Transplacental passage and breast milk concentrations of hydralazine

Summary The concentrations of real and apparent (= real hydralazine + acid-labile hydrazones) hydralazine in maternal and umbilical plasma obtained at delivery of 6 women treated with hydralazine and atenolol for pregnancy hypertension were measured by gas chromatography. In one of the patients, the concentrations of the same substances were subsequently measured in breast milk. Apparent hydralazine reached higher levels in umbilical than in maternal blood. The concentration of real hydralazine seemed to be at least as high in the fetus as in the mother. On the other hand, even though the fraction of real (i.e. presumably active) hydralazine was greater in milk than in plasma, the total concentration was smaller, and the estimated dose per milk feed of 75ml would not exceed 0.013mg. Thus, hydralazine treatment of the pregnant woman would expose her fetus to effective concentrations of the drug, but breast feeding would not result in a clinically relevant concentration in the infant.     

Differential Effects of Sulfate and Sulfobutyl Ether of β-Cyclodextrin on Erythrocyte Membranes in Vitro

The hemolytic activity of -cyclodextrin (-CyD) on rabbit erythrocytes was reduced by the introduction of negatively-charged groups onto the hydroxyls of -CyD; the membrane disrupting abilities decreased in the order of -CyD > 2-hydroxypropyl--CyD (HP--CyD) > sulfobutyl--CyD (SB--CyD) >> -CyD sulfate (S--CyD). Under pre-hemolytic concentrations, both -CyD and SB--CyD induced shape changes of membrane invagination on the erythrocytes. In sharp contrast, S--CyD showed biphasic effect on the shape of the erythrocytes; i.e. the crenation at relatively low concentrations and the invagination at higher concentrations. The S--CyD-induced membrane crenation arose from a direct action on the membranes rather than cell metabolism-mediated effects. Unlike -CyD, S--CyD was found to bind to the erythrocytes and may be confined to the outer surface of the membrane bilayer, which may expand the exterior layer relative to the cytoplasmic half, thereby inducing the cells to crenate. On the other hand, the membrane invagination mediated by the three - CyDs was initiated by extracting specific membrane lipids from the cells, depending upon their inclusion abilities, subsequently leading to the lysis of the cells. These results indicate that SB--CyD and S--CyD interact with the erythrocyte membranes in a differential manner and possess lower membrane disrupting abilities than the parent -CyD and HP--CyD.     

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     

The sympathetic axons innervating the sinus node of the rabbit possess presynaptic opioid к- but not μ- or δ-receptors

Summary The postganglionic sympathic nerves of rabbit isolated hearts were stimulated with pulses delivered at 5 Hz and train durations of 1–5 s. Ethylketocyclazone 0.01–1 mol/l and fentanyl 1 and 10 mol/l but not morphine 1 and 10 mol/l, Met-enkephalin 1 and 4 mol/l or d-Ala², d-Leu⁵-enkephalin 0.5 and 5 mol/l diminished the stimulation-evoked increase in heart rate. The effect of ethylketocyclazocine 0.1 mol/l was antagonized by naloxone 1 and 10 mol/l. In contrast, the effect of fentanyl was not changed by naloxone 10 mol/l. Ethylketocyclazocine 0.03 and 1 mol/l did not reduce the tachycardia elicited by exogenous noradrenaline. The results suggest that, under in vitro conditions, only presynaptic opioid - but not - or -receptors inhibit the release of noradrenaline from the sympathetic neurones innervating the sinus node.     

Evidence for a vasoconstriction-mediating receptor for UTP,distinct from the P2 purinoceptor,in rabbit ear artery

Summary 1. The mechanism of uridine 5-triphosphate-(UTP-)induced vasoconstriction was studied in the rabbit ear artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. 2. Noradrenaline, adenosine 5'-triphosphate (ATP) and UTP caused concentration-dependent vasoconstriction. ATP and UTP were approximately equipotent. 3. The vasoconstrictor effect of UTP 300 mol/l was enhanced by a mixture of atropine, diphenhydramine and methysergide (1 mol/l each) and not affected by indometacin 10 mol/l. 4. Prazosin (0.01 –1 mol/l) and phentolamine (1–10 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by up to 34%. Prazosin 1 mol/l failed to diminish the vasoconstrictor effect of UTP 300 mol/l after the sympathetic nerves had been destroyed with 6-hydroxydopamine. 5. , -Methylene-ATP (10–50 ol/l) elicited transient vasoconstriction. Subsequently, vasoconstrictor responses to ATP 100 or 300 pmol/1 were reduced by 88%, whereas responses to UTP 100 gmol/1 were enhanced, responses to UTP 300 mol/l decreased by only 32% and responses to UTP 1000 gmol/1 reduced by 74%. After in vitro-denervation with 6-hydroxydopamine or in the presence of phentolamine 1 mol/l throughout, a, -methylene-ATP (10–50 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by 44% and 43%, respectively. 6. We suggest that, in the rabbit ear artery, the non-adrenergic and , -methylene-ATP-resistant vasoconstrictor response to UTP is mediated by a separate receptor mechanism, distinct from the P₂ purinoceptor. Send offprint requests to K. Starke     

Differential effects of six structurally related benzodiazepines on some ethological measures of timidity,aggression and locomotion in mice

The effects were compared of three 2' chloro-phenyl-benzodiazepines (triazolam, clonazepam and lorazepam) and three corresponding 2' deschloro-phenylderivatives (alprazolam, nitrazepam and oxazepam, respectively) on the incidence of six ethological elements in both timid and aggressive singly-housed male mice, treated with drugs in paired interactions with untreated non-aggressive males. Alprazolam and oxazepam reduced defensive upright postures and escapes at doses which did not reduce rearing and actually increased walking, while their chlorinated counterparts (triazolam and lorazepam, respectively) decreased incidence of defenses and escapes mostly at doses decreasing locomotor acts involving a similar movement (rears and walks, respectively). Alprazolam and oxazepam also reduced attacks at doses not reducing rears, in contrast to triazolam and lorazepam which reduced attacks only at doses suppressing rearing. Nitrazepam stimulated sniffing partners much more than its chlorinated counterpart clonazepam. The 2 deschloro-phenyl-benzodiazepines were more potent in reducing defensive-escape activities than attacks or locomotion. Yet, none of the benzodiazepines tested produced a complete inhibition of timid defensive-escape behavior at non-sedative doses. The present study suggests that 2 deschloro-phenyl-benzodiazepines are less sedative with respect to their anxiolytic activity.     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Viscoelastic Properties of Bioadhesive,Chlorhexidine-Containing Semi-Solids for Topical Application to the Oropharynx

Purpose. This study examined the viscoelastic properties of bioadhesive, chlorhexidine-containing semi-solid formulations, designed for topical application to the oropharynx. Methods. Oscillatory rheometry was performed using a Carri-Med CSL²-100 rheometer at 20.0 ± 0.1° C in conjunction with parallel plate geometry (2 cm diameter, 0.5 mm sample thickness). Samples were subjected to a constant strain (6.5 × 10^–3 rad) and defined viscoelastic parameters, namely storage modulus (G), loss modulus (G), loss tangent (tan ) and dynamic viscosity (), measured over a defined frequency range (0.01-1.0 Hz). Results. As the oscillatory frequency was increased, G G of all formulations increased, whereas both and tan significantly decreased. The magnitude of increase of G and G as a function of frequency was relatively small, indicating that, in general, the formulations were non-cross-linked elastic systems. Increasing concentrations of HEC, PVP and PC significantly increased G, G, yet decreased tan observations that may be attributed to the physical state of each polymer in the formulations. Formulation elasticity increased (i.e. tan decreased) as a result of increased entanglement of polymeric chains of dissolved components (i.e. HEC and PVP) and the restrained extension of swollen, cross-linked chains of PC. Additionally, in formulations where the saturation solubility of PVP was exceeded and/or insufficient 'free-water' was available for maximal swelling of PC, formulation elasticity increased as a result of the increasing mass of dispersed solid particles of PVP and/or PC. Formulation increased due to the attendent effects of polymer chain entanglement and polymer state on overall formulation viscosity. Conclusions. Following application to the oropharynx, the formulations will behave as elastic systems. Thus, these formulations would be expected to offer advantageous clinical properties, e.g., prolonged drug release, increased bioadhesion. However, it is noteworthy that the final choice of formulation for clinical evaluation will involve a compromise between viscoelastic characteristics and acceptable textural properties, e.g. ease of product application. This study has shown the applicability of oscillatory rheometry for both the characterisation and selection of candidate, topical bioadhesive formulations for clinical evaluation.     

Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

SK&F 104078, a post-functionally selective α2-adrenoceptor antagonist in the human saphenous vein in vitro

Summary The present study investigated the effects of SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3methyl-1H,2,3,4,-tetrahydro-3-benzazapine) at pre- and post functional ₂-adrenoceptors in the human isolated saphenous vein. Noradrenaline (0.001–100 mol/l) produced concentration-dependent contractions of the human saphenous vein which were competitively antagonised by the ₁-adrenoceptor antagonist prazosin (0.01–1.0 mol/l) and the ₂-adrenoceptor antagonist, rauwolscine (0.01–1.0 mol/l), indicating the presence of both post functional ₁- and ₂-adrenoceptors in this preparation. The selective ₂-adrenoceptor agonist, UK-14,304 (0.01–100 mol/l) also produced concentration-dependent contractions of the human saphenous vein which were antagonised by both rauwolscine (0.1 mol/l) and prazosin (0.1 mol/l). In the presence of angiotensin II (0.05 mol/l), which itself produced a transient contraction, rauwolscine (0.1 mol/l) produced a rightward shift of the UK-14,304 concentration-response curve while prazosin (0.1 mol/l) had no effect. SK&F 104078 (10.0 mol/l) under these conditions also produced a rightward shift of the concentration-response curve to UK-14,304, but was at least 100-fold less potent than rauwolscine. At pre functional ₂-adrenoceptors, exogenous noradrenaline (0.01 and 0.1 gmol/l) induced a concentration-dependent inhibition of stimulation-evoked [7-³H]-noradrenaline release from the human saphenous vein in vitro, which was antagonised by rauwolscine (0:1 mol/l) and tolazoline (10.0 mol/l) but not by SK&F 104078 (10.0 gmol/l).Rauwolscine (0.1 mol/l) produced a small increase in stimulation-evoked [7-³H]-noradrenaline release while both tolazoline and SK&F 104078 failed to produce any enhancement in release in the absence of exogenous agonist atconcentrationsupto10 gmol/l.Insummary, noradrenaline and UK-14,304 contracted the human isolated saphenous vein by an action at both postfunctional ₁- and ₂-adrenoceptors. These data demonstrate that SK&F 104078 discriminates between post- and pre-junctional ₂-adrenoceptors in the human isolated saphenous vein. Send offprint requests to M. V. Sennitt at the above address     

Phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex

Summary Possible antagonist effects of phentolamine at presynaptic serotonin autoreceptors were studied in slices of the occipito-parietal cortices of the rabbit and the rat. The slices were preincubated with ³H-serotonin and then superfused and stimulated electrically with single pulses or pulse trains. Nitroquipazine 1 mol/l, a compound that inhibits the high affinity neuronal uptake of serotonin, was present in the superfusion medium in all one pulse-experiments as well as in experiments in which the effect of unlabelled serotonin was examined.In rabbit cortical slices, unlabelled serotonin reduced the single pulse-evoked overflow of tritium. Its concentrationresponse curve was not changed by the selective ₂-adrenoceptor antagonist idazoxan 1 mol/l but was shifted to the right by phentolamine 1 and 10 mol/l. Phentolamine 10 mol/l also shifted to the right the concentration-inhibition curve of the selective 5-HT₁-receptor agonist 5-carboxamidotryptamine. When the slices were stimulated by trains of 30 pulses at 3 Hz, phentolamine 1 and 10 mol/l but not 0.1 mol/l increased the evoked overflow of tritium, the maximal increase amounting to 178%; its effect was enhanced in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l (a drug combination that, when given alone, slightly increased the evoked overflow of tritium). The serotonin receptor antagonist metitepin at concentrations of 0.01–1 mol/l also increased the overflow of tritium elicited by 30 pulses/3 Hz, the maximal increase amounting to 280%; its effect was potentiated in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l but was abolished or almost abolished in the presence of nitroquipazine 1 mol/l plus phentolamine 10 mol/l (a drug combination that, given alone, greatly increased the evoked overflow of tritium). When slices were stimulated by trains of 360 pulses at 3 Hz, there was no apparent antagonism of phentolamine 10 mol/l against the inhibitory effect of unlabelled serotonin. In rat brain cortex slices, unlabelled serotonin reduced the overflow of tritium elicited by 4 pulses delivered at 100 Hz. Again, phentolamine 10 mol/l shifted the concentration-response curve to the right.It is concluded that phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex with pA₂ values of 6.44 and 5.95, respectively. Previous failures to detect the antagonistic effect against exogenous agonists were probably due to stimulation conditions that led to marked endogenous autoinhibition of serotonin release. At least the major part of the increase by phentolamine of the release of serotonin is due to autoreceptor blockade rather than blockade of the presynaptic a2-adrenoceptors at the cortical serotoninergic axons.Send offprint requests to N. Limberger at the above address     

Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain

Summary Levocabastine is a potent antihistamine drug, structurally unrelated to neurotensin. In rat and mouse brain but not in other animal species, it inhibited 60% of the [³H]neurotensin binding displaced by unlabelled neurotensin or neurotensin (8–13).The levocabastine-sensitive site or site 1 displayed high affinity properties for levocabastine (IC₅₀=25 nM) and was highly stereospecific (IC₅₀-value higher than 10 M for one of the isomers). Binding to the site 1 in rat brain corresponded to the [³H]neurotensin binding displaceable by 1 M levocabastine, whereas binding to the site 2 corresponded to the binding displaced by 1 M neurotensin when the site 1 was occluded by 1 M levocabastine.Both site 1 and site 2 appeared to be saturable. Scatchard plots obtained in rat bulbus olfactorius allowed to calculate a K _D-values of 7.1 nM and a B _max-values of 37.2 fmol/mg original tissue for site 1, while site 2 displayed a K _D-value of 0.7 nM and a B _max-value of 16.3 fmol/mg original tissue. The regional distributions of both sites showed marked differences. The site 1 was homogeneously distributed throughout all rat brain areas, whereas the amount of site 2 binding was markedly different in separate brain areas: bulbus olfactorius and substantia nigra had the highest amounts (8.9 and 7.8 fmol/mg tissue) while cerebellum had the lowest (0.4 fmol/mg tissue).In spite of its high affinity and stereospecificity, site 1 has to be considered as an acceptor or recognition site for [³H]neurotensin because of its species-link, low saturability and homogeneous distribution in all rat brain areas.On the other hand, site 2 had the characteristics of a physiological receptor: high affinity, saturability in the low nanomolar range and marked regional distribution in rat brain. Site 2 corresponds therefore most probably to the physiological neurotensin receptor. The foregoing experiments provide evidence for the presence of a drug displaceable, non-specific (=unrelated to a physiological receptor) neurotensin binding site in rat brain; levocabastine should be an important tool to occlude this site in order to reveal, by means of in vitro binding assays, the specific neurotensin binding site in rat brain.     

The selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate,discriminates between smooth muscle and neuronal P2X purinoceptors

The effects of the putative selective P_2X purinoceptor agonist, ,-methylene-l-adenosine 5-triphosphate (me-l-ATP), were determined at rat neuronal and smooth muscle P_2X purinoceptors.Me-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 M. In contrast, the archetypal P_2X purinoceptor agonist, , methylene ATP (meATP;1–100 M), produced concentration-related depolarisation responses with a mean EC₅₀ value of 10.8 M. The depolarising effects of meATP were not attenuated by me-l-ATP (100 M). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 M), but not me-l.-ATP (1–300 M), evoked rapid ( < 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, me-d-ATP and meATP competed with high affinity for [³H]Lx meATP binding sites, with mean pIC₅₀ values of 7.7 and 8.3, respectively. However, me-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 M (mean pIC₅₀ value 4.1).In prostatic segments of the rat vas deferens, me-l-ATP (1–100 M) and meATP (0.3–100 M) each produced concentration-related contractile responses with mean EC₅₀ values of 17.1 and 3.6 M, respectively. Me-l-ATP (1–10 M) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [³H]meATP exhibited high affinity for me-l-ATP, meATP and me-d-ATP with mean PIC₅₀ values of 7.7, 8.4 and 7.3, respectively.These results indicate that me-l-ATP exhibits neither agonist nor antagonist properties at P_2X purinoceptors on rat vagal neurones and possesses only very low affinity for [³H]meATP binding sites in rat brain. In contrast, me-l-ATP is a potent, high affinity agonist at smooth muscle P_2X purinoceptors of the rat vas deferens. This selective agonist action of me-l-ATP suggests that P_2X purinoceptors in smooth muscle and neurones are different and represent distinct P_2X purinoceptor subtypes.     

Analysis of receptor-G protein interactions in permeabilized cells

Receptor-induced binding of the stable GTP analogue, guanosine 5-[-thio]triphosphate (GTP [S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus -toxin, binding of GTP [S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.Overall, this study indicates that receptor-stimulated GTP [S] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.     

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.     

Nicotine and smoking: Effects upon subjective changes in mood

A Mood Adjective Check List and an activation scale were used to measure subjective reports on mood changes in 24 male habitual smokers before and after smoking cigarettes with known content of nicotine, at different times of day and rates of puffing. Ratings on pleasantness were dose related. Aggression and anxiety showed effects attributable to circadian influence and slight decreases in both factors occurred after smoking the highest nicotine cigarette. The MACL scores were greatly affected by the experimental procedure. Levels of inner tension were found related to the nicotine inhaled. The heuristic value of the concept of activation in these studies is suggested.This work was supported by the Tobacco Research Council, and carried out at the Institute of Psychiatry, London.     

New therapeutic targets for rheumatoid arthritis

New insights into the pathogenesis of rheumatoid arthritis (RA) and consequently new targets of therapy are covered in a broad overview fashion. Shortterm significant beneficial effect on RA disease activity has been established in a small but rapidly growing number of doubleblind placebocontrolled trials now including recombinant human IL1 receptor antagonist, chimeric (mouse/human) monoclonal antibodies (mAb) against TNFa (cA2), humanised (human/mouse) antiTNFa mAb (CDP571) and recombinant human TNFreceptorFc fusion protein (TNFR : Fc). Placebocontrolled trials of antiT cells agents such as chimeric antiCD4 mAb (cMT412) and antiCD5 immunoconjugate, did not demonstrate clinical benefit. A placebocontrolled study of the antiT cell derived cytokine IL2 (DAB486IL2) showed only modes clinical improvement. Other antiT cell approaches such as autologous T cell vaccination and induction of tolerance by oral type II collagen have been unsuccessful. The one controlled trial with an antiinflammatory cytokine, recombinant human IFNg, showed modest clinical benefits. Controlled trials with IL4 and IL10 and with antiadhesion molecules are awaited.     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

Determinants of change in the intention to use benzodiazepines

Objective: To examine attitudes and beliefs associated with changes in the intention to use benzodiazepines during the sixmonth period after first benzodiazepine use.Design: Populationbased 6month followup with 3 measurement points (baseline, 2 weeks after inclusion, 6 months after inclusion).Setting: Starting or initial benzodiazepine users were included during a period of 4 months from November 1994 in the only pharmacy of a Dutch community of 13,500 people.Measures: Variables proposed by the Model of Planned Behaviour and the Health Belief Model. Drug exposure data from automated pharmacy records.Results: At baseline, the intention to use benzodiazepines was primarily predicted by the perceived norm of the general practitioner regarding benzodiazepine use, and by the participants' own attitudes. After fourteen days, the determinants of change in the intention to use benzodiazepines were the initially perceived norm of the general practitioner and the change in the severity of participants' illnesses. After six months, the change in the severity of the illness and the perceived health benefits of benzodiazepines at the time of inclusion were the main determinants of the change in the intention to use benzodiazepines between the second and third measurement point. The intention to use benzodiazepines showed a decrease during followup. The three intention measures were significant predictors of actual benzodiazepine use during the year following baseline assessment.Conclusion: The study sheds light on interesting determinants of decrease or increase in the intention to use during the sixmonth period after first benzodiazepine use.