Showing papers in "Naunyn-schmiedebergs Archives of Pharmacology in 1980"

Characterization of adenosine receptors in rat brain by (-)[3H]N6-phenylisopropyladenosine.

Abstract: (−)N6-Phenylisopropyladenosine, a potent agonist in adenosine-responsive cellular systems, has been labeled with tritium to high specific activity (26 Ci/mmol) and used to identify adenosine binding sites in rat brain membranes. (−)[H3]N6-Phenylisopropyladenosine binding was studied by a vacuum filtration technique. The binding was rapid, rapidly reversible, dependent on pH and temperature and stereospecific since the (−)isomer of N6-phenylisopropyladenosine was 40-fold more potent than the (+)isomer in competition experiments. The stereospecific binding sites were saturable and bound 0.8 pmol of (−)N6-phenylisopropyladenosine per mg of membrane protein. The dissociation constant (KD) of (−)N6-phenylisopropyladenosine for these sites was 5–12 nM as determined independently by saturation and kinetic binding studies. Endogeneous ligands seem to occupy the binding sites since pretreatment with adenosine deaminase increased the specific binding. Adenosine and several adenosine derivatives were studied for their ability to compete with (−)[3H]N6-phenylisopropyladenosine binding. (−)N6-Phenylisopropyladenosine-5′-monophosphate, N6-phenyladenosine, N6-benzyladenosine, 2-chloroadenosine and adenosine were most potent in displacing the radioligand from its binding sites and the IC50-values ranged from 0.3–7 μM. Physiologically inactive compounds such as inosine, hypoxanthine, adenine and the ribose-modified analogues 2′-deoxyadenosine and 2′,5′-dideoxyadenosine did not substantially inhibit binding at concentrations up to 100 μM. The adenosine antagonists isobutylmethylxanthine (IC50 3.2 μM), theophylline (IC50 7.6 μM) and caffeine (IC50 99 μM) competed for the binding sites of (−)[3H]N6-phenylisopropyladenosine in a manner which parallels their known pharmacological activity whereas other phosphodiesterase inhibitors were ineffective. The (−)[3H]N6-phenylisopropyladenosine binding sites in rat brain membranes appear to be equivalent to adenosine receptor sites on the cell surface which have recently been classified as R-site adenosine receptors.

A comparison of pre- and post-junctional potencies of several alpha-adrenoceptor agonists in the cardiovascular system and anococcygeus muscle of the rat. Evidence for two types of post-junctional alpha-adrenoceptor.

Abstract: The pre- and post-junctional effects of five α-adrenoceptor agonist drugs were examined in the pithed rat. On pre-junctional receptors on the cardioac-celerator nerves, oxymetazoline, guanabenz and xylazine had effects which were qualitatively similar to the established effects of clonidine; phenylephrine had not. These pre-junctional effects could be antagonised by yohimbine. Post-junctional effects of the agonists, assessed as the diastolic arterial pressor response or contraction of the anococcygeus, showed an unexpected pattern. The effects of phenylephrine were almost abolished by prazosin (1 mg/kg) whereas the effects of the other four agonists, clonidine, oxymetazoline, guanabenz and xylazine were only moderately antagonised, guanabenz and xylazine being particularly resistant; this resulted in an order of agonist potency for the “prazosin-resistant” responses which was similar to that for pre-junctional α-adrenoceptor agonism on the cardiac or vas deferens sympathetic nerves. Yohimbine (1 mg/kg) produced greater antagonism of the pressor effects of guanabenz and xylazine than did prazosin. It is postulated that at least two types of post-junctional α-adrenoceptors exist in rat tissues, one of which is similar to α1-adrenoceptors previously demonstrated and another which has similarities to the prejunctional α2-adrenoceptors. Noradrenaline released from vasopressor nerves or administered intravenously could be shown to produce post-junctional effects which were partly resistant to prazosin, suggesting a physiological role for each of these α-adrenoceptors.

Three classes of dopamine receptor (D-2, D-3, D-4) identified by binding studies with 3H-apomorphine and 3H-domperidone.

Abstract: Dopamine receptors subtypes were studied in homogenates from rat brain areas, mainly the corpus striatum, using the two highly selective ligands 3H-apomorphine and 3H-domperidone. The clearly biphasic inhibition of the specific binding of these two ligands by some agents allowed us to define four distinct classes of binding site. 3H-apomorphine labels two classes of site displaying a large difference in affinity for domperidone, i.e. class I sites well recognized (IC50=5 nM) and class II sites poorly recognized (IC50=10 μM). 3H-domperidone also labels two distinet classes of site displaying a large difference in affinity for apomorphine and dopamine, i.e. class III sites well recognized by these agents (IC50=5 and 35 nM, respectively) and class IV sites poorly recognized (IC50=790 nM and 14 μM, respectively). The two classes I and III represent a single pharmacological class of dopaminergic receptors (labelled by either 3H-apomorphine or 3H-domperidone) as indicated by 1) their almost identical pharmacological specificities (high correlation between Kd or Ki values for a variety of dopaminergic agonists and antagonists); 2) their similar capacity in striatum as well as in other brain regions; 3) the identical decrease in capacity following kainate lesions; 4) their similar sensitivity to GTP and thermal denaturation. Because the pharmacological specificity of these sites excludes the possibility that they represent the recognition sites of the dopamine-sensitive adenylate cyclase, i.e. D-1 receptors, we propose to term them D-2 receptors. Class II and IV sites also differ from D-1 receptors as shown by drug specificity and the effect of kainate. We propose to term class II sites D-3 receptors and class IV sites D-4 receptors.

Specific 3H-imipramine binding in human platelets. Influence of age and sex.

Abstract: Human platelets have been shown to possess high-affinity binding sites for 3H-imipramine. These binding sites have a similar affinity and drug specificity to those already described in rat brain. The platelets from healthy volunteers show no difference in 3H-imipramine binding between the sexes but there is a decrease in maximal 3H-imipramine binding with increasing age of the donor.

Modulation by endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit

Abstract: Slices of the caudate nucleus of rabbits were preincubated with 3H-choline and then superfused. Stimulation by electrical pulses at 3 Hz or by 25 mmol/l potassium elicited an increase in tritium outflow which was calcium-dependent and, in the case of electrical stimulation, tetrodotoxin-sensitive. The dopamine receptor agonist apomorphine (0.01-1 mumol/l) decreased, whereas the antagonist haloperidol increased the electrically evoked overflow of tritium. Nomifensine and cocaine, used at concentrations known to inhibit the re-uptake of dopamine, also reduced the evoked overflow of tritium, and this reduction was antagonized by haloperidol. Combined pretreatment with reserpine and alpha-methyltyrosine methylester (alpha-MT), which lowered dopamine levels by 99.5%, increased the electrically evoked overflow, as did bretylium which is shown here to block action potential-induced release of dopamine. The facilitation by haloperidol and bretylium as well as the inhibition by nomifensine and cocaine were diminished or abolished after pretreatment with reserpine plus alpha-MT. Apomorphine decreased, and haloperidol increased, the potassium-evoked overflow of tritium; the effects were not changed by tetrodotoxin. The results indicate that the striatal dopamine receptors which, when activated, depress the release of acetylcholine, are akin to the D-2 type. Endogenous dopamine also acts on the receptors as shown by several manipulations with known effects on dopaminergic transmission. A large fraction of these dopamine receptors may be located on the cholinergic axon terminals.

Dendrotoxin from the venom of the green mamba, Dendroaspis angusticeps. A neurotoxin that enhances acetylcholine release at neuromuscular junction.

Abstract: The venom of the green mamba, Dendroaspis angusticeps has previously been shown to produce neuromuscular facilitation by increasing acetylcholine release. After gel filtration and ion-exchange chromatography of the whole venom, a basic polypeptide with facilitatory actions was isolated. This polypeptide, named dendrotoxin, has 59 amino acid residues, probably with only 3 disulphide bonds and a blocked N-terminus.

Direct evidence for an axonal site of action of capsaicin.

Abstract: 1. Local application of capsaicin to the sciatic nerve of rats induced a long-lasting increase in the nociceptive threshold as tested by the hot-plate method, and prevented neurogenic inflammation in the lateral part of the dorsal skin of the rat's paw. 2. Application of capsaicin to the saphenous nerve prevented the neurogenic inflammatory response, induced either by antidromic electrical stimulation of the saphenous nerve or by painting the skin with mustard oil, in the medial part of the dorsal skin of the paw. 3. The functional impairment induced by local capsaicin treatment of saphenous or sciatic nerves was strictly confined to the skin area supplied by the corresponding nerve. 4. It is suggested that local capsaicin treatment of peripheral nerves selectively damages the chemosensitive nerve fibres presumably by depleting their substance P content.

Serotonin-receptor-mediated modulation of Ca2+-dependent 5-hydroxytryptamine release from neurones of the rat brain cortex.

Abstract: Rat brain cortex slices were incubated with 3H-5-hydroxytryptamine and subsequently superfused. Tritium overflow from slices superfused with physiological salt solution was stimulated by rectangular electrical pulses (normal frequency 3 Hz), and overflow from slices superfused with Ca2+-free solution containing 25 mM K+ was stimulated by introduction of (usually) 1.3 mM CaCl2. In most of the experiments the neuronal uptake of 5-hydroxytryptamine (5-HT) was blocked by paroxetine. 1. Electrically evoked 3H overflow was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid, whereas Ca2+-evoked overflow was not affected by tetrodotoxin, but inhibited by Mg2+ ions. 2. Both electrically and Ca2+-evoked 3H overflow were reduced by unlabelled 5-HT and increased by methiothepin; the potencies of the drugs in modifying evoked overflow were similar with both methods of stimulation. Methiothepin caused a substantial shift to the right of the concentration-response curve of unlabelled 5-HT for its inhibitory effect. By contrast, metergoline produced only a very slight shift of this curve. 3. The inhibitory effect of unlabelled 5-HT on electrically evoked 3H overflow increased with decreasing Ca2+ concentration (1.3–0.65 mM; 3 Hz) or with decreasing frequency of stimulation (10–0.3 Hz; 1.3 mM Ca2+). 4. When 3H overflow was stimulated by introduction of various Ca2+ concentrations (0.65–4.55 mM) into the Ca2+-free, K+-rich solution, the data obtained yielded a straight line in the double reciprocal plot (Km=2.5 mM Ca2+). Straight lines were also obtained, when the effects of unlabelled 5-HT and methiothepin on 3H overflow evoked by different Ca2+ concentrations were analyzed; these lines and that calculated for the controls had a common intercept on the ordinate, whereas the Km values were considerably different from each other (in the presence of unlabelled 5-HT and methiothepin, 5.4 and 1.2 mM Ca2+, respectively).

Evidence suggesting a transmitter or neuromodulatory role for substance P at the first synapse of the baroreceptor reflex

Abstract: There is evidence that the undecapeptide substance P is the transmitter released from pain fibres in the dorsal horn of the spinal cord. This, in turn, suggested to us the possibility of a similar role for substance P in another type of primary sensory structure, namely the baroreceptor neurones which terminate in the bulbar nucleus of the solitary tract (NTS). Substance P, injected into a lateral brain ventricle of urethane-anaesthetized rats, caused dose-dependent increases in blood pressure and heart rate. By contrast, local application of substance P to the region of the NTS, using small pieces of filter paper soaked in substance P-containing solution, resulted in hypotension and bradycardia. In cats anaesthetized with urethane, the same procedure also decreased blood pressure, heart rate and spontaneous sympathetic nervous activity. Release of substance P from nerve endings, through local application of capsaicin to the NTS, produced cardiovascular effects which were indistinguishable from those of substance P. A combined cannula-electrode with an uninsulated tip enabled identification of sites in the NTS of rats and cats, where electrical stimulation elicited decreases in blood pressure, heart rate and spontaneous sympathetic nervous activity. These sites were considered to contain the first synapse of the baroreceptor reflex. Subsequent microinjection of substance P through the cannula into these defined areas resulted in effects which were similar to those of the preceding electrical stimulation. The results suggest a transmitter or neuromodulatory role for substance P at the first synapse of the baroreceptor reflex in the NTS.

Tetanus toxin blocks the neuromuscular transmission in vitro like botulinum A toxin.

Abstract: 1. The blocking effect of tetanus toxin on the neuromuscular junction of the mouse phrenic nervehemidiaphragm preparation exposed to the toxin (0.05–20 μg/ml) in the organ bath was studied and compared with the action of botulinum A toxin. 2. The time course of the paralysis of the diaphragm could be divided into a latent and a manifest period. Still during the latent period the effect of the toxin became progressively resistant to washing and, with some delay, to antitoxin. 3. Between 25 and 41°C the time until paralysis strongly depended on temperature with Q10 of about 2.7. 4. Procedures increasing the transmitter release shortened, and procedures depressing it prolonged the time until paralysis. 5. 4-Aminopyridine and guanidine temporarily restored the contraction of the partially paralyzed diaphragm, indicating the persistence of activatable calcium and acetylcholine pools. Raising the external Ca2+-concentration and application of the Ca-Ionophore A 23187 were ineffective in the doses applied. 6. About 80 min after exposure to the toxin (10 μg/ml), the m.e.p.p. activity decreased by a factor of 30. Parallel to this, paralysis of nerve evoked muscle contraction developed. 7. Neuraminidase treatment did not prevent tetanus toxin poisoning. 8. The paralysis is produced by tetanus toxin itself and not by contaminants as shown by the parallel decrease of toxicity and paralysis following treatment with either antitoxin or brain homogenate, or by the use of spontaneously inactivated toxin. 9. Tetanus toxin was compared with botulinum A toxin as to the shape of its dose-response curve, time course of paralysis, temporary reversal by 4-aminopyridine and behaviour against Ca-ionophore. In any case, both toxins were indistinguishable, albeit botulinum A neurotoxin was calculated to be about 2000 times more potent than tetanus toxin.

Effect of antidepressant drugs on serotonergic and adrenergic receptors

Abstract: We examined eight tricyclic antidepressants (doxepin, amitriptyline, clomipramine, imipramine, trimipramine, nortriptyline, desipramine and protriptyline) and four nontricyclic antidepressants (mianserin, nomifensin, iprindole and zimelidine) in terms of their effects on serotonergic and adrenergic receptors by direct binding assays.3H-WB-4101 (0.22 nM) served as a label for postsynaptic alpha receptors while3H-clonidine (0.2 nM) served to label presynaptic alpha receptors in the frontal cortex of the calf.3H-dihydroalprenolol (3H-DHA) (0.5 nM) was used to label the beta1 adrenoceptors in the calf frontal cortex and beta2 adrenoceptors in the calf cerebellar cortex.3H-d-Lysergic acid diethylamide (3H-LSD) (2 nM) and3H-serotonin (3H-5HT) (0.5 nM) were used to label the serotonergic receptors in the calf frontal cortex.

Alpha-adrenoceptor-mediated modulation of 5-hydroxytryptamine release from rat brain cortex slices.

Abstract: Slices of rat brain cortex preincubated with 3H-5-hydroxytryptamine were superfused with physiological salt solution and stimulated electrically, or they were superfused with Ca2+-free solution containing 25 mM K+ and stimulated by introduction of 1.3 mM CaCl2 for 2 min. These findings suggest that the terminal serotoninergic fibres of rat brain cortex possess alpha-adrenoceptors, activation of which by exogenous or endogenous noradrenaline leads to inhibition of the release of 5-hydroxytryptamine.

1-Methyl-beta-carboline (harmane), a potent endogenous inhibitor of benzodiazepine receptor binding.

Abstract: The interaction of several beta-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several fold higher affinity for the benzodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related beta-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

Alpha 2-adrenoceptors modulating insulin release from isolated pancreatic islets.

Abstract: Using rat isolated pancreatic islets, we investigated the effects of various α-adrenoceptor blocking agents on adrenaline-induced inhibition of glucose-stimulated insulin release. Yohimbine was about 100 times more potent than prazosin in antagonizing the inhibitory effect of adrenaline. At concentrations of 10 μM, phentolamine was about as effective as an antagonist as yohimbine, whereas dihydroergotamine, WB-4101 and phenoxybenzamine were less effective and prazosin produced very little antagonism. These results strongly suggest that postsynaptic α2-adrenoceptors modulate insulin release from pancreatic islets.

3H-Prazosin binds specifically to 'alpha 1'-adrenoceptors in rat brain.

Abstract: Prazosin is know to block postsynaptic α-adrenoceptors. In this study 3H-prazosin has been used to label biochemically central α-adrenoceptors. In rat brain membranes 3H-prazosin bound specifically in a rapid, reversible and saturable manner to a single class of high affinity sites. The relative order of potencies for inhibition of 3H-prazosin binding was WB4101 > ARC 239 > phentolamine ≫ piperoxane > yohimbine which is a characteristic of the α1 type of adrenoceptors. In contrast, the relative order of potencies for inhibition of 3H-clonidine binding was yohimbine > piperoxane > WB4101 > ARC239 > prazosin which is a characteristic of the peripheral ‘α1’-and ‘α1’-adrenoceptors. These results indicate that 3H-prazosin binds to central ‘α1’ and 3H-clonidine to ‘α’2-receptor and confirm the presence of two classes of α-adrenoceptors in rat brain membranes.

Titration of human brain monoamine oxidase -A and -B by clorgyline and L-deprenil.

Abstract: The interaction of clorgyline andl-deprenil with the-A and-B forms of human brain monoamine oxidase (MAO) has been studied. Both compounds inhibit cerebrocortical MAO in a manner consistent with a ‘suicide’ inactivation of the enzyme. The interaction of clorgyline with the-A form of the enzyme appears to take place almost entirely at specific binding sites, and the conditions required for this inhibitor to ‘titrate’ the concentrations of MAO-A have been elucidated.l-Deprenil has also been used to titrate the concentration of the-B form of MAO in cerebrocortical homogenates, but there is a considerable degree of non-specific binding of this compound. The two inhibitors have been used to titrate the concentrations of the two enzyme forms in frontal cortex homogenates from different age groups. There was a significantly higher MAO-B activity for the age range 73–95 years than for the age range 2–63 years. No significant differences between the two age groups were found for MAO-A. The activity of MAO-A in the samples correlated very well with the concentration of this enzyme form. Titration of the B-form of the enzyme withl-deprenil indicated an increased enzyme concentration with age, although other factors, such as the non-specific binding of this compound, could contribute to this effect.

Regulation of adenylate cyclase activity in hamster adipocytes. Inhibition by prostaglandins, alpha-adrenergic agonists and nicotinic acid.

Abstract: In ghosts of hamster adipocytes, the regulation of adenylate cyclase (ATP: pyrophosphate lyase, cyclizing; EC 4.6.1.1) activity by prostaglandins, α-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 α-adrenergic agonists) and 60% (by prostaglandins and nicotinic acid). The order of potency for the inhibitory prostaglandins (PG) was PGE1 ≧ PGE2>PGF2α≅PGI2>PGD2>6-keto PGF1α. The IC50 values obtained were about 0.007, 0.06, 0.3 and 1 μM for PGE1, PGF2α, PGD2 and 6-keto PGF1α, respectively. α-Adrenergic agonists, studied in the presence of the β-adrenergic blocking agent, propranolol (30 μM), inhibited the fat cell enzyme with the order of potency (1)-adrenaline > (1)-α-methylnoradrenaline ≅ (1)-noradrenaline > clonidine ≅ tetryzoline > (1)-phenylephrine. The IC50 values obtained for (1)-adrenaline and (1)-noradrenaline were about 3 and 10 μM, respectively. The inhibitory effect of (1)-adrenaline was blocked by the α-adrenergic antagonists with the potency order yohimbine ≅ phentolamine > prazosin. These findings suggest that an α2 of receptors is involved in this catecholamine-induced inhibition. Nicotinic acid (10 μM) reduced adenylate cyclase activity by about 60% with half-maximal effectiveness at about 0.6 μM. The nicotinic acid derivatives, nicotinamide, β-pyridylcarbinol and NAD (up to 100 μM), had no effect on enzyme activity.

An improved assay of cyclic 3',5'-nucleotide phosphodiesterases with QAE-Sephadex columns.

Abstract: A method for the assay of cyclic nucleotide phosphodiesterases is described which employs chromatographical separation of cyclic nucleotides and nucleosides on QAE A-25 Sephadex columns and 5′-nucleotidase as an auxiliary enzyme. The assay allows quantitative recovery of adenosine, guanosine and their metabolites from the anion exchanger and thus is suitable for use in crude phosphodiesterase preparations containing purine catabolizing enzymes. in comparative studies, this method was found to be considerably more sensitive than previous reported methods because of lower assay blanks and higher recoveries of the nucleoside catabolites. The method is suitable for kinetic analysis of crude enzyme preparations from micromolar to millimolar substrate concentrations.

Effect of adenosine on sinoatrial and ventricular automaticity of the guinea pig.

Abstract: In isolated spontaneously beating right ventricular strips and right atrial preparations of guinea pigs adenosine was found to exert a concentration-dependent suppressing effect on the pacemaker activity. Responsiveness to adenosine was approximately 30-fold higher in ventricular than in atrial preparations. A decrease in the rate of slow diastolic (phase 4) depolarization of Purkinje and sinoatrial nodal fibers proved to be a major determinant of the adenosine-induced alteration in pacemaker activity. It is suggested that adenosine might exert its depressant effect on ventricular automaticity via direct excitation of purine receptors located in the specialized pacemaker fibres of the ventricular tissue.

Parallel bioassay of physalaemin and kassinin, a tachykinin dodecapeptide from the skin of the African frog Kassina senegalensis.

Abstract: 1. Kassinin, a tachykinin dodecapeptide isolated from the skin of the African frog Kassina senegalensis was submitted to parallel bioassay with physalaemin, eledoisin and substance P, three major representatives of the tachykinin peptide family. Bioassay was carried out on blood pressure, salivary secretion and isolated or in situ smooth muscle preparations. 2. As expected, kassinin possessed the entire spectrum of biological activity peculiar to the tachykinins. However, among the examined tachykinins kassinin was the poorest stimulant of salivary secretion and the weakest hypotensive agent, while displaying very powerful stimulant effects on different smooth muscle preparations, especially on isolated preparations of urinary bladder. 3. Kassinin differed from the other tachykinins also for its more gradual and sustained action on several in situ and isolated preparations. The peptide most similar to kassinin in its spectrum of activity was eledoisin. 4. Emphasis is laid on the possibility to dissociate the effects of the tachykinins on different target systems through changes in the N-moiety of the tachykinin molecule.

Vasoconstriction mediated by postsynaptic α2-adrenoceptor stimulation

Abstract: The postsynaptic α-adrenoceptors involved in vasoconstriction brought about by B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepin) administered i.v. to pithed, normotensive rats were characterized. The rate of onset of the hypertensive response to i.v. B-HT 933 is slower than that induced by (−)-phenylephrine, an agonist of α1-adrenoceptor. The antagonism of the α-adrenoceptor blocking drugs rauwolscine, yohimbine and corynanthine was quantified towards B-HT 933-induced increases in diastolic pressure. Rauwolscine (pA2=7.06) and yohimbine (pA2=6.83) were effective antagonists, whereas corynanthine proved much less potent (pA2=5.03). On the basis of the reported selectivity of yohimbine and its two diastereoisomers rauwolscine and corynanthine for α1- and α2-adrenoceptor, it is concluded that the postsynaptic α-adrenoceptors triggered by B-HT 933 are of the α2-type. B-HT 933 identifies a subclass of postsynaptic α2-adrenoceptor in vascular smooth muscle distinct from postsynaptic α1-adrenoceptor. Both types of α-adrenoceptors are likely to be involved in the mediation of vasoconstriction.

beta-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: dissociation of stimulation and blockade.

Abstract: The cardiac stimulant actions of nine β-adrenoceptor blocking agents were examined in kitten papillary muscles and in isolated atria of kittens and guinea pigs to determine to what extent these drugs behaved as classical partial agonists. In many ways the agents do appear to comprise a spectrum of partial agonists with widely differing efficacies. However, in one respect the actions of some of the β-blockers did not fit into the classical mold. Several β-blockers were found to exert stimulant effects only in concentrations appreciably higher than those required for substantial β-adrenoceptor blockade. These observations suggest that more than one type of β-adrenoceptor may be involved in the production of sympathomimetic effects on cardiac muscle.

A comparative study of the pharmacology of inhibitors of GABA-metabolism

Abstract: Four catalytic inhibitors of GABA-aminotransferase (GABA-T), viz. gabaculine, gamma-acetylenic GABA, gamma-vinyl GABA, and ethanolamine O-sulphate (EOS), as well as the unspecific enzyme inhibitor aminooxyacetic acid (AOAA), sodium valproate (VPA), and GABA itself were studied for anticonvulsant, biochemical, and toxic effects in mice. Elevations of the electroconvulsive threshold by 30 V were produced at the time of their maximal effect by the i.p. injection (AOAA s.c.) of 13 mg/kg AOAA, 37 mg/kg gabaculine, 65 mg/kg gamma-acetylenic GABA, 125 mg/kg VPA, 1,440 mg/kg EOS, 1,900 mg/kg gamma-vinyl GABA and 2,800 mg/kg GABA. At these doses, all drugs except GABA and VPA increased the clonic pentetrazole threshold to a similar extent, but differed in their increases in the brain content of GABA, which varied from 70% (EOS) to 300% (gamma vinyl GABA) as a consequence of decreases in the activity of GABA-T. The activity of the GABA-synthesizing enzyme glutamate decarboxylase was decreased only by gamma-acetylenic GABA. When determining the anticonvulsant effect of the different drugs against the convulsant ED 97 of pentetrazole, 3-mercaptopropionic acid, strychnine and maximal electroshock seizures, gabaculine, AOAA, VPA and in part gamma-vinyl GABA and GABA were efficacious enough to allow the determination of ED50 values, whereas gamma-acetylenic GABA and EOS showed no clear activity in any of these seizure models. Gabaculine and AOAA at their anticonvulsant ED50 were toxic or lethal. All inhibitors of GABA-T except EOS caused numerous side effects which cast doubt on the specificity of these drugs. The present results indicate that inhibitors of GABA-T hardly seem to be suited for treatment of convulsive disorders in human but are useful tools in studies of experimental epilepsy.

Adenosine receptor activation by adenine nucleotides requires conversion of the nucleotides to adenosine

Abstract: Adenine nucleotides cause adenosine receptor-mediated increases in cyclic AMP in the VA13 human fibroblast line. Levels of adenosine accumulated in the medium are insufficient to account for the responses to adenine nucleotides. Since rapid conversion of the nucleotides to adenosine by 5′-nucleotidase in the vicinity of the receptor might account for the responses, six experimental methods were developed to distinguish between “local conversion” and direct action of the nucleotides. Results of all six methods favored local conversion. (1) 5′-Nucleotidase inhibitors blocked the accumulations of cyclic AMP elicited by AMP, ADP, and ATP, but did not affect the response to adenosine. The most potent inhibitor of both conversion of AMP and response to AMP was α,β-methylene-ADP (APCP). (2) Adenosine deaminase blocked the responses to AMP, ADP, ATP, and adenosine-containing coenzymes. (3) Theophylline, a specific competitive adenosine antagonist, was an insurmountable inhibitor of the increases in cyclic AMP caused by AMP, ADP, and ATP. The insurmountability was presumably due to substrate sataration of the converting enzyme 5′-nucleotidase. (4) Although ADP and ATP had partial agonist-like dose-response curves, they did not inhibit the response to adenosine. (5) Nine cell lines which responded to adenosine were tested for response to AMP. Cell lines with high levels of 5′-nucleotidase had large responses to AMP, those with intermediate levels of 5′-nucleotidase had large or intermediate responses to AMP, and those with low 5′-nucleotidase levels did not respond to AMP. (6) Inhibition of the uptake of labelled adenosine was used as an indicator of unlabelled adenosine concentrations near the cell membrane. Unlabelled AMP inhibited uptake nearly as effectively as unlabelled adenosine. APCP reversed the inhibition by AMP but not the inhibition by adenosine.

Pharmacological characterisation of pre- and postsynaptic alpha-adrenoceptors in dog saphenous vein.

Abstract: 1. The pre- and postsynaptic α-adrenoceptors in the dog saphenous vein have been characterised according to their sensitivity to agonists and antagonists. 2. In the presence of uptake 1 and uptake 2 inhibitors, adrenaline, noradrenaline and α-methylnoradrenaline reduced the overflow of radioactivity during field stimulation of saphenous veins pre-loaded with 3H-noradrenaline. Phenylephrine and methoxamine were very weak or inactive. Yohimbine and phentolamine considerably enhanced the stimulationevoked overflow of radioactivity, but prazosin had little effect even at high concentrations. These results identify the presynaptic α-adrenoceptors as belonging to the α2-subtype. 3. Clonidine (up to 10−6 M) did not inhibit the stimulation-evoked overflow of radioactivity; instead it antagonised the effects of exogenous noradrenaline. However, at concentrations of 10−8 M and above clonidine did reduce radioactivity overflow if the uptake inhibitors were omitted from the bathing fluid. These results demonstrate the partial agonist activity of clonidine at presynaptic α2-adrenoceptors. 4. The postsynaptic α-adrenoceptors in the saphenous vein resemble α2-adrenoceptors identified in other tissues. However, α-methylnoradrenaline was more potent, and phenylephrine less potent, than expected. Furthermore, yohimbine exhibited a higher blocking potency at the postsynaptic α-adrenoceptors than has generally been found. 5. In contrast to their effects in other tissues, α-agonist did not greatly reduce contractile responses of the saphenous vein to nerve stimulation, and yohimbine did not enhance them. These effects are discussed in relation to the balance between the pre- and postsynaptic effects of the drugs.

Inhibiton of \(\dot V_{\max } \) of the action potential by propafenone and its voltage-, time- and pH-dependence in mammalian ventricular myocardium

Abstract: In microelectrode experiments on isolated papillary muscles of guinea-pigs, the effect of propafenone (1×10−5 mol/l–1×10−4 mol/l) on the fast Na+ action potential was analyzed. Propafenone caused, without changing the resting potential, a concentration dependent decrease of $$\dot V_{\max } $$ . At continuous stimulation with a rate of 12/min, complete inhibition of $$\dot V_{\max } $$ appeared in response to 1×10−4 mol/l. Transformation of the concentration-response curve into the Hill form revealed a Hill coefficient of 1.8. Some minor changes of the action potential configuration occurred consisting of a plateau shift to less positive potentials and of an abbreviation of action potential duration. Propafenone did not alter the linear dependence of $$\dot V_{\max } $$ on the logarithm of external Na+ concentration. Propafenone shifted the curve relating $$\dot V_{\max } $$ to membrane potential (h ∞-curve) to more negative potentials suggesting an interference with the inactivation process of the fast Na+ system. This effect developed on the quiescent, non-stimulated preparation and leads to the occurrence of a resting-state block being, consequently, potential dependent. Repetitive stimulation enhanced the propafenone-induced inhibition indicating a use-dependent block. During a train of 5–7 (at 12/min) or 15–20 (at 120/min) action potentials, a continuous decline of $$\dot V_{\max } $$ occurred until a new steady-state was attained. The interstimulus interval determined strength and kinetics of development (τdevelopment) of the use-dependent block. It was 22% and 8.4s, respectively at 5000 ms, but 70% and 1.8 s, respectively, at 500 ms. A slight intensification could also be induced by a decrease of membrane potential whilst pH or temperature changes remained ineffective. After cessation of continuous stimulation, the use-dependent block disappeared with a time constant of 15.5 s. Neither an increase of pH from 7.4 to 8.4 nor a decrease to 6.4 had any influence on the action of propafenone (pK a=9.0). Resting-state and usedependent block were of an identical amount regardless of the pH in the medium.

Requirement for sulfhydryl groups in the differential effects of magnesium ion and GTP on agonist binding of muscarinic cholinergic receptor sites in rat atrial membrane fraction.

Abstract: 1. The binding of agonist (carbachol) and antagonist (atropine) to the rat heart atrial muscarinic cholinergic receptor sites was investigated. 2. Divalent cations (Mg2+ or Ca2+), in low concentrations, modestly increased the agonist binding affinity of the receptor site without any effect on the antagonist binding affinity. 3. Guanine nucleotides (e. g. GTP), on the other hand, decreased the agonist binding affinity (but not the antagonist binding affinity), and the extent of GTP effect depended on the absence or presence of divalent cation (Mg2+) in the binding assay. 4. Pretreatment of atrial membranes with N-ethylmaleimide (NEM) altered the agonist binding curve (obtained with varying concentrations of carbachol) such that the Hill coefficient (nH) became very close to 1.0, whereas the corresponding nH values for control (untreated) or dithiothreitol (DTT)-treated membranes were much less than 1.0; NEM or DTT treatments failed to show any effect of antagonist binding curve. 5. NEM treatment abolished both divalent cation-induced and guanine nucleotide-induced alterations in the agonist binding affinity of the receptor site. 6. Monovalent cations in low concentrations did not mimic the effects of Mg2+ or Ca2+ on agonist binding. Instead, concentration dependent decreases in both agonist and antagonist binding affinities and abilities were observed. Neither NEM nor DTT treatments failed to alter the monovalent cation effects on carbachol and atropine binding. 7. These observations indicate a likely involvement of-SH groups in the opposing effects of Mg2+ and guanine nucleotides (GTP) on cardiac muscarinic receptor-agonist interaction. The results further suggest some subtle in vitro differences in the brain and heart muscarinic receptor sites with regard to the influence by divalent cations and guanine nucleotides on the receptor-agonist interaction.

Effect of 2-nicotinamidethyl nitrate (SG-75) on the membrane potential of left atrial muscle fibres of the dog. Increase in potassium conductance

Abstract: 1. 2-Nicotinamidethyl nitrate (SG-75) (10−6–10−4 mol/l) and methacholine (MeCh) (10−7–10−5 mol/l) hyperpolarized the resting membrane potential, and produced increases in amplitude and maximum rate of rise and a decrease in duration of the action potential of left atrial muscle fibres of the dog. 2. Changes in membrane potentials produced by SG-75 were not modified by atropine at a concentration (3×10−7 mol/l) which greatly antagonized those induced by MeCh. 3. The decrease in resting membrane potential for a 10-fold increase in extracellular K+ concentration ([K+]0) was greater in the presence than in the absence of SG-75 or MeCh. 4. SG-75- and MeCh-induced changes in membrane potentials were not affected by the replacement of NaCl by Na isethionate in perfusion fluid. 5. SG-75- and MeCh-induced changes in membrane potentials were not modified by ouabain (10−6 mol/l). 6. The “slow response” of left atrial muscle fibres was obtained by increasing the [K+]0 to 27 mM in the presence of isoprenaline (10−6 mol/l). SG-75 and MeCh primarily produced a decrease in duration of the “slow response”. At higher concentrations SG-75 and MeCh hyperpolarized the resting membrane potential and decreased the amplitude and maximum rate of rise of the “slow response”. Unlike SG-75 and MeCh, verapamil primarily decreased the amplitude and maximum rate of rise of the “slow response” without changes in duration of the “slow response” and resting membrane potential. 7. The above results suggest that the mechanism of action of SG-75 on left atrial muscle fibres may be an increase in potassium conductance of the membrane without mediation through muscarinic receptors.

Effect of chronic tricyclic antidepressant treatment on the serotoninergic autoreceptor

Abstract: Chronic treatment with tricyclic antidepressant (TCA) drugs has been shown to enhance the responsiveness of rat forebrain neurons to serotonin (5-HT). In the present study, imipramine (5 mg and 10 mg/kg), iprindole (2.5 mg/kg), desipramine and femoxetine (5 mg/kg) were administered daily for 14 days. The response of dorsal raphe neurons to intravenous injection of LSD (4 μg/kg) and to microiontophoretic applications of 5-HT and LSD was assessed 24h after the last dose. The responsiveness to intravenous LSD and the effectiveness of microiontophoretic applications of 5-HT and LSD were not altered by TCA drug pretreatments. Furthermore, the treatments did not change the mean firing rate of these 5-HT neurons. Those results suggest that chronic treatment with TCA drugs does not alter the sensitivity of the 5-HT autoreceptor. Thus, the effect of the previously reported increase of postsynaptic neuron responsiveness to 5-HT would not be dampened by a decreased activity of the presynaptic neurons.

K+-evoked release of met-enkephalin from rat striatum in vitro: effect of putative neurotransmitters and morphine.

Abstract: The effects of drugs on the K+-evoked release of met-enkephalin from superfused rat striatal slices were investigated using a specific radioimmunoassay GABA, at concentrations of 50μM and 100 μM, and the GABA agonist muscimol (50 μM), significantly inhibited the release The inhibitory effect of GABA was reversed by picrotoxin suggesting that GABA inhibition is mediated by GABA receptors Selected concentrations of the dopamine agonists apomorphine and ergonovine, as well as of haloperidol, acetylcholine, carbachol, noradrenaline, glutamic acid and substance P, had no effect on the release of metenkephalin Increases in the evoked release (80%) and striatal enkephalin content (60%) were found in rats after chronic haloperidol administration, pointing to an increase in the synthesis and utilization of striatal enkephalin No differences were found between the release from slices from morphine-tolerant/dependent and naive rats or after addition of naloxone to slices derived from tolerant/dependent animals Selected concentrations of morphine and naloxone had no effect on release suggesting the absence of a mechanism for the regulation of enkephalin release involving autoreceptors