibition and storage depletion were shorter for noradrenaline (12 h) than for dopamine (16 h). Further, the ED₅₀ for synthesis inhibition of dopamine (0.057 mmoles/kg) was half of the ED₅₀ for synthesis inhibition of noradrenaline (0.117 mmoles/kg). This might suggest different sensitivities towards -MT or different availabilities of -MT in the two neuron populations.At the three highest doses of -MT there were signs of interference with the uptake process for tyrosine from plasma into the brain. This was indicated by increased plasma levels and decreased brain levels of tyrosine (-³H).     

Different changes in striatal dopamine metabolism induced by nicotine in mice kept at different ambient temperatures

Summary Further information about the nicotine-induced changes in striatal dopamine metabolism in hypothemic mice was searched by measuring the contents of dopamine and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; 3-methoxytyramine, 3-MT; and homovanillic acid, HVA) after blocking the synthesis of dopamine by-methyl-p-tyrosine (-MT). This method gave a possibility to study the effect of nicotine on the metabolism of dopamine in two pools (the cytoplasmic newly-synthesized dopamine and the granular dopamine). 3 mg/kg of (–)nicotine was given s.c. four times, at 110, 80, 50 and 20 min, and-MT (250 mg/kg i.p.) at 60 min before sacrifice. To prevent the peripheral effects of nicotine all mice were given hexamethonium (10 mg/kg i.p.) at 140 min before sacrifice. Hexamethonium did not alter striatal dopamine metabolism. Experiments were performed at 20–22°C at which temperature nicotine induced hypothermia or at 32–34°C.The-MT-induced proportional decrease of 3-MT content was clearly less than that of dopamine content. On the contrary the-MT treatment decreased the DOPAC content proportionally more than the dopamine content. Thus DOPAC could not be solely formed from the same dopamine pool as 3-MT. These results indicate that 3-MT reflects best the metabolism of the granular dopamine and DOPAC that of the newly-synthesized dopamine.In hypothermic mice nicotine administration reduced the-MT-induced depletion of the dopamine content. Nicotine also decreased the 3-MT content and this effect was not altered by-MT. These results indicate that nicotine prevents the metabolism and release of the granular dopamine. The nicotine-induced decrease of the HVA content did not occur in-MT treated mice, which suggests that nicotine also diminished the release of the newly-synthesized dopamine.In mice kept at 32–34°C nicotine tended to enhance the-MT-induced depletion of the dopamine content and nicotine alone did not decrease the 3-MT content. Thus in these mice nicotine tended to increase the release of the granular dopamine. The nicotine-induced increases of HVA and DOPAC contents in these mice were reduced by-MT, which indicates that nicotine increased also the metabolism and probably the release of the newly-synthesized dopamine.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

Effects of some α-adrenoceptor agonists and antagonists on the guinea-pig ileum

Summary The purpose of the present study was to further characterize the -adrenoceptors located on parasympathetic fibres. Segments of guinea-pig ileum were stimulated by transmural electrical pulses, and the ensuing contractions, which are due to the release of acetylcholine from postganglionic parasympathetic fibres, were monitored. Clonidine and tramazoline, which are thought to act preferentially on presynaptic -adrenoceptors, reduced the contractions, whereas phenylephrine and methoxamine, postsynaptic -adrenoceptor agonists, were ineffective. Contractions induced by acetylcholine were not changed by clonidine but were abolished by atropine. Yohimbine, piperoxan, phentolamine and thymoxamine reversed or prevented the inhibitory effect of clonidine. Prazosin and AR-C239 did not antagonize this effect. The inhibitory effect of tramazoline was antagonized by piperoxan but not by AR-C239 or by prazosin. Naloxone did not alter the action of clonidine, and piperoxan did not change the inhibitory effect of morphine.In conclusion, these experiments suggest the presence on cholinergic postganglionic fibres of both opiate receptors and -adrenoceptors. The latter appear to resemble more closely ₁-adrenoceptors than ₁-adrenoceptors.     

The relationship between amphetamine antagonism and depletion of brain catecholamines by alpha-methyl-p-tyrosine in rats

Summary The time-course and the dose-response relationship for the antagonistic effect of alpha-methyl-p-tyrosine methyl ester HCl H 44/68 (-MT) on damphetamine (10.6 moles/kg) induced increase in motor activity was studied. The effect of amphetamine was gradually reduced from 30–60 min to a minimum at 1–4 h after the administration of 0.407 mmoles/kg of -MT. From (4-) 8 h the amphetamine response started to reappear and the original response was restored completely at 16 h after -MT. The dose-response curve showed, that between 0.051–0.41 mmoles/kg of -MT, given 1 h before amphetamine, there was a gradual reduction of the amphetamine response; doses above 0.41 mmoles/kg did not cause any further effect.The antiamphetamine action of -MT was compared with its time-and dose-dependent effects of inhibition of synthesis and reduction of stores of brain catecholamines. It was found, that the antiamphetamine action was more closely correlated with the reduction of the levels of brain dopamine, than with the brain noradrenaline levels. Further, the inhibition of catecholamine synthesis per se did not appear to be a sufficient condition for -MT induced antagonism of amphetamine These finding support the view that amphetamine is dependent on a substantial portion of the brain pool of dopamine and possibly noradrenaline rather than on very small, newly synthesized pools of these neurotransmitters.     

Effects of low and high doses of l-Dopa on the tetrabenazine or α-methyltyrosine-induced suppression of behaviour in a successive discrimination task

The effect of the administration of l-Dopa, 10 or 100 mg/kg i.p., on the -methyltyrosine methylester HCl (-MT; 250+50 mg/kg i. p.) or tetrabenazine (TBZ; 2 mg/kg i.p.)-induced suppression of a successive discrimination-conditioned avoidance task has been studied. It was found that administration of the high l-Dopa dose resulted in a reversal of the -MT- or TBZ-induced suppression of the avoidance behaviour, although the discrimination was lost. Restoration of the avoidance behaviour and the correct discrimination was obtained with the low l-Dopa dose after -MT but not after TBZ. The difference in the behavioural response to l-Dopa after -MT as compared to the response obtained after TBZ is discussed in terms of the availability of the catecholamines formed from l-Dopa for release by the nerve impulses. Biochemical determinations of brain noradrenaline and dopamine were made in parallel. Further, a simple and reliable method for the aquisition of a successive discrimination in the rat is described.     

Effect of haloperidol on reflex activation of rat α-motoneurones

Summary Effects of haloperidol on rat flexor and extensor -motoneurones were studied in ventral roots of laminectomized rats under halothane anesthesia. The -motoneurones were activated by tetanic stimulation of low-threshold afferents (group I and II), either of the ipsilateral peroneal nerve (flexor -motoneurones) or gastrocnemius-soleus nerve (extensor -motoneurones).Haloperidol, given in the doses of 0.075, 0.15 and 0.30 mg/kg i.p. inhibited the reflex activation of flexor -motoneurones; higher doses seemed to be more effective than lower ones. Apomorphine (2 mg/kg s.c.) partially antagonized the inhibitory action of haloperidol with some latency. Higher doses of haloperidol (0.15–0.60 mg/kg i.p.) also inhibited the reflex activation of extensor -motoneurones; this inhibitory effect was, at least for a short time, antagonized by apomorphine (2 mg/kg s.c.).The threshold for reflex activation both of flexor and extensor -motoneurones was raised by haloperidol and lowered by a subsequent administration of apomorphine.Our results suggest that akinesia and catalepsy, induced in rats by haloperidol might be, at least in part, due to a decrease in sensitivity of -motoneurones to proprioceptive stimuli.     

Enhancement by α-fluoromethylhistidine of the thiopental sleep-prolonging action of Δ9-tetrahydrocannabinol

The effect of -fluoromethylhistidine (-FMH), a specific inhibitor of histidine decarboxylase, on the potentiation of thiopental-induced sleep by ⁹-tetrahydrocannabinol (THC), which inhibits the histamine turnover in the brain, was examined in mice and rats. The sleeping time after injection of thiopental sodium (40 mg/kg, IV) was prolonged by THC (10 mg/kg, IP, 1 h before) to approximately twice the control value. -FMH (50 mg/kg, IP) administered alone had no significant influence on the thiopental sleeping time. However, -FMH given 1 or 3 h before THC treatment markedly enhanced the THC potentiation of thiopental-induced sleep. Such an enhancement by -FMH was not observed when -FMH was administered 15 h before THC treatment. The brain histamine level decreased by 60% during the first 4 h after -FMH injection and remained low until 15 h after the treatment. The thiopental sleep-potentiating action of morphine, chlorpromazine and diazepam was not affected by pretreatment with -FMH. The transient enhancing effect of -FMH on the THC potentiation of thiopental-induced sleep suggests that the histaminergic system is one of the activating transmitter systems in the brain.     

Enzyme kinetics of zearalenone biotransformation: pH and cofactor effects

The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3- and 3-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as -zearalenol (-ZOL) formation increased in the presence of NADPH, whereas -zearalenol (-ZOL) production only increased in the presence of NADH (P<0.001). The optimal pH for -ZOL production was pH 5.6 and that for -ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5-androstanedione>androstanedione>pregnenolone on -ZOL formation, whereas -ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3- and 3-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.     

A comparison of the central actions of prostaglandins A1, E1, E2, F1α, and F2α in the rat

Prostaglandins (PGs) injected into the right lateral brain ventricle (i.v.c.) of the rat increased the sleeping time induced by hexobarbital, chloral hydrate, and ethanol. PGE₁ and PGE₂ intensified chlorpromazine-induced catalepsy, inhibited amphetamine hyperactivity, and significantly depressed the amphetamine-induced stereotypy. NA concentrations were decreased by PGE₁ and PGE₂ and were increased by PGF₂. PGF₂ increased both 5-HT and 5-HIAA levels in rat brain. Total ACh concentrations were increased by PGF₁ and PGF₂. PGE₁, PGE₂, and PGF₂ enhanced the turnover of NA, DA, and 5-HT. PGE₂ counteracted the decreased activity induced by -MT and abolished the hypothermic action of -MT. PGF₂ had little effect on the activity of PCPA pretreated rats, whereas the higher doses of PGF₂ increased body temperature in these animals.     

Storage and release of false transmitters after infusion of (+)- and (−)-α-methyldopamine

Summary Rabbits were given an infusion of 10 mg/kg (–)- or 30 mg/kg (+)--methyldopamine and killed after 135 min. The noradrenaline content of the heart was decreased to 26±5 and 34±2%, respectively, of the control value. After infusion of the (+)-isomer the missing noradrenaline was replaced by (–)--methylnoradrenaline. Electrical stimulation of the sympathetic nerves or infusion of acetylcholine plus atropine caused an output of noradrenaline and (–)--methylnoradrenaline from the isolated heart. The two amines were released in the same proportion as they were stored in the heart and the total output of both amines equalled the output of noradrenaline from control hearts. Nerve stimulation caused frequency-dependent increases in the rate and tension of cardiac contraction. There was no significant difference between the frequency-response curves obtained from untreated hearts and those treated with (+)--methyldopamine.After pretreatment with (–)--methyldopamine the noradrenaline lost from the heart was substituted by the infused amine;-methylnoradrenaline was not detected. Nerve stimulation or infusion of acetylcholine plus atropine caused an output of noradrenaline and (–)--methyldopamine at the same proportion as the amines were stored. Electrical stimulation of the sympathetic nerves caused an undiminished output of noradrenaline although the cardiac noradrenaline was decreased. It is suggested that the (–)--methyldopamine released during nerve stimulation interferes with the membranal re-uptake and therefore increases the overflow of the catecholamines. Pretreatment with (–)--methyldopamine shifted the frequencyresponse curves for rate and tension to the right.It concluded that treatment with (+)--methyldopamine failed to inhibit adrenergic transmission because the cardiostimulant activity of its metabolite, (–)--methylnoradrenaline, does not differ from that of noradrenaline, and the false transmitter released fully balances the decrease of noradrenaline output. In contrast, admixture of a large proportion of a weak cardiostimulant amine such as (–)--methyldopamine inhibits the actions of the physiological transmitter released by nerve stimulation probably by a postjunctional effect.This work was supported by the Deutsche Forsehungsgcmeinschaft. We wish to thank Dr. phil. et reed. Petra Netter (Institut für Medizinische Statistik und Dokumentation, Mainz) for her advice in the statistical analysis, and Dr. C. A. Stone (Merck, Sharpe and Dohme) for the donation of (–)- and (+)--methyldopamine. The technical assistance of Miss B. Hering and Miss H. Zappe is gratefully acknowledged.     

Circadian variations in the effect of α-methyl-p-tyrosine on avoidance learning

Circadian variations in -methyl-p-tyrosine's (MT) disruption of active avoidance acquisition were studied. MT administered between 11.0 and 13.00 h, 17.00 and 19.00 h and 23.00 and 01.00 h impaired learning, but MT administered between 05.00 and 07.00 h did not impair learning. The time of MT ineffectiveness corresponds with the reported circadian peak in brain catecholamines, suggesting that the circadian susceptibility rhythm of MT depends upon circadian fluctuations of brain catecholamines.     

Disposition Kinetics of α-Tocopherol in Apolipoprotein B Knockout Mice

Purpose. We examined the effects of apolipoprotein B (apoB) on the disposition kinetics of -tocopherol by using apoB knockout mice. Methods. The concentrations of -tocopherol in plasma and tissues were measured by gas chromatography-mass spectrometry. Results. In apob (–/–) mice, the endogenous levels of -tocopherol in plasma and tissues (except liver) were significantly lower, and the liver concentration was significantly higher than those in wild-type mice. After single i.v. administration of -tocopherol (25 mg/kg), the area under the plasma concentration-time curve (AUC) and the distribution volume at steady state were significantly decreased, whereas the total clearance of -tocopherol was significantly increased in apob (–/–) vs. wild-type mice. -Tocopherol was highly distributed to the liver, compared with other tissues. After an oral administration of -tocopherol (100 mg/kg), the intestinal absorption of -tocopherol was very low in apoB knockout mice, as the value of AUC_0-32h for apob (–/–) mice (17.7 ± 8.3 g h/mL) was significantly less than that for apob (+/+) wild-type mice (96.5 ± 15.8 g h/mL, mean ± SD of five experiments, p < 0.01). The biliary excretion of -tocopherol was significantly greater in apob (+/–) mice than in apob (+/+) mice. Conclusions. These results show that apoB plays a role in hepatic secretion and intestinal absorption of -tocopherol.     

The effect of intracerebroventricular administration of catecholamines and their antagonists on rectal temperature of Mastomys natalensis

Summary Noradrenaline (NA), adrenaline (ADR), isoprenaline (ISO) and dopamine (DA) were given through a chronically implanted cannula in the lateral cerebral ventricle of Mastomys natalensis. Low doses of NA (0.05–0.25 g) reduced rectal temperature while larger doses (0.35 g upwards) produced dose-dependent hyperthermia. The hypothermic effect was antagonised by -adrenceptor and the hyperthermia by -adrenoceptor antagonists. -Methyl noradrenaline produced less hyperthermia but it antagonised the hyperthermic effect of NA. Adrenaline (0.1–10 g) was ineffective per se but when given after tolazoline it produced hyperthermia and after propranolol it produced hypothermia. The dose-dependent hyperthermia with isoprenaline (0.1–10 g) was blocked by propranolol and MJ-1999. Dopamine (0.5–20 g) and its agonists apomorphine, amantadine and BS 9641 produced hyperthermia which was antagonised by haloperidol and pimozide but not by - or -adrenoceptor antagonists. Noradrenaline (1.0 g) produced hypothermia at ambient temperature of 10°C and 16°C. It had no effect at 20°C which seems to be the thermoneutral zone for mastomys. The hyperthermic effect at 33°C was less than at 24°C. Dopamine (10 g) response was attenuated at 33°C and unaffected at other ambient temperatures. It is concluded that - and -adrenoceptors and DA-receptors exist in the central thermoregulatory mechanism in mastomys. The -receptors are concerned with lowering the body temperature whereas the -receptors and DA-receptors are involved in raising it.Communication No. 2841 from the Central Drug Research Institute, Lucknow     

Opposite interactions between α- and β-endorphin fragments with dopamine mediated responses on the rat rectum in vitro

Summary Dopamine causes a dose-dependent contraction of the rat rectum in vitro followed by a relaxation. This contraction can be inhibited by apomorphine and phenylephrine. This inhibition can be attenuated by the -endorphin (E) fragments 2–17 (des-Tyr¹--endorphin, DTE) and 6-17 (des-enkephalin--endorphin, DEE). E 6-17 seems to be the shortest sequence with full activity in this respect since a shorter fragment (E 10-17) was less effective. The atypical neuroleptics oxypertine, sulpiride, and clozapine, the classic neuroleptic haloperidol and metoclopramide have a similar action to DEE. The peptides and atypical neuroleptics do not affect the dopamine response per se while the classic neuroleptics haloperidol and metoclopramide enhance the dopamine response.The effects of the -type endorphins are opposite to those of the -type endorphins, since des-Tyr¹--endorphin (DTE, E 2-16) and des-enkephalin--endorphin (DEE, E 6-16) enhance the phenylephrine-induced decreased responsiveness to dopamine. Structure-activity studies revealed that the active moiety of the -endorphin fragments probably resides in the 6–9 region. In addition the -type endorphins directly inhibit the dopamine response.It is concluded that the rat rectum may be used to analyse neuroleptic-like action. In this model - and -endorphin fragments may directly or indirectly influence the interaction of dopamine with the rectum. Because of the strong similarities between the effects of -type endorphins and that of neuroleptics the results support the purported neurolepticlike action of -type endorphins. The influence of -type endorphins and -type endorphins on the apomorphine or phenylephrine induced decreased responsiveness to dopamine, although opposite, seems to be mediated by an influence on different dopamine sensitive systems.     

Effect of K+ channel blockers on the α2-adrenoceptor-coupled regulation of electrically evoked noradrenaline release in hippocampus

Summary The question whether presynaptic ₂-adrenoceptors regulating noradrenaline release in hippocampus directly couple to tetraethylammonium chloride (TEA) or -dendrotoxin (-DTX)-sensitive K⁺ channels was investigated. Hippocampal slices, prelabelled with [³H] noradrenaline, were superfused in the presence of (+)-oxaprotiline and electrically stimulated with 4 pulses delivered at 100 Hz, in order to avoid autoinhibition due to released noradrenaline.TEA enhanced the evoked [³H]noradrenaline release in rabbit hippocampus in a concentration-dependent manner, yielding an approximately 4-fold increase at 30 mmol/l, whereas the spontaneous outflow of tritium was only slightly affected at this concentration. The ₂-adrenoceptor agonist clonidine, at 10–100 nmol/l inhibited the evoked [³H]noradrenaline release between 77% and 96%. The inhibitory effect of the ₂-agonist was distinctly diminished in the presence of 30 mmol/l TEA but was restored in low Ca²⁺/high Mg²⁺ buffer. Therefore, the diminution of the ₂-agonist effect by TEA observed in experiments with normal Ca²⁺ can be explained by an increase of the Ca²⁺ availability for the release process due to the prolongation of action potentials. In rabbit hippocampus -DTX (10–200 nmol/l) did neither affect the evoked release of [³H]noradrenaline nor its ₂-agonist-induced modulation. However, in rat hippocampus -DTX significantly increased the evoked transmitter release and diminished the effect of clonidine.Taken together, the present data for the rabbit hippocampus exclude the possibility that activation of presynaptic ₂-adrenoceptors inhibits depolarization-evoked [³H]noradrenaline release by inducing an outward K⁺ current through TEA- or -DTX-sensitive K⁺ channels. However, there are species differences between the rabbit and the rat so that in the rat the ₂-adrenoceptors could actually be coupled to K⁺ channels — provided that the release-enhancing properties of -DTX do not account for the ₂-antagonism observed.Correspondence to C. Allgaier at the above address     

Exploring the pharmacology of the pro-convulsant effects of α2-adrenoceptor antagonists in mice

The effects of selective and specific ₂-adrenoceptor antagonists on electroshock seizure threshold in mice were investigated. Idazoxan, at low doses, efaroxan, RX811059 and RX821002 significantly lowered seizure threshold. The ₁-agonist St 587 and the -agonist isoprenaline were also pro-convulsant. On the other hand the ₂-agonists clonidine and UK 14,304 produced small increases in seizure threshold. Anticonvulsant effects were also produced by low doses of the noradrenaline uptake inhibitor desipramine. This compound increases levels of noradrenaline in the synaptic cleft which could subsequently act at post-synaptic ₂-adrenoceptors. The pro-convulsant action of ₂-adrenoceptor antagonists could be explained in terms of two mechanisms: a) blockade of endogenous noradrenaline which may normally exert a tonic anti-convulsant influence on seizure threshold, through post-synaptic ₂-receptors and/or b) increased activation of ₁- and -adrenoceptors by elevated synaptic noradrenaline levels following blockade of pre-synaptic ₂-adrenoceptors. Of the ₂-antagonists tested, idazoxan was unusual in that high doses were not pro-convulsant; this difference may be explained by ₁-adrenoceptor mediated actions or be related to its recently described affinity at a non-adrenoceptor site — a function for which is currently unknown.     

Evaluation of GABAergic neuroactive steroid 3α-hydroxy-5α-pregnane-20-one as a neurobiological substrate for the anti-anxiety effect of ethanol in rats

Rationale Acute systemic ethanol administration is known to elevate plasma and cerebral levels of neuroactive steroid 3-hydroxy-5-pregnane-20-one (3, 5-THP; allopregnanolone) to a concentration sufficient to potentiate GABA_A receptors. We have earlier demonstrated that 3, 5-THP mediates the antidepressant-like effect of ethanol in Porsolt forced swim test.Objective The aim of the present study is to explain the relationship between endogenous GABAergic neurosteroids and anxiolytic effect of ethanol in Sprague–Dawley rats.Method The mediation of 3, 5-THP in the anti-anxiety effect of ethanol was assessed by pharmacological interactions of ethanol with various endogenous neurosteroidal modulators and using simulated physiological conditions of altered neurosteroid content in elevated plus maze (EPM) test.Results Pretreatment of 3, 5-THP (0.5–2.5 g/rat, i.c.v.) or neurosteroidogenic agents such as 3, 5-THP precursor progesterone (5 or 10 mg/kg, i.p.), 11- hydroxylase inhibitor metyrapone (50 or 100 mg/kg, i.p.) or the GABA_A receptor agonist muscimol (25 ng/rat, i.c.v.) significantly potentiated the anti-anxiety effect of ethanol (1 g/kg, i.p.). On the other hand, the GABAergic antagonistic neurosteroid dehydroepiandrosterone sulphate (DHEAS) (1 mg/kg, i.p.), the GABA_A receptor blocker bicuculline (1 mg/kg, i.p.), the 5-reductase inhibitor finasteride (50×2 mg/kg, s.c.) or the mitochondrial diazepam binding inhibitory receptor antagonist PK11195 (1 mg/kg, i.p.) reduced ethanol-induced preference of time spent and number of entries into open arms. Anti-anxiety effect of ethanol was abolished in adrenalectomized (ADX) rats as compared to sham-operated control. This ADX-induced blockade was restored by prior systemic injection of progesterone, signifying the contribution of peripheral steroidogenesis in ethanol anxiolysis. Socially isolated animals known to exhibit decreased brain 3, 5-THP and GABA_A receptor functions displayed reduced sensitivity to the effects of ethanol and 3, 5-THP in EPM test.Conclusions Our results demonstrated the contributory role of neuroactive steroid 3, 5-THP in the anti-anxiety effect of ethanol. It is speculated that ethanol-induced modulation of endogenous GABAergic neurosteroids, especially 3, 5-THP, might be crucial pertinent to the etiology of trait anxiety (tension reduction) and ethanol abuse.     

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein l (hu-MIP-l), murine-macrophage inflammatory protein l (mu-MIP-l), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-l, mu-MIP-l, or hu-IL-8 expression vector. The production of hu-MIP-1 reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 10⁵ cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-l, mu-MIP-l, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-l and mu-MIP-l. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-l showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-l gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-l gene might be useful as an effective therapy for the treatment of certain tumors.     

In vivo studies on alpha-adrenergic receptor subtypes in human veins

Summary We studied in vivo responsiveness of venous ₁ and ₂-adrenoceptors, measuring the diameter changes in superficial veins in response to -adrenergic agonists and antagonists in healthy human volunteers. The dorsal hand vein technique was used because it permits complete dose-response studies of venous constriction without confounding reflex alterations.Local infusions of all agonists studied induced dose-dependent contraction of the hand vein; the maximal effects (Emax) were: norepinephrine (88% ± 10%), methox amine (97% ± 5%), phenylephrine (95% ± 6%), clonidine (54% ± 12%), and azepexole (68% ± 26%). Clonidine reduced the norepinephrine-induced venoconstriction by 11% ± 10%. Oral doses of 1 mg prazosin antagonized the venoconstriction induced by norepinephrine, methoxamine, and clonidine, but not by azepexole. Yohimbineantagonism was observed against all agonists studied. Inhibition by yohimbine of clonidine-induced venoconstriction was irreversible over 60–180 min.Results show that the in vivo effects on veins of -adrenergic agonists are in good agreement with results from in vitro experiments. Agonists with ₁- and ₂-adrenoceptor subtype selectivity cause venoconstriction in vivo, but ₂-receptor mediated constriction is intrinsically weaker. Clonidine acts as a partial antagonist against norepinephrine, presumably on postsynaptic ₂-receptors. At high doses, ₂-adrenoceptor subtype selectivity of clonidine and yohimbine appear to be partially lost in vivo. Send offprint requests to H. G. Eichler at the above address