Radioimmunoassay of β-endorphin basal and stimulated levels in extracted rat plasma

Summary A sensitive radioimmunoassay for -endorphin is described. Antibodies against human -endorphin which exhibit a high avidity for the C-terminal of the peptide were raised in rabbits following the injection of thyroglobulin-coupled human -endorphin (h-E) as immunogen. Methionineenkephalin, - -endorphin, as well as ACTH peptides did not cause interference in the radioimmunoassay. -Lipotropin, however, showed a 50% cross-reactivity. The sensitivity of the assay is 25 pg/0.5 ml tube volume for -endorphin. -Endorphin was extracted with a high recovery from the rat plasma using silicic acid and -endorphin levels as low as 100 pg/ml could be measured.Basal levels of -endorphin-like immunoreactivity in plasma of rats were about 400 pg/ml. -Endorphin levels in adrenalectomized rats and in animals chronically treated with the cortisol synthesis blocker metyrapone were found to be markedly increased (about 7-fold). Exposure of the rats to electrically induced foot-shocks caused a similar increase of immunoreactive -endorphin in plasma. A significant increase was also seen after insulin injection.     

Über die Wirkung einiger herzwirksamer Bisguanylhydrazone auf den Veratrineffekt am Frosch-Sartoriusmuskel

Zusammenfassung Die Wirkung einiger Guanylhydrazone sowie von Tyramin auf die Veratrine-Response des Froschsartoriusmuskels wurden untersucht. Von den drei herzglykosidartigen Bisguanylhydrazonen zeigten BG 60 und BG 31 den unspezifischen Veratrinantagonismus des Chinidins. BG 85 hingegen beeinflußte die VR herzglykosidartig. Von den drei tyraminartigen Substanzen BG 81, MG 41 und Tyramin wirkten die letzten zwei chinidinartig, während BG 81 trotz seiner tyraminartigen Wirkung die VR herzglykosidartig beeinflußte.

---

Summary A series of guanylhydrazones was synthesized, some of the substances had a cardiac glycoside-like activity on the mammalian heart whereas other ones acted tyramine-like. The effect of some guanylhydrazones of each kind of action on the veratrine response (VR) of the frog's sartorius muscle should be investigated. Following substances were synthesized: 1. with cardiac glycoside-like activity 3,3-dimethyl-4,4-diacetyldiphenylbisguanylhydrazone (BG 60), progesteronebisguanylhydrazone (BG 31), p-aceto-m-methyl-phenyl-acetonyl-ether-bisguanylhydrazone (BG 85) 2. with sympathomimetic activity: p-aceto-phenyl-acetonyl-ether-bisguanylhydrazone (BG 81) and p-hydroxybenzaldehyd-monoguanylhydrazone (MG 41). The characteristic influence of the cardiac glycosides on the VR was only to be seen with BG 85 and BG 81. BG 60, BG 31 and BG 41 showed a quinidine-like effect on the VR. That means that from 3 guanylhydrazones with cardiac glycoside-like activity on the mammalian heart two of them had a quinidine-like effect on the VR and only one of them showed the typical cardiac glycoside-like effect on the VR. On the other hand among the guanylhydrazones with tyramine-like activity on the mammalian heart there is one, BG 81, which influenced the VR like a cardiac glycoside.

     

Structure-activity relationship studies on mescaline: II. Tolerance and Cross-tolerance between mescaline and its analogues in the rat

Summary Using the CAR shuttle box technique, with male hooded rats as subjects, the development of tolerance to mescaline, 3,4-dimethoxyphenyl-ethylamine (DMPE) and NN-dimethylmescaline (DMM) and possible cross-tolerance between these drugs was studied. It was found that seven successive daily doses of mescaline induced tolerance to its inhibitory effect, but that the excitatory effect was increased. Tolerance developed to the predominantly inhibitory effect of DMPE, and to the excitatory effect of DMM.Partial cross-tolerance existed between DMPE and mescaline, and when the procedure was reversed, between mescaline and DMPE. Cross-tolerance was shown in the same manner between DMM and mescaline, and mescaline and DMM.The problems of quantification and interpretation of these results is discussed. It was concluded, on the basis of subsidiary experiments, that the inhibitory effect of mescaline was probably not caused by ataxia or by a peripheral effect.     

Regulation of adenylate cyclase activity in hamster adipocytes

Summary In ghosts of hamster adipocytes, the regulation of adenylate cyclase (ATP: pyrophosphate lyase, cyclizing; EC 4.6.1.1) activity by prostaglandins, -adrenergic agonists and nicotinic acid was studied. These three classes of antilipolytic agents caused adenylate cyclase inhibition without an apparent lag phase. Maximal inhibitions observed ranged between about 45% (by -adrenergic agonists) and 60% (by prostaglandins and nicotinic acid). The order of potency for the inhibitory prostaglandins (PG) was PGE₁ PGE₂>PGF₂PGI₂>PGD₂>6-keto PGF₁. The IC₅₀ values obtained were about 0.007, 0.06, 0.3 and 1 M for PGE₁, PGF₂, PGD₂ and 6-keto PGF₁, respectively. -Adrenergic agonists, studied in the presence of the -adrenergic blocking agent, propranolol (30 M), inhibited the fat cell enzyme with the order of potency (1)-adrenaline > (1)--methylnoradrenaline (1)-noradrenaline > clonidine tetryzoline > (1)-phenylephrine. The IC₅₀ values obtained for (1)-adrenaline and (1)-noradrenaline were about 3 and 10 M, respectively. The inhibitory effect of (1)-adrenaline was blocked by the -adrenergic antagonists with the potency order yohimbine phentolamine > prazosin. These findings suggest that an ₂ of receptors is involved in this catecholamine-induced inhibition. Nicotinic acid (10 M) reduced adenylate cyclase activity by about 60% with half-maximal effectiveness at about 0.6 M. The nicotinic acid derivatives, nicotinamide, -pyridylcarbinol and NAD (up to 100 M), had no effect on enzyme activity.Inhibition of the hamster adipocyte adenylate cyclase by the antilipolytic agents required the presence of both GTP, which reduced basal activity by about 80% at 10 M, and sodium ions, which specifically activated the GTP-affected from of the enzyme. Inhibition was also observed in the presence of ACTH, which in a GTP-dependent manner increased adenylate cyclase activity. Pretreatment of the enzyme preparation with NaF (10 mM) partially reduced the inhibitory effect, and preactivation with the stable GTP analogue, guanylyl 5-imidodiphosphate (100 M), abolished the adenylate cyclase inhibition by the antilipolytic agents.Abbreviations PG prostaglandin - GMP-P(NH)P guanylyl 5-imidodiphosphate Some of the data were presented in abstract form (Aktories et al., 1979a)     

Effects of locally applied dopamine to the nucleus accumbens on the motor activity of normal rats and following α-methyltyrosine or Reserpine

The motor activity of rats was investigated following bilateral application of various doses (0–80 g) of dopamine to the nucleus accumbens. A high dose (80 g) of dopamine increased the motor activity of normal as well as -methyltyrosine- and reserpine-treated rats. It also increased the late motor activity (6–9 min) of normal rats, probably due to stimulation of postsynaptic dopamine receptors. Lower doses (10–40 g) of dopamine suppressed initial (0–3 min) motor activity of normal rats, perhaps due to stimulation of dopamine autoreceptors on the dopamine nerve terminals in the nucleus accumbens with a subsequent inhibition of dopamine neurotransmission. An intermediate dose (40 g) of dopamine was able to restore the motor activity of -methyltyrosine-treated but not of reserpine-treated rats at all time intervals. This difference, indicating a restoration of the normal pattern of habituation by dopamine only in animals pretreated with -methyltyrosine, suggests that normal behaviour is dependent on release of dopamine by nerve impulses.     

A method to shorten the training phase of drug discrimination

Rats were trained to discriminate drug from no drug in a two-lever, food-reinforced task. One group was trained with cocaine (10 mg/kg) and a second group was trained with pentylenetetrazol (20 mg/kg). A method designed to shorten the time required for the training phase of drug discrimination experiments was assessed in subgroups for each drug. In one subgroup, single training sessions were conducted daily. In the other subgroup, a second session (either drug or saline) was conducted on days for which the first condition was saline. The training conditions were presented in an irregular sequence, with the same condition occurring in no more than two consecutive sessions. Rats trained by the accelerated method learned the discrimination in fewer days, with no decrement in acquisition per session, suggesting that drug discrimination training can be accomplished more rapidly by reducing inter-session interval.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Effects of chlordiazepoxide on discrimination performance

Chlordiazepoxide (0, 5, and 10 mg/kg), administered on eight successive sessions, significantly impaired the reinforcement-cued discrimination performance of male Sprague-Dawley rats. This performance partially recovered on two postdrug (saline) session. The discrimination impairment was due to less inhibition of responding during no-go phases and lower response rates during go phases of the task by the drugged than control animals.     

(−)δ9 THC as an hypnotic

(–) ⁹ THC was found to significantly decrease the time it takes to fall asleep in physically healthy insomniacs. Once asleep, interruptions of sleep were not significantly altered over the whole night. The (–) ⁹ THC tended to be associated with some decrease in awakenings in the first half of the night.The primary side effect experienced by the subjects at all dose levels in the Pre-Sleep phase was temporal disorganization and mood alterations. There was an increase in intensity of side effects and number of subjects affected with increasing dosage.The most significant side effect, however, was a hangover phenomenon, or continued high the next day, with some residual of temporal disorganization. It increased in intensity and duration with increase in dosage. This hangover seems severe enough to eliminate the consideration of the 30 mg dose range of (–) ⁹ THC for clinical use as an hypnotic.Dr. Cousens is a 3rd Year Resident in Psychiatry at the Napa State Hospital, Napa, California     

The effect of nicotine on the swimming speed of pre-trained rats through a water alley

Summary In sessions of ten runs each, swimming time of rats through a 4 m long water alley was measured. Four doses of nicotine (0.05; 0.1; 0.2; 0.4 mg/kg given intraperitoneally 30 minutes before testing) were tested in sessions with a braking load on the tails of the animals either in all 10 runs of a session, or in every second run, or in none of the 10 runs. Regardless of the swimming condition, nicotine produced a considerable, and at doses of 0.1 mg/kg and over, significant decrease of performance in the first two runs. From the third to the 10th run, the changes caused by nicotine were smaller and differed depending on the swimming conditions.A dose of 0.1 mg nicotine/kg improved performance in the without-load-sessions and the without-load-runs of the alternating sessions, while both 0.1 and 0.2 mg/kg improved performance of the with-load-runs of the alternating sessions. Performance in the without-load-sessions and the without-load-runs was depressed by 0.4 mg/kg and that in the with-load-sessions by 0.2 and 0.4 mg/kg.     

Differential inhibition of biphenyl hydroxylation in perfused rat liver

Summary A differential inhibition of biphenyl hydroxylation by -naphthoflavone and metyrapone was observed in isolated pefused rat liver. -Naphthoflavone inhibited 2- and 4-hydroxylation in livers from -naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in -naphthoflavone-stimulated livers.Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.     

Identification of the neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery

Summary Vasoconstriction or excitatory junction potentials (e.j.ps) evoked by nerve stimulation (15 field pulses at 2 Hz every 3 min) were recorded in rabbit isolated jejunal arteries. The resting diameter of the arteries and its decrease in response to stimulation was measured by a photoelectric method. Vasoconstriction was insensitive to prazosin 0.1 or 1 mol/l. Yohimbine 1 mol/l considerably enhanced, whereas ,-methylene ATP (,-meATP) 1 mol/l abolished the contractile response. In order to test the effect of exogenously applied transmitter candidates, noradrenaline (0.1–1 mol/l) and ATP (10–30 mol/l) were added in concentrations which evoked a vasoconstriction comparable to that induced by electrical stimulation. The action of noradrenaline was prevented by prazosin 0.1 mol/l, but was unaffected by both yohimbine 1 mol/l and ,-meATP 1 mol/l. ,-meATP 1 mol/l depressed the effect of ATP. The e.j.ps evoked by a train of 15 pulses showed facilitation up to the third response and thereafter depression; a partial summation was also observed. Prazosin 0.1 mol/l did not change the e j.p. amplitudes. By contrast, when yohimbine 0.1 or 1 mol/l was added to the prazosin-containing medium, both the late e j.ps in the train and the summation were enhanced in a concentration-dependent manner. ,-meATP 1 mol/l almost abolished the e.j.ps. In conclusion, in rabbit jejunal arteries, stimulation of postganglionic sympathetic nerves may release noradrenaline together with ATP which is probably the sole neuroeffector transmitter under our conditions. Transmitter release seems to be modulated by the activation of presynaptic ₂-adrenoceptors. Under the stimulation conditions of the present experiments the released transmitter does not activate postsynaptic ₁-adrenoceptors. Send offprint requests to P. Illes     

The Mechanism of Uptake of Biodegradable Microparticles in Caco-2 Cells Is Size Dependent

Purpose. To study the uptake of biodegradable microparticles in Caco-2 cells. Methods. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 m, 1 m, and 10 m containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 g/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. Results. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 m diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 m and 6 fold greater than the 10 m diameter microparticles. Similarly in terms of number the uptake of 0.1 m diameter microparticles was 2.7 × 10³ fold greater than the 1 m and 6.7 × 10⁶ greater than the 10 m diameter microparticles. The efficiency of uptake of 0.1 m diameter microparticles at 100 g/ml concentration was 41% compared to 15% and 6% for the 1 m and the 10 m diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 m) uptake increased with concentration in the range of 100 g/ml to 500 g/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37°C compared to at 4°C. Conclusions. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 m) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.     

Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

Purpose. The present study addresses how maltosyl--cyclodextrin (G₂--CyD) impacts upon the -chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G₂--CyD on the activity of the protease. Methods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of -chymotrypsin with G₂--CyD in the buffer solution was examined by differential scanning calorimetry. Results. G₂--CyD decelerated the -chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1–3 tripeptide and the 4–9 hexapeptide fragments. This deceleration can be explained solely by a nonproductive encounter between a complex of the substrate with G₂--CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G₂--CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of -chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of -chymotrypsin was observed in the presence of G₂--CyD, suggesting reduced proteolytic activity upon binding to G₂--CyD. Conclusions. These results suggest that G₂--CyD at higher concentrations inhibits the proteolytic action of -chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.     

EEG spectral analysis for the evaluation of the central effects of δ6-tetrahydrocannabinol in rabbits

The effects of ⁶-THC on the frequency spectrum of the hippocampal EEG were examined with the assistance of an electronic integrator. Six rabbits bearing chronically implanted cortical and hippocampal electrodes were dosed intravenously with ⁶-THC (2.5% solution in polyethylene glycol 300; doses of 10, 50, 100, 250 and 1000 g/kg) once a week. A comparison of epochs of arousal in the pre-drug control and post-drug periods demonstrated that a dose as low as 50 g/kg produced a consistent, statistically significant diminution in power of the 7, 8, 9 Hz frequencies of the hippocampal EEG, with 8 Hz the most sensitive. Doses above 250 g/kg produced an overall decrease in the power spectrum from 7 to 27 Hz.Spectral analysis of the EEG offers a method which is sufficiently sensitive to reveal a central effect of ⁶-THC in rabbits at doses equivalent to the human threshold for psychic effects.Presented, in part, at the VIIIth International Congress of Electroencephalography and Clinical Neurophysiology, Marseilles, France, September 1–7, 1973.M. D. W. was supported by the Medical Research Council of Canada during the tenure of this work.The technical assistance of Mr. E. Deodati is gratefully acknowledged.     

Different effects of vinblastine on the polymerization of isotypically purified tubulins from bovine brain

Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an - and a -subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, _II, _III and _IV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except _II tubulin was resistant to 1M vinblastine. Vinblastine at low concentrations (<10M) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for _II. The tau-induced assembly of unfractionated tubulin and _III were equally sensitive to 1M vinblastine whereas _II and _IV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, _II and _IV could be easily aggregated by 20M vinblastine whereas such as aggregation of microtubules obtained from _III and tau required approximatedly 40M vinblastine. Our results suggest that among the tubulin isotypes, _II is the most sensitive to vinblastine in the presence of MAPs while _III is the most resistant and this intrinsic resistance of _III dimers persists in the polymeric form of _III tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.     

Opioid δ agonists and endogenous enkephalins induce different emotional reactivity than μ agonists after injection in the rat ventral tegmental area

The possible role of opioid receptor heterogeneity in the biphasic changes in locomotion (activation and inhibition) induced by non-selective opiates such as morphine, has been investigated by measuring the behaviour of rats exposed to different environments after injection into the ventral tegmental area, of selective (DAGO) or (DTLET, DSTBULET, BUBU) opioid agonists and of kelatorphan, a complete inhibitor of enkephalin metabolism. agonists or kelatorphan-induced hyperactivity in a familiar (actimeter), unfamiliar (four-hole box) and a fear inducing (open-field) environment. These effects were suppressed by naloxone and selective antagonists (ICI 174, 864 2 mg/kg SC, naltrindole 7 nmol in the ventral tegmental area). Moreover, the agonists and endogenous enkephalins protected by kelatorphan did not affect the emotional state of rats measured in an elevated plus maze. Infused into the ventral tegmental area, DAGO also enhanced locomotion in the actimeter but in contrast to agonists and kelatorphan, the agonist decreased activity in the open-field and the four-hole box. The hypoactivity observed in these tests could be related to an enhanced emotionality produced by receptor stimulation, as shown by the significant decrease in the number of visits and time spent in open arms of the elevated plus maze. Naloxone (0.3 mg/kg SC) but not selective antagonists, blocked the various responses induced by DAGO.     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address     

Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin

This study investigated the vasorelaxant activity, superoxide radicals (O₂^–)-scavenging capacity and cyclic nucleotide phosphodiesterase (PDE)-inhibitory effects of hesperidin and hesperetin, two flavonoids mainly isolated from citrus fruits. Hesperetin concentration-dependently relaxed the isometric contractions induced by noradrenaline (NA, 1 M) or by a high extracellular KCl concentration (60 mM) in intact rat isolated thoracic aorta rings. However, hesperetin (10 M–0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 M). Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 M), tetraethylammonium (TEA, 2 mM) or nifedipine (0.1 M) did not significantly modify the vasorelaxant effects of this flavonoid. Hesperetin (10 M–0.1 mM) did not affect the basal uptake of ⁴⁵Ca²⁺ but decreased the influx of ⁴⁵Ca²⁺ induced by NA and KCl in endothelium-containing and endothelium-denuded rat aorta. Hesperetin (10 M–0.1 mM) did not scavenge O₂^– generated by the phenazine methosulfate (PMS)-reduced -nicotinamide adenine dinucleotide (NADH) system. Hesperetin (0.1 mM) significantly reversed the inhibitory effects of NA (1 M) and high KCl (60 mM) on cyclic nucleotide (cAMP and cGMP) production in cultured rat aortic myocytes. Hesperetin preferentially inhibited calmodulin (CaM)-activated PDE1 and PDE4 isolated from bovine aorta with IC₅₀ values of about 74 M and 70 M respectively. In contrast, the 7-rhamnoglucoside of hesperetin, hesperidin (10 M–0.1 mM), was inactive in practically all experiments, although it inhibited basal and cGMP-activated PDE2 isolated from platelets (IC₅₀ values of 32±4 M and 137±34 M respectively). These results suggest that the vasorelaxant effects of hesperetin are basically due to the inhibition of PDE1 and PDE4 activities.     

An accelerated stability study of 5‐flucytosine in intravenous solution

The stability of the antimycotic drug flucytosine (5FC) and the extent of 5fluorouracil (5FU) formation in 5FC intravenous solution was studied in an accelerated stability experiment. 5FC intravenous solution (10 mg/ml) was heated at 40, 60, 70, 80 and 90 C for a maximum of 131 days. At appropriate time intervals samples were taken and the concentrations of 5FC and 5FU were determined using a newly developed, stability indicating HPLCUV method. Heating the 5FC intravenous solution at 40, 60, 70, 80 and 90 C lead to 5FC decomposition of respectively 0, 8.9, 14.4, 52.5 and 61.6%. The Arrhenius plot of the 5FC decomposition is described by: Lnk5-FC decomposition = 80.1892 * 1/T 0.2396 and the 5FU formation is described by Lnk5FU formation = 13087 * 1/T + 34.4028. It is concluded that 5FC is very stable in intravenous solution at regular storing temperatures and can therefore be stored at ambient temperatures for several years before the critical limit of 95% 5FC is reached. However, the toxic and teratogen degradation product 5FU may be present in considerable amounts in the product, due to both impurities in the raw material and the formation from 5FC upon sterilisation and storage.