5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates

Effects of eight small aromatic isothiocyanatosulphonates, of the aliphatic 2-isothiocyanatoethene-1-sulphonate (IES), and of the parent amines were studied on contractions of the rat vas deferens elicited by ,-methy-lene ATP (,-MeATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.The aromatic isothiocyanato-sulphonates all reduced contractions of the rat vas deferens elicited by ,-methy-lene ATP. The antagonism was non-competitive, with depression of the maximum of the concentration-response curve of ,-MeATP and incomplete reversibility. The IC₅₀ values were between 11 and 54 M. In the guineapig taenia coli, the aromatic compounds shifted the concentration-response curve of ADPS to the right in a surmountable manner (one exception), and where three concentrations were tested, the Arunlakshana-Schild regression was linear and its slope did not differ from 1. The apparent K _d values were between 10 and 214 M. The removal of ATP from the medium by vas deferens tissue was decreased by the aromatic isothiocyanates with IC_25% values between 25 and 464 M. IES and the parent amines were inactive or almost inactive (parent amines not tested on ATP breakdown).The results indicate that the isothiocyanato residue as well as the aromatic core are essential for P₂-purinoceptor blockade. At the P_2X-purinoceptor, potency increases with the size of the molecules but is independent of the position of the isothiocyanato and sulphonate substituents. No simple structure-activity relationship for the P_2Y-purinoceptor and the ATP-degrading ecto-nucleotidases can be derived beyond the apparent lack of a major influence of the position of the substituents. 2-Isothiocyanatonaphthalene1-sulphonate (-INS) seems to be interesting because of relatively high P_2X-selectivity versus both the P_2Y-purinoceptor and ecto-nucleotidases.     

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

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

Evidence for the existence of two forms of α2A-adrenoceptors in the rat

Summary The _2A-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [³H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a K _d of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for _2A-, _2B- or _2C-adrenoceptor selective drugs, indicated that the sites labelled by [³H]-RX821002 in the spleen consisted of a single population of _2A-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (K _d = 35 nmol/1) and low affinity (K _d = 8900 nmol/1) _2A-adrenoceptor sites in the proportions 57% and 43%, respectively. The K _d ^Sfor a number of ₂-adrenoceptor subtype selective drugs, measured in competition with [³H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their _2A-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the K _d of guanoxabenz being about 4000 nmol/l for both tissues. Drug K _d ^Sfor kidney _2A-adrenoceptors were also determined using [³H]-RX821002. For nearly all drugs tested, the K _d ^Swere highly correlated with those found for the _2A-adrenoceptors in the other rat tissues. However, for guanoxabenz, the data indicated that it competed with [³H]-RX821002 at a single _2A-adrenoceptor site with a K _d of 39 nmol/1. When the rat _2A-adrenoceptor gene RG20 was transiently expressed in COS-7 cells and its ligand binding properties probed using [³H]-RX821002, the drug K _d ^Sobtained were also highly correlated with those found for the _2A-adrenoceptors in the spleen, cerebral cortex, spinal cord and kidney of the rat. For the RG20 encoded receptor, the guanoxabenz competition curves were steep and monophasic and modelled best into one site fits, with the Kd of guanoxabenz being 5200 nmol/1.It is suggested that guanoxabenz can differentiate between two forms of _2A-adrenoceptors in the rat: _2A1 and _2A2. The _2A1-form is present in the spleen and kidney where it shows a high apparent affinity for guanoxabenz. The _2A2-form shows a low apparent affinity for guanoxabenz and is present in the spleen, cerebal cortex and spinal cord. The _2A2-form of the rat ₂-adrenoceptor appears to be encoded by the RG20 gene. The _2A, and _2A2-adrenoceptor forms do not represent high and low affinity receptor forms for agonists because assays included EDTA, Gpp(NH)p and Na⁺, which eliminated the high affinity receptors for agonists.     

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

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

Peripheral effects of opioid drugs on capsaicin-sensitive neurones of the guinea-pig bronchus and rabbit ear

Summary The effect of a potent opioid agonist, [d-Met², Pro⁵]-enkephalinamide was investigated on two responses involving capsaicin-sensitive afferent neurones, namely, atropine-resistant contractions of the guinea-pig bronchus evoked by electrical field stimulation and the nociceptor stimulation to intraarterial injections of acetylcholine or capsaicin into the vascularly isolated rabbit ear. The hypotheses to be tested were whether (a) opioid receptor activation may inhibit mediator release from primary afferent neurones and (b) the opioid could exert an analgesic effect at a peripheral site of action. Non-cholinergic contractions of the guinea-pig isolated main bronchi due to electrical stimulation were concentration-dependently inhibited by [d-Met², Pro⁵]-enkephalinamide (10 nM–1 M). This effect was abolished by naloxone (1 M). Naloxone alone induced no change in the stimulation-evoked contractions of the bronchus, indicating that no endogenous opioid control was present. Substance P and neurokinin A induced bronchial contractions that were not influenced by [d-Met², Pro⁵]-enkephalinamide. This indicates that [d-Met², Pro⁵]-enkephalinamide inhibits electrically-evoked bronchial contractions by reduced mediator release from capsaicin-sensitive sensory nerve endings, since these contractions are most probably brought about by tachykinins, released from afferent neurones. Capsaicin-induced bronchial contractions were in contrast to electrical stimulation not influenced by [d-Met², Pro⁵]-enkephalinamide which suggests a different site of action. The activation of sensory neurones in the rabbit ear by i. a. injection of acetylcholine and capsaicin was not reduced under infusion of [d-Met², Pro⁵]-enkephalinamide (1 and 10 M) or lofentanil (1 and 10 M). The enhancement of the effect of acetylcholine by infusion of prostaglandin E₂ (0.15 M) also remained unchanged under infusion of 10 M [d-Met², Pro⁵]-enkephalinamide. A peripheral analgesic action of the two opioid agonists studied is therefore not indicated. Send offprint requests to F. Lembeck     

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the α2B-adrenoceptor subtype

Summary The present study aimed at relating the presynaptic ₂-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described _2A- and _2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for _2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be _2B selective) were evaluated on [³H]noradrenaline and [^H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [³H]yohimbine binding to human platelet membranes (containing only _2A receptors) and to neonatal rat lung membranes (containing only _2B receptors).The affinity pattern of these compounds at _2A-adrenoceptors in binding studies was oxymetazoline > = idazoxan > PmP > prazosin; at _2B-adrenoceptors it was idazoxan > = prazosin > PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K⁺-evoked [³H]noradrenaline and [³H]serotonin release, IC₅₀ 18 and 7 nM, respectively; in the same conditions, the IC₅₀ values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan > PmP > prazosin, either on [³H]serotonin release.These results indicate that presynaptic ₂ auto- or heteroreceptors do not belong to the _2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the _2A-adrenoceptor subtype. Send offprint requests to M. Gobbi at the above address     

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157–2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 M) decreased MLC phosphorylation and the amplitude of contractions induced by high K⁺ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca²⁺ levels ([Ca²⁺]_i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 M) and prostaglandin F₂ (PGF₂, 10 M) without a change in the [Ca²⁺]_i. On the other hand, wortmannin (1 or 10 M) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF₂ to a level seen at rest. In the absence of external Ca²⁺, caffeine (20 mM) induced a transient increase in [Ca²⁺]_i and force with an increase in MLC phosphorylation. Wortmanmn completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca²⁺]_i. In the absence of external Ca²⁺, phenylephrine induced a small transient increase in [Ca²⁺]_i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca²⁺]_i and MLC phosphorylation. Wortmannin (1 M) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca²⁺]_i nor the sustained contraction. Wortmannin inhibited the Ca²⁺-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca²⁺-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca²⁺ or through the release of Ca²⁺ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF₂. induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Inhibition of N-methyl-d-aspartate (NMDA) and l-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol

Summary The influence of ethanol on stimulation-evoked ³H-transmitter release was examined in slices of the rat brain cortex and corpus striatum preincubated with ³H-noradrenaline and ³H-choline, respectively. ³H-Transmitter release was stimulated by NMDA, l-glutamate, electrical impulses, reintroduction of Ca²⁺ ions (Ca²⁺-evoked release; after superfusion with Ca²⁺-free, K⁺-rich solution) or veratridine. In cortical slices preincubated with ³H-noradrenaline and superfused with Mg²⁺-free, otherwise physiologically composed salt solution, ethanol inhibited the NMDA- or l-glutamate-induced tritium overflow (IC₅₀ 45 and 37 mmol/l, respectively). In contrast, the tritium overflow in response to electrical stimulation, reintroduction of Ca²⁺ ions or veratridine was not affected by ethanol at concentrations up to 320 mmol/l; these experiments were carried out in cortical slices superfused with solution containing a physiological Mg²⁺ concentration. Ethanol also failed to inhibit Ca²⁺-evoked release in the absence of Mg2⁺ ions. In the presence of 1 mol/l veratridine, but not in its absence, NMDA induced tritium overflow even when cortical slices were superfused with salt solution containing a physiological Mg²⁺ concentration; again, ethanol inhibited this NMDA-evoked tritium overflow (IC₅₀ 73 mmol/l). In striatal slices preincubated with ³H-choline and superfused with Mg²⁺-free physiological salt solution, the NMDA-evoked tritium overflow was also, although at lower potency, inhibited by ethanol (IC₅₀ 192 mmol/l).In spite of the differences between the IC₅₀ values of ethanol determined for the inhibition of cortical noradrenaline and striatal acetylcholine release, it may be concluded that the NMDA receptor-ion channel complex is one of the sites of action underlying the ethanol-induced inhibition of neurotransmitter release. Since in the brain cortex the NMDA-induced ³H-noradrenaline release appears to be mediated by an excitatory interneurone activated by NMDA, this neuronal system may be involved in the cortical actions of ethanol.     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.     

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     

Possible involvement of both N- and L-type voltage-dependent Ca channels in adrenergic neurotransmission of canine saphenous veins in low Ca2+ plus tetraethylammonium medium

Summary The involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca²⁺ + 20 mM tetraethylammonium (low Ca²⁺ + TEA) medium has been studied by examining the effects of -conotoxin GVIA (-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked ³H overflow from canine saphenous veins preloaded with [³H]-noradrenaline. Nisoldipine (10 and 30 M) and nifedipine (30 M) reduced significantly the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 M nisoldipine and 10 tM nifedipine. -CTX inhibited markedly the TNS-evoked ³H overflow in both normal Krebs and low Ca²⁺ + TEA media, the inhibition by -CTX being ten times more potent in low Ca²⁺ + TEA medium. Nisoldipine (30 M), when combined with 1 nM -CTX, produced a further significant inhibition of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. However, no additional inhibition by 30 M nisoldipine was observed when -CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 M) did not affect the inhibitory effect of 10 nM -CTX on the evoked ³H overflow. The low Ca²⁺ + TEA medium increased the spontaneous ³H overflow, which was not influenced by -CTX and dihydropyridines. These results suggest that in low Ca²⁺ + TEA medium but not normal Krebs one, Ca²⁺ entry via both N- and L-type VDCCs may be involved in adrenergic neurotransmission in the canine saphenous veins. Correspondence to Y. Takata at the above address     

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X₁ receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y₁ or P2Y₁₂ receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X₁ receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X₁ antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on meATP (P2X₁ receptors)-induced or ADP (P2Y₁ receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y₁ & P2Y₁₂ receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8,8,8-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X₁ antagonist reported to date, blocking meATP-induced Ca²⁺ increases and shape change in a concentration-dependent manner, with a pA₂ of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X₁ receptor displayed the following order : NF864>NF449NF110>NF023=MK-HU1=suramin. A different antagonistic profile was observed for ADP-induced Ca²⁺ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X₁ responses, with the order NF110>NF449NF864MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X₁ receptors in thrombosis and haemostasis, NF864 being the most promising compound.     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above     

Role of basolateral amygdala dopamine in modulating prepulse inhibition and latent inhibition in the rat

Rationale The dopamine (DA) projection to the basolateral amygdala (BLA) modulates nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) DA transmission. Given the involvement of the BLA, and of NAc and mPFC DA, in select forms of information processing, we sought to determine the role of BLA DA in modulating prepulse inhibition (PPI) and latent inhibition (LI).Objective The effects of BLA D₁ (SCH 23390) and D₂/D₃ (raclopride) receptor blockade on PPI and LI were examined.Methods Separate groups of male Long–Evans rats received bilateral intra-BLA infusions of SCH 23390 (3.2 or 6.4 g/0.5 l per side), raclopride (2.5 or 5.0 g/0.5 l per side) or saline prior to testing. In two experiments, the effects of BLA DA receptor antagonism on PPI of the acoustic startle response (ASR) and LI of conditioned taste aversion were determined. A control group received bilateral intra-striatal infusions of SCH 23390 or raclopride prior to PPI testing.Results Intra-BLA SCH 23390 or raclopride had no effect on the ASR. Intra-BLA SCH 23390 enhanced and raclopride disrupted PPI, both in a dose-related manner. Intra-striatal SCH 23390 or raclopride had no effect on PPI or ASR magnitude. Finally, BLA DA receptor blockade had no effect on LI.Conclusions These results indicate that PPI is modulated by BLA DA and suggest that this modulation occurs independently of changes in NAc and/or mPFC DA transmission. They also suggest that BLA DA is not involved in modulating LI and add to evidence indicating that PPI and LI are mediated by different neural substrates.     

Inhibition by carbamazepine of various ion channels-mediated catecholamine secretion in cultured bovine adrenal medullary cells

The effects of carbamazepine (CBZ) on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, catecholamine secretion and cyclic GMP production were examined in cultured bovine adrenal medullary cells. 1 CBZ (40–120 mol/l) inhibited ²²Na⁺ influx evoked by carbachol in a concentration-dependent manner. CBZ inhibited carbachol-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion at concentrations similar to those which suppressed ²²Na⁺ influx. 2 CBZ (4–120 mol/l) inhibited veratridine-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion. 3 CBZ (12 or 40–120 mol/l) suppressed 56 mmol/1 K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, respectively. 4 Combination of CBZ with nitrendipine or -agatoxin-IVA produced further inhibition of 56 mmol/l K⁺ - evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, compared to the effect of CBZ alone, whereas CBZ plus -conotoxin-GVIA did not produce any further inhibition. 5 CBZ (40 mol/1) attenuated the production of cyclic GMP caused by muscarine. These results suggest that CBZ at therapeutic concentrations (16–48 mol/l: 4–12 g/ml) inhibits catecholamine secretion by interfering with nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and N-type voltage-dependent Ca²⁺ channels, and may have an antimuscarinic effect in adrenal medullary cells.     

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris

Summary The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with ¹⁴C-choline. The stimulation-evoked ¹⁴C-overflow was doubled in the presence of atropine 0.1 mol/l but unaffected by the agonist (±)-methacholine (50 mol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mol/l). peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M₂-selective antagonists methoctramine (10 mol/l) and gallamine (100 µmol/l). increased both the ¹⁴Goverflow and the peak contractions evoked by field stimulation. In contrast, the M₃-selective antagonist hexahydrosiladifenidol (0.1–10 mol/l) failed to affect the evoked ¹⁴C-release but concentration-dependently (1–10 mol/l) reduced the iris contractions. Pirenzepine (10 mol/l) enhanced the evoked ¹⁴C-overflow and inhibited the peak contractions (0.1–10 mol/l; maximal effect at 10 mol/l). The low potency of the antagonist at both receptor sites indicates that an M₁ muscarine receptor is not involved. The results are consistent with the idea of M₂ muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M₃-like receptors inducing the contraction of the sphincter muscle. Send offprint requests to I. T. Bognar at the above address     

Selective blockade of nicotinic acetylcholine receptors by pimobendan,a drug for the treatment of heart failure: reduction of catecholamine secretion and synthesis in adrenal medullary cells

Pimobendan, a Ca²⁺ sensitizer, is used clinically in the treatment of chronic heart failure. Although chronic heart failure is associated with activation of the sympathetic nervous system, it remains unknown whether pimobendan affects the function of sympathetic neurons and the adrenal medulla. Here, we report the inhibitory effects of pimobendan on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Pimobendan decreased the catecholamine secretion (IC₅₀=29.5 M) elicited by carbachol, an agonist at nicotinic acetylcholine receptors, but not that elicited by veratridine, an activator of voltage-dependent Na⁺ channels, or by high K⁺, an activator of voltage-dependent Ca²⁺ channels. Pimobendan also inhibited carbachol-induced influx of ²²Na⁺ (IC₅₀=25.9 M) and ⁴⁵Ca²⁺ (IC₅₀=26.0 M), but not veratridine-induced ²²Na⁺ influx or high K⁺-induced ⁴⁵Ca²⁺ influx. The reduction of catecholamine secretion caused by pimobendan was not overcome by increasing the concentration of carbachol. UD-CG 212, an active metabolite of pimobendan, lowered carbachol-induced catecholamine secretion with a concentration/inhibition curve similar to that of pimobendan. In experiments in situ, pimobendan suppressed both basal and carbachol-stimulated ¹⁴C-catecholamine synthesis (IC₅₀=5.3 and 4.9 M) from [¹⁴C] tyrosine [but not from l-3, 4-dihydroxyphenyl [3-¹⁴C] alanine ([¹⁴C]DOPA)], as well as tyrosine hydroxylase activity (IC₅₀=3.8 and 4.3 M). These findings suggest that pimobendan inhibits carbachol-induced catecholamines secretion and synthesis through suppression of nicotinic acetylcholine receptors.