Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

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     

Activation of β2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats

Summary The aim of the present study was to investigate -adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with ³H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25–20 Hz) increase in the S-1 outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mol/l). The non-selective -adrenoceptor agonists isoprenaline (0.l gmol/1) and adrenaline (0.01 and 0.1 mol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) were blocked by the selective ₂-adrenoceptor antagonist ICI 118551 (0.1 mol/l) but not by the selective ₁-adrenoceptor antagonist atenolol (0.3 mol/l). The cell-permeable CAMP analogue 8-bromo-cAMP (300 mol/l) enhanced S-1 outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mol/l) and adrenaline (0.1 mol/l) did not enhance S-1 outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mol/l) alone in both strains. However, the facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) but not that of adrenaline (0.01 mol/l) were significantly greater in SHR than in WKY. The results suggest that stimulation of prejunctional ₂-adrenoceptors by adrenaline even in the absence of a-adrenoceptor blockade enhances noradrenaline release in kidney cortex of young SHR and WKY. This ₂-adrenoceptor mediated effect may possibly be dependent on cAMP formation. The greater facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) in SHR as compared to WKY are in accord with receptor binding studies which show a higher density of ₂-adrenoceptors in SHR than in WKY kidney cortex.Abbreviations SHR Spontaneously hypertensive rats - WKY WistarKyoto rats - cAMP 3-5-cyclic adenosine monophosphate - S-I stimulation induced Send offprint requests to: L. C. Rump     

Pharmacological evidence for the possible coexistence of multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle

Summary Contractile responses to neurokinin and neurokinin were characterized and compared with those to substance P (a SP-P agonist) and eledoisin (a SP-E agonist) in isolated rabbit iris sphincter. Neurokinin a and neurokinin as well as substance P and eledoisin produced atropine- and tetrodotoxin-resistant contractions of the iris sphincter in nanomolar concentrations, and the rank order of sensitivity was eledoisin > substance P = neurokinin = neurokinin . After prolonged cold-storage of the preparations, responses to capsaicin, a releaser of tachykinins from sensory nerve endings, were nearly absent, but responses of considerable magnitude to carbachol and the tachykinins persisted. On wash-out of the tachykinins, responses faded at characteristic rates (neurokinin a > eledoisin > neurokinin substance P). From the Schild analyses, [abetd-Arg¹, abetd-Pro², abetd-Trp^7,9, Leu¹¹]-substance P, a potent substance P antagonist, competitively antagonized the response to substance P, had no significant effect on the response to neurokinin , and antagonized the response to neurokinin and eledoisin in a more complex manner. Taken together, these results suggest that there coexist multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle. Send offprint requests to I. Takayanagi at the above address     

Different muscarinic receptors mediate autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat isolated perfused heart

Summary Experiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [¹⁴C]-choline. The effects of muscarinic receptor antagonists on the [14C]overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4–10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart.Stimulation of the vagus nerves (1200 pulses) caused an increase in [¹⁴C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 mol/l and abolished by tetrodotoxin 0.3 mol/l or perfusion with Ca²⁺-free solution. The fractional rate of evoked [¹⁴C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01–1 mol/l) as well as in that of the M₂-selective compounds methoctramine (0.1 mol/l) and AF-DX 116 (0.1–1 mol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 mol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 mol/l) or hexahydrosiladifenidol (0.1 mol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 mol/l).The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M₂ receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M₂-selective antagonists. Send offprint requests to I. T. Bognar at the above address     

Effects of nitrous oxide on decision-strategy and sustained attention

The significance of central noradrenergic, dopaminergic and serotonergic neural systems for the locomotor stimulant effects of methylphenidate was investigated in the rat. In order to study the role of brain catecholamines, rats were pretreated with reserpine (2.5 mg/kg) followed 24 hrs later by treatment with -methyltyrosine (25 mg/kg) or U-14,624 (75 mg/kg), a dopamine--hydroxylase inhibitor. In these experiments, methylphenidate stimulated motor activity was antagonized by -methyltyrosine and enhanced after treatment with U-14,624, suggesting that release of newly synthesized dopamine is important to a locomotor stimulant action of methylphenidate. Evidence implicating brain serotonin in the actions of methylphenidate was obtained in rats pretreated with pargyline or p-chlorophenylalanine (PCPA). Administration of pargyline 1 hr prior to methylphenidate was found to reduce the locomotor activity induced by methylphenidate and this was antagonized by pretreatment with low doses of PCPA. Higher doses of PCPA caused a significant elevation of methylphenidate induced activity which could be reduced by 5-hydroxytryptophan. Destruction of serotonergic neurons with 5,7-dihydroxytryptamine also potentiated methylphenidate induced locomotion. These latter findings suggest that serotonergic fibers have an inhibitory function in brain. These results are discussed in relation to the possible mechanism by which methylphenidate may act in hyperkinesis.     

Assessment of the muscarinic receptor subtype involved in the mediation of pilocarpine-induced purposeless chewing behaviour

Purposeless chewing behaviour in rats was enhanced by intraperitoneal administration of the muscarinic agonists pilocarpine (1.0–8.0 mg/kg). RS 86 (0.5–0.8 mg/kg), oxotremorine (1–2 mg/kg) and arecoline (2–32 mg/kg), but not by nicotine (0.1–3.2 mg/kg). Chewing behaviour was also induced by the ICV administration of the muscarinic agonists carbachol (12.5–100 g) and pilocarpine (50–200 g), but not by the putative M-1 receptor agonist McN-A-343 (50–200 g) or AH 6405 (100–200 g). The muscarinic receptor antagonists scopolamine (0.01–0.1 mg/kg SC), benzhexol (0.075–2.5 mg/kg SC), secoverine (1–10 mg/kg SC), and dicyclomine (1.25–10 mg/kg SC) antagonised purposeless chewing behaviour induced by pilocarpine (4 mg/kg IP). AF-DX 116 (2.5–100 mg/kg SC), an M-2 antagonist, partially inhibited the actions of pilocarpine (4 mg/kg IP). Based on ED₄₀ values the rank order of potency following IP administration was scopolamine > benzhexol > secoverine > dicyclomine > AF-DX 116. The ICV administration of the muscarinic antagonists N-methylscopolamine (2.5–10 g) and oxyphenonium (10–40 g) antagonised chewing behaviour induced by pilocarpine (4 mg/kg IP) in a dose-related manner. The M-2 antagonist 4-DAMP (40–160 g ICV), as well as AF-DX 116 (40–160 g ICV), also inhibited the effects of pilocarpine (40–160 g ICV). The putative M-1 receptor antagonist pirenzepine (80–320 g ICV) did not antagonise chewing behaviour induced by pilocarpine (4 mg/kg IP). Based on ED₄₀ values, the rank order of potency of muscarinic antagonists administered ICV was N-methylscopolamine > oxyphenonium > 4-DAMP > AF-DX 116 > pirenzepine. Comparisons of the actions of muscarinic antagonists in vivo, with their published actions in vitro suggest that pilocarpine-induced chewing behaviour is mediated through central M-2 receptors rather than via central M-1 sites.     

Effect of catechol-O-methyl-transferase (COMT) inhibition on the vascular and metabolic responses to noradrenaline,isoprenaline and sympathetic nerve stimulation in canine subcutaneous adipose tissue

Summary The effect of COMT-inhibitors (U-0521, H 22/54) on lipolysis and on vascular responses induced by noradrenaline, isoprenaline and nerve stimulation in canine adipose tissue in situ has been studied.COMT inhibition, before or after -adrenoceptor blockade, did not influence the -adrenoceptor mediated vasoconstriction due to nerve stimulation (2–5 Hz). Vasoconstriction due to close i.a. noradrenaline was unaffected at a dose of 2×10^–10 moles but at a dose of 10^–9 moles the peak vasoconstriction was enhanced but the duration of the response was not changed. Prior -adrenoceptor blockade did not alter the responses to U-0521.After -adrenoceptor blockade with Hydergine (120–300 g i.a.) a vasodilatation was induced by noradrenaline 2×10^–10 mole. This response and that produced by isoprenaline (2×10^–11 or 10^–10 moles) were enhanced after U-0521-treatment, but the vasodilating response to nerve stimulation (2 Hz) after -adrenoceptor blockade was unaffected.Lipolysis induced by nerve stimulation or noradrenaline in vivo was increased 50% and that induced by isoprenaline was increased 100% by COMT inhibition. U-0521 did not alter basal lipolysis in vitro but enhanced lipolysis induced by 0.1 M noradrenaline significantly, both in the presence and in the absence of theophylline.The present results shows that COMT influences the effective concentration of isoprenaline more than that of noradrenaline. In the vasculature the effects of exogenous noradrenaline is influenced to a larger degree than those induced by nerve stimulation. However, COMT blockade increased lipolysis induced by equieffective doses of exogenous noradrenaline and nerve stimulation to an equal extent. These results are in agreement with the idea that COMT is of physiological importance mainly at receptor sites that are not in close contact with sympathetic nerve endings and supports the idea that vascular -adrenoceptors are innervated receptors. Vascular -adrenoceptors on the other hand seem to be humoral receptors unrelated to sympathetic nerve endings and preferentially stimulated by circulating noradrenaline. The -adrenoceptors on the fat cells may be of both types, i.e. innervated and humoral.     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Steady-state concentrations of choline and acetylcholine in rat brain parts during a constant rate infusion of deuterated choline

Summary An intravenous infusion of deuterated choline at constant rate for 6 min (5 or 25 moles kg^–1 min^–1) significantly increases the concentration of choline in plasma, occipital cortex and striatum. Both 5 and 25 moles kg^–1 min^–1 increase the concentration of acetylcholine in cortex but only 25 moles kg^–1 min^–1 increases the acetylcholine content in striatum. In contrast, 1 mole kg^–1 min^–1 does not change the choline or acetylcholine content in cortex or striatum. A single pulse injection of choline (200 moles kg^–1) causes a significant increase in the concentration of choline in striatum 30 sec following injection. The choline content returns to normal values within 2 min. These studies show that when a pulse injection of a non-tracer dose of radioactive choline is used to measure brain acetylcholine turnover rate the maintenance of steady state must be verified within seconds after the pulse injection of radioactive choline. When constant infusion of deuterated choline is used to measure turnover rate of acetylcholine in the brain of rats, a dose of 1 mole kg^–1 min^–1 appears to be a maximal infusion rate.     

Viscoelasticity of Anionic Polymers and Their Mucociliary Transport on the Frog Palate

The influence of formulation variables on the rheology of polyanionic formulations and the relationships between viscoelastic properties and mucociliary transport rate were investigated. Polymeric samples were oscillated from 0.001 to 5 Hz using either a "cone and plate or a "coaxial cylinder measuring system. The mucociliary transport rates of polymeric samples were determined and compared movement of charcoal powder on the frog palate. For the linear polymeric solutions, sodium carboxymethylcellulose and sodium alginate, the elastic modulus (G) increased with increasing amplitudes during frequency scan. However, the G or viscous modulus (G) of partially cross-linked polyacrylic acid (cPAA) samples did not change significantly under oscillation. Both G and G of cPAA samples were significantly influenced by the amount of salt present in the formulation. The rheology of 2% (w/w) cPAA in 90:10 (w/w) propylene glycol:alcohol changed from a viscous fluid to a coarse suspension after neutralization. The pH increased gradually when the nonaqueous formulation reacted with water and the maximum dynamic moduli were obtained after incorporating 20% (w/w) water in the formulation. A negative correlation was found between the G of linear polyanionic samples and the relative transport rate. However, the lowest mucociliary transport rate was observed when the loss tangent (G/G) was around 0.4–0.5.     

Different types of opioid receptors mediating analgesia induced by morphine,DAMGO, DPDPE,DADLE and β-endorphin in mice

Summary The effects of intracerebroventricular (i.c.v.) administration of d-Phe-Cys-Tyr-d-Try-Orn-Thr-Pen-Thr-NH₂ (CTOP), a selective mu-opioid receptor antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) and (N,N-Bisallyl-Tyr-Gly-Gly--(CH₂S)-Phe-Leu-OH (ICI 154129), selective delta-opioid receptor antagonists on blocking analgesia induced by -endorphin, morphine, d-Ala²-NMePhe⁴-Gly-ol-enkephalin (DAMGO), d-Ala²-d-Leu⁵-enkephalin (DADLE) and d-Pen²-enkephalin (DPDPE) administered i.c.v. were studied in male ICR mice. The analgesia was assessed by the tail-flick and paw-licking (hot-plate) tests. The potencies of opioid agonists injected i.c.v. for producing analgesia were DAMGO > DADLE > -endorphin > morphine > DPDPE. Intracerebroventricular administration of CTOP (0.05 g) selectively antagonized inhibition of the tail-flick and paw-licking response induced by morphine, DAMGO or DADLE but not -endorphin or DPDPE. ICI 174864 (5 g) and ICI 154129 (5 g) injected i.c.v. selectively antagonized analgesia induced by DPDPE or DADLE but not -endorphin, morphine or DAMGO injected i.c.v. These results indicate that analgesia induced by morphine and DAMGO is mediated by the stimulation of mu-opioid receptors while analgesia induced by DPDPE is mediated by the stimulation of delta-opioid receptors. DADLE-induced analgesia is mediated by the stimulation of both mu- and delta-opioid receptors. Analgesia induced by -endorphin is mediated by neither munor delta-opioid receptors.Abbreviations i.c.v. intracerebroventricular - i.t. intrathecal - CTOP d-Phe-Cys-Tyr-d-Try-Orn-Thr-Pen-Thr-NHZ - DAMGO d-Ala²-NMePhe²-Gly-ol-enkephalin - DADLE d-Ala²-d-Leus-enke-phalin - DPDPE dd-Pen²-dd-Pen⁵-enkephalin - ICI 174864 (Allyl)2Tyr-Aib-Aib-Phe-Leu-OH - ICI 154129 (N,N-Bisallyl-Tyr-Gly-Gly-(CH₂S)-Phe-Leu-OH     

Short-, medium-, and long-term effects of prenatal oxazepam on neurobehavioural development of mice

A benzodiazepine (oxazepam) was given to nulliparous mice on days 12–16 of pregnancy, and the development and young adult behaviour of the offspring were studied. Experiment 1, using 5, 15, and 50 mg/kg doses given PO twice daily, showed a dose-dependent retardation of postnatal development of several responses such as righting, bar holding, limb placing, and auditory startle. These changes were maximal in the first 2 postnatal weeks and then were markedly attenuated, or disappeared, being apparently related to a temporary retardation of body growth. A reduction of locomotor activity at 60 days was found only in the 50 mg/kg group. The effects of the 15 mg/kg dose on postnatal body growth and neurobehavioural development were replicated in Experiments 2 and 3. Moreover, in these experiments prenatal oxazepam reduced open field activity at 14–16 days and attenuated the hyperactivity induced by dl-amphetamine sulphate (2 mg/kg IP). On the other hand activity, habituation, and response to a scopolamine challenge (2 mg/kg IP) at 21–23 days were not significantly different from those of appropriate controls. Experiment 3, using a cross-fostering procedure, showed that postnatal maternal effects were not responsible for the changes so far mentioned. Experiment 2 also investigated the acquisition of several go-no go avoidance discriminations in a shuttle-box, using either light (L) or buzzer noise (N) as the go signal, a compound no go signal (NL in the L-go groups and LN in the N-go groups), and either an extinction or a passive avoidance contingency during the no go signal (4 weeks of training, starting at 60 days). The main effect of prenatal oxazepam was an impairment of active avoidance responding, while the treatment effects on overall discrimination performance were less marked and limited to the later stages of training.     

Involvement of central β-adrenoceptors in the regulation of yawning responses

Summary A behavioral study was performed in an attempt to understand the role of central -adrenoceptors in yawning in rats. Yawning was evoked by apomorphine and piribedil, mixed dopamine D₁/D₂-receptor agonists, but not by SK&F 38393 [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8diol], a dopamine Dr-receptor angonist. The apomorphine-induced yawning was increased by pindolol, propranolol, indenolol, alprenolol and bukumolol which block the central -adrenoceptors, but not by the peripheral -adrenoceptor antagonists, carteolol and atenolol. These -adrenoceptor antagonists given alone did not elicit yawning. Conversely, the yawning was inhibited by salbutamol, a -adrenoceptor agonist, without being affected by prazosin, an -adrenoceptor antagonist. The combined administration of SK&F 38393 and the -adrenoceptor antagonists did not induce yawning. The yawning elicited by either apomorphine or piribedil in combination with pindolol was suppressed by spiperone and YM-09151-2 [cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-methylaminobenzamide], dopamine D₂-receptor antagonists, and scopolamine, a muscarinic receptor antagonist, but not by SCH 23390 [R(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol], a dopamine D₁-receptor antagonist. Physostigmine or pilocarpine induced yawning, which was also enhanced by pindolol and propranolol. This enhanced yawning was inhibited by scopolamine, but not by spiperone, YM-09151-2 and SCH 23390. The present results suggest that the \-adrenoceptor blockade facilitates the occurrence of yawning induced by dopaminergic and cholinergic agonists. Therefore, the central adrenergic neuron systems may participate in the regulation of yawning responses.Send offprint requests to K. Yamada at the above address     

A pharmacokinetic model-independent approach for estimating dose required to give desired steady-state trough concentrations of drug in plasma

A maintenance dose designed to give a desired minimum concentration of drug in plasma at steady-state can be determined in a model-independent manner assuming that concentration-time data needed for the calculation are obtained after absorption and distribution are complete. Using a few concentration-time points obtained after the first dose, numerical values of and Z, a parameter consisting of different pharmacokinetic parameters for different models, can be obtained. An administration interval () can be chosen based on . Using the values of , Z, and , a maintenance dose is calculated. This approach will allow calculation of a maintenance dose when drug is present in plasma at the time the first monitored dose is given.     

Is there a need for more precise definitions of bioavailability?

Summary After evaluation of the present definitions in a set of particular cases, it was agreed that there was no need for more precise definitions and that the current ones were adequate in the majority of cases. However, it was felt that the present definitions might be improved, in particular in view of the existence of non-systemically acting drugs and future targeted drugs. Thus, the FDA definition might be modified as follows: Bioavailability means the rate and extent to which the active drug ingredient or therapeutic moiety from a drug product becomes available at the site of drug action or in a biological medium believed to reflect accessibility to a site of action.     

Cardiovascular effects of selective agonists and antagonists of histamine H3 receptors in the anaesthetized rat

The cardiovascular responses to a series of selective histamine H₃ receptor agonists, (R) -methylhistamine, imetit and immepip and selective antagonists, thioperamide, clobenpropit and clophenpropit, were studied in anaesthetized rats. At 0.003–1 mol/kg i.v. doses, H₃ agonists failed to produce any significant change in the basal blood pressure and heart rate. Larger doses of (R) -methylhistamine increased the blood pressure and heart rate and higher doses of imetit caused vasodepressor responses and reduced heart rate, whereas immepip proved virtually inactive. While (R) -methylhistamine-induced effects were not blocked by histamine H₁-, H₂- and H₃-receptor antagonists, they were however reduced by idazoxan and propranolol, which indicates that the mechanisms involved are adrenergic. The effects induced by imetit are not related to histamine H₃ receptors but are mediated by indirect (via 5HT₃ receptors) cholinergic mechanisms, since these effects were prevented by 1 mg/kg i.v. atropine and by 0.1 mg/kg i.v. ondansetron. Similarly, the H₃ antagonists per se failed to change basal cardiovascular function up to 10 mol/kg i.v. and only at 30 mol/kg i.v. were marked decreases observed in the blood pressure and heart rate with a significant reduction in the effects of noradrenaline. These data indicate that in anaesthetized rats, histamine H₃ receptor activation or blockade has no effect on basal cardiovascular function. The effects recorded after the administration of large doses of (R) -methylhistamine and imetit are clearly unrelated to histamine H₃ receptors and should be taken into account when using these compounds as H₃ ligands for in vivo experiments.