Concentration andpH dependent steady-state volume of distribution of methotrexate estimated by a simple physiologically based method

The effects of plasma concentration and pH on the steady-state volume of distribution, V_ss,of methotrexate (MTX) were studied in five conditioned male beagle-mongrel dogs. Steady-state plasma MTX concentrations of approximately 1, 20, and 100g/ml were targeted for by i.v. bolus doses followed by i.v. infusions. An isotonic solution of sodium bicarbonate or ammonium chloride was simultaneously infused for the purpose of inducing plasma pH change, while the infusion of an isotonic solution of sodium chloride served as a control. Plasma and urine concentrations of MTX were quantitated by a sensitive high-performance liquid chromatographic method, and the V_ss of MTX was estimated by a recently reported physiologically based method of Chiou and Lam. Statistically significant (p<0.05) concentration and plasma pHdependent V_ss of MTX were observed. Concentration dependence of V_ss was noted in sodium chloride and ammonium chloride infused dogs, but not in bicarbonate treated dogs. There was an average 50.0 and 44.8% increase in V_ss at 1 g/ ml relative to the two higher concentrations (20 and 100 g/ ml) for dogs treated with ammonium and sodium chloride, respectively. However, V_ss of MTX at the targeted concentrations of 20 and 100 g/ml was relatively constant. Plasma pHdependence of V_ss was observed only at the plasma concentration of 1 g/ml, and on the average, ammonium chloride and sodium chloride treatments resulted in 50.0 and 31.3% higher V_ss,respectively, when compared with the bicarbonate treatment. These phenomena appear to be adequately explained by the reported tissue uptake kinetics of MTX.This research was supported in part by a grant from the National Cancer Institute, CA-29754.Abstracted from a dissertation submitted in 1984 by Chung Y. Lui to the Graduate College, University of Illinois at Chicago, in partial fulfillment of Doctor of Philosophy Degree requirements.     

Cyclic nucleotide phosphodiesterases of human and rat gastric mucosa

Summary Cyclic adenosine-3,5-monophosphate (cAMP) phosphodiesterases (PDE) were partially purified from human and rat gastric mucosa. Drugs known to affect the cyclic nucleotide system and/or gastric secretion were tested for effects on the PDE-activities from both species.In rat gastric mucosa PDE-activity can be detected in the 100 000×g sediment (K _m =8.3 M; V _max=3.2 nmoles cAMP hydrolyzed/mg protein x min) and the cytoplasma (K _m =5.6 M; V _max=2.6 nmoles cAMP hydrolyzed/mg protein x min).The most effective inhibitors of the particle-bound activity are papaverine (K _i =4 M, non-competitive) and 3-isobutyl-1-methylxanthine (K _i=14 M, competitive). There was only a modest competitive inhibition by theophylline (K _i =495 M). PDE-activity in the cytoplasma was inhibited competitively by these three drugs (papaverine: K _i =6.5 M; 3-isobutyl-1-methylxanthine: K _i =37 M; theophylline: K _i =152 M.In human gastric mucosa PDE-activity can be detected in the particular fraction (K _m =23.9 M; V _max=1.2 nmoles cAMP hydrolyzed/mg protein x min), and the soluble fraction (K _m =12.1 M; V _max=2.4 nmoles cAMP hydrolyzed/mg protein x min).PDE-activity in the 100 000×g sediment was inhibited by papaverine (K _i =5.6 M, non-competitive), 3-isobutyl-1-methylxanthine (K _i =16 M, non-competitive), theophylline (K _i =165 M, non-competitive), and N₆-2-O-dibutyryl-cAMP (K _i =746 M, competitive).Inhibition in the 100 000×g supernatant was noncompetitive with 3-isobutyl-1-methylxanthine (K _i =7.1 M and papaverine (K _i =8.5 M), but competitive with N₆-2-O-dibutyryl-cAMP (K _i =170 M), and theophylline (K _i =225 M). This study indicates that PDE-activities of the two species are qualitatively similar, but quantitative differences exist.     

Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors

Summary Effects of dynorphin-(1–13), Leu⁵-enkephalin,d-Ala²,d-Leu⁵-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S₁, S₂). Drugs were administered before S₂. Bremazocine 10 g/kg+2g/kg/h and 100 g/kg+20 g/kg/h, dynorphin 1 and 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 10 and 30 g/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 g/kg/min) was given twice for 3 min each (N₁, N₂). Drugs were administered before N₂. Bremazocine 100 g/kg+20 g/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 30 g/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 g/kg/min and DADLE 10g/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 g/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 g/kg/min or DADLE 10 g/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure. They act at peripheral sites, presumably prejunctional opioid receptors at postganglionic sympathetic axons. Dynorphin on the one hand, and Leu⁵-enkephalin and DADLE on the other hand, appear to act at different receptors, dynorphin probably at a - and DADLE and Leu⁵-enkephalin at a -receptor.     

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery

Summary The muscarinic agonists acetylcholine (150 mol/l), carbachol (1–10 mol/l) and McN-A-343 (1–50 mol/l, selective for M₁ receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [³H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, nonselective), pirenzepine (1 and 5 mol/l, M₁-selective), methoctramine (1 and 5 mol/l, M₂-selective) and pfluoro-hexahydro-sila-difenidol (0.1–1 mol/l, M₃-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methylatropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) methoctramine (6.20). These results demonstrate the existence of facilitatory M₁ receptors modulating noradrenaline release in blood vessels. Correspondence to M. Salaices at the above address     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.     

Liquid Dose Pulmonary Instillation of Gentamicin PulmoSpheres® Formulations: Tissue Distribution and Pharmacokinetics in Rabbits

Purpose. To assess the pharmacokinetics and biodistribution of gentamicin, delivered as PulmoSpheres® formulations in rabbit serum and lung tissue following intratracheal instillation in a perflubron vehicle. Methods. Rabbits were anesthetized, intubated, and mechanically ventilated with O₂(FiO₂ = 0.50). Animals were then given 5 mg/kg gentamicin either intravenously, intramuscularly (IM), or intratracheally (IT) gentamicin PulmoSpheres® formulation, instilled in 1.8 ml/kg of liquid perflubron vehicle. Serum and lung lobe sections were collected at multiple time points and assayed for gentamicin content. Results. Serum gentamicin levels peaked at 64.7 g/ml, 11.2 g/ml, and 5.0 g/ml following intravenous, IM, and IT administration, respectively. Absolute bioavailability at 8 h for IM administration was 76.8% and 57.0% when delivered IT. Although peak lung levels of drug were reached within 1 h, total lung gentamicin concentration after IT administration was more than two orders of magnitude greater than that achieved following IM administration (680,540 vs. 4,985 g min, respectively) with significant levels of the antibiotic remaining in the lung even after 1 week. Conclusions. High levels of gentamicin in lung tissue can be achieved by instillation of a gentamicin PulmoSpheres® formulation in a perflubron vehicle, termed liquid dose installation, without reaching toxic systemic levels allowing for increased local delivery of agents such as gentamicin at the site of the infection.     

Calcium channel modulation by dihydropyridines modifies sufentanil-induced antinociception in acute and tolerant conditions

Summary The study was aimed at elucidating the possible participation of l-type Ca²⁺ channel in the acute analgesic effect of an opiate and the development of tolerance to this action. Sufentanil, a selective p agonist, and two dihydropyridines, the Ca²⁺ antagonist nimodipine and the Ca²⁺ agonist Bay K 8644, were selected. The tail-flick test was used to assess the nociceptive threshold. In naive rats, nimodipine (200 g/kg) potentiated the analgesic effect of sufentanil reducing the ED₅₀ from 0.26 to 0.08 g/kg. Similar results were observed with its (–)-enantiomer Bay N 5248, while the (+) enantiomer Bay N 5247 was ineffective. Tolerance to the opiate was induced by chronic subcutaneous administration of sufentanil with minipumps (2 g/h, 7 days). In these conditions the dose-response curve to sufentanil was displaced to the right and the ED₅₀ was increased to 1.49 g/kg. In tolerant rats, nimodipine preserved its potentiating ability and prevented the displacement to the right of the sufentanil dose response-curve (ED₅₀ = 0.48 g/kg). When nimodipine was pumped (1 g/h, 7 days) concurrently with sufentanil, the development of tolerance to the opioid was not disturbed. However, the expression of tolerance was abolished and even the effect of acutely administered sufentanil was markedly potentiated (ED₅₀ = 0.03 g/kg). Similar experiments were performed with Bay K 8644. In naive rats, Bay K 8644 at a low dose (20 g/kg) that behaves as a calcium agonist, antagonized the analgesic effect of sufentanil (ED₅₀ = 0.58 g/kg), whereas at a high dose (200 g/kg) it potentiated this action (ED₅₀ = 0.15 g/kg). In tolerant rats, Bay K 8644 (20 g/kg) preserved its antagonizing ability inducing a displacement to the right of the sufentanildose-response curve (ED₅₀ = 4.2 g/kg). When Bay K 8644 was pumped (1 g/h, 7 days) concurrently with sufentanil, it enhanced the expression of tolerance to the opiate (ED₅₀ = 3.8 g/kg). These results suggest that the calcium fluxes through the l-type channel in neurones are functionally linked to the activation of the opiate receptor: the blockade of the channel increased the potency of sufentanil, whereas its activation reduced the potency of the opiate. In chronic experiments, DHPs concurrently administered with sufentanil did not affect the development of tolerance to the opiate. However, nimodipine prevented the expression of this phenomenon. Even more, the animals became hypersensitive to the opiate suggesting that the adaptative mechanisms induced by chronic opiate could be affected by chronic nimodipine.This work was supported by grants from Universidad de Cantabria-Caja Cantabria (1988) and Bayer AG, Wuppertal, FRGPredoctoral Fellow: Fondo de Investigaciones Sanitarias de la Seguridad Social.Send offprint requests to: M. A. Hurlé at the above address     

Toxicity of Organic Compounds to Marine Invertebrate Embryos and Larvae: A Comparison Between the Sea Urchin Embryogenesis Bioassay and Alternative Test Species

This study investigated the toxic effects of the insecticides lindane and chlorpyrifos, the herbicide diuron, the organometallic antifoulant tributyltin (TBT), and the surfactant sodium dodecyl sulfate (SDS) on the early life stages of Paracentrotus lividus (Echinodermata, Euechinoidea), Ciona intestinalis (Chordata, Ascidiacea), Maja squinado and Palaemon serratus (Arthropoda, Crustacea) in laboratory acute toxicity tests. The assays studied embryogenesis success from fertilized egg to normal larvae in P. lividus (48 h incubation at 20 °C) and C. intestinalis (24 h incubation at 20 °C), and larval mortality at 24 and 48 h in M. squinado and P. serratus. For P. lividus, the median effective concentrations (EC₅₀) reducing percentages of normal larvae by 50% were: 350 g l^–1 for chlorpyrifos, 5500 g l^–1 for diuron, 4277 g l^–1 for SDS, and 0.309 g l^–1 for TBT. For C. intestinalis, the EC₅₀ values affecting embryogenesis success were 5666 g l^–1 for chlorpyrifos, 24,397 g l^–1 for diuron, 4412 g l^–1 for lindane, 5145 g l^–1 for SDS, and 7.1 g l^–1 for TBT. The median lethal concentrations (LC₅₀) for M. squinado larval survival were 0.84 g l^–1 (24 h) and 0.79 g l^–1 (48 h) for chlorpyrifos, 2.23 g l^–1 (24 h) and 2.18 g l^–1 (48 h) for lindane, and 687 g l^–1 (48 h) for SDS. For P. serratus the LC₅₀ values obtained were 0.35 g l^–1 (24 h) and 0.22 g l^–1 (48 h) for chlorpyrifos, 3011 g l^–1 (24 h) and 3044 g l^–1 (48 h) for diuron, 5.20 g l^–1 (24 h) and 5.59 g l^–1 (48 h) for lindane, and 22.30 g l^–1 (24 h) and 17.52 g l^–1 (48 h) for TBT. Decapod larvae, as expected, were markedly more sensitive to the insecticides than sea urchins and ascidians, and SDS was the least toxic compound tested for these organisms. Lowest observed effect concentrations (LOEC) of TBT for sea urchin and ascidian embryos, chlorpyrifos and lindane for crustacean larvae, and SDS, were similar to those found in many coastal areas indicating that there would be a risk to invertebrate embryos and larvae from exposure in the field to these pollutants.     

Photoelectric measurement of neurogenic vasoconstriction in jejunal branches of the rabbit mesenteric artery reveals the presence of presynaptic opioid δ-eceptors

Summary The perivascular nerves of rabbit mesenteric arteries were stimulated with 15 pulses at 2 Hz, and decreases in external diameter were measured by means of a photoelectric device. Both extra- and intraluminally added [Met⁵]-enkephalin 1 mol/l depressed vasoconstriction, although with the second mode of application a larger inhibition occurred. Therefore, in the subsequent experiments all opioids were added into the lumen. [Met⁵]enkephalin 0.1 mol/l had no effect. [d-Pen², l-Pen⁵]enkephalin 3 mol/l was less potent than [Met⁵]enkephalin 1 mol/l. ICI 174864 1 mol/l was also without effect when given alone, but antagonized the action of [Met⁵]enkephalin 1 mol/l.Ethylketocyclazocine, dynorphin A(1–13), normorphine and DAGO, all 1 mol/l, were ineffective. [Met⁵]enkephalin 1 mol/l did not change the vasoconstriction evoked by the application of noradrenaline (0.1 –3 mol/l). It is concluded that in the mesenteric artery action potential-induced transmitter release, and in consequence vasoconstriction can be inhibited by the activation of presynaptic opioid -receptors. Send offprint requests to P. Illes at the above address     

Crisnatol mesylate: Phase I dose escalation by extending infusion duration

Summary Crisnatol mesylate is a rationally designed cytotoxic arylmethylamino-propanediol with broad spectrum cytotoxic activity. A phase I study with an unconventional escalation scheme was developed using a constant drug infusion rate (mg/m²/hr) and prolonging the infusion duration from 6 to 96 hours. Sixty-five patients received crisnatol at doses from 18 mg/m² in 6 hrs to 3400 mg/m² in 72 hours. The dose-limiting toxicity in two of five patients at 2700 mg/m² and two of three patients at 3400 mg/m² was neurologic and consisted of a syndrome of confusion, agitation, and disorientation. Phlebitis mandated the use of a central line. The mean terminal phase half-life (T_1/2 ) was 3.3 hours with a total body clearance (CL) of 22.8 L/hr/m² and a volume of distribution (Vd_ss) of 53 L/m². The median steady-state peak plasma concentration (C_ss) at 2700 mg/m²/72 hours was 2.7 g/ml and at 3400 mg/m²/72 hours was 3.8 g/ml. No responses were seen. The maximum tolerated dose (MTD) on this schedule is 2700 mg/m²/72 hours in patients with no liver disease and good performance status.     

Monoamines modulate the electrically-evoked efflux of 3H-choline from slices of guinea pig nucleus basalis magnocellularis

The influence exerted by monoamines on acetylcholine release was studied in electrically stimulated slices of guinea pig nucleus basalis magnocellularis (nbM) prelabelled with ³H-choline (3H-Ch).Noradrenaline, 30 M, and clonidine, 1 M, reduced the evoked ³H-Ch efflux by about 50%, but phenylephrine, 100 M. did not; idazoxan, 0.1 M. but not prazosin, 1 M, antagonized these effects. pointing to the involvement of alpha₂ receptors. Apomorphine, 1 or 30 M. reduced ³H-Ch efflux from nbM slices as well. The effect was shared by quinpirole, 1 or 10 M, but not by 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benz-azepine (SKF 38393). 10 M, and was antagonized by sulpiride, 1 M, but not by R-(+)-8-chloro-2,3,4,5-tetra-hydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SCH 23390). 1 M, suggesting the involvement of the D₂ receptor subtype.5-hydroxytryptamine (5-HT) 0.3–30 M, and alphamethyl-5-HT, 10 M, significantly increased ³H-Ch efflux from nbM slices; the 5-HT₂ antagonist ritanserin, 1 M. prevented this response. 2-methyl-5-HT, 1–30 M, inhibited the evoked ³H-Ch efflux and its effect was prevented by the 5-HT₃ antagonist 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222). 1 M.These findings indicate that i) catecholamines inhibit nbM neurons through alpha₂ and D₂ receptors and that ii) a complex serotonergic modulation of cholinergic function exists in the nbM, involving the activation of various receptor subtypes. which can mediate opposite responses. Correspondence to: A. Siniscalchi at the above address     

Evidence for an A2 adenosine receptor in guinea pig lung

Summary Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP levels in lung slices about 4-fold over basal values with an EC₅₀ of 0.32 mol/l. N⁶-R-(–)-Phenylisopropyladenosine (R-PIA) was 5-fold less potent than NECA. 5-N-Methylcarboxamidoadenosine (MECA) and 2-chloroadenosine had EC₅₀-values of 0.29 and 2.6 mol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig lung can therefore be classified as A₂ receptors. Several xanthine derivatives antagonized the NECA-induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; K _i 0.14 mol/l) was the most potent analogue, followed by 8-phenyltheophylline (K _i 0.55 mol/l), 3-isobutyl-1-methylxanthine (IBMX; K _i 2.9 mol/l) and theophylline (K _i 8.1 mol/l). In contrast, enprofylline (1 mmol/l) enhanced basal and NECA-stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [³H]NECA. The K _D for [³H]NECA was 0.25 mol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (K _i 0.14 mol/l) was the most potent inhibitor of [³H]NECA binding, followed by NECA (K _i 0.19 mol/l) and 2-chloroadenosine (K _i 1.4 mol/l). These results correlate well with the EC₅₀-values for cyclic AMP formation in lung slices. However, the K _i-values of R-PIA and theophylline were 240 and 270 mol/l, and DPX and 8-phenyltheophylline did not compete for [³H]NECA binding sites. Therefore, a complete characterization of A₂ adenosine receptors by [³H]NECA binding was not achieved. In conclusion, our results show the presence of adenylate cyclase-coupled A₂ adenosine receptors in lung tissue which are antagonized by several xanthines.     

Detection of benzodiazepine receptor occupancy in the human brain by positron emission tomography

Benzodiazepine receptor occupancy in the brain following oral administration of clonazepam (CZP) with a dose of 30 g/kg in six healthy young men and a further dose of 50 g/kg in one of the subjects was estimated by carbon-11 labeled Ro15-1788 and positron emission tomography (PET). The effects of CZP on the latency of auditory event-related potentials (P300) were also studied. Overall brain ¹¹C uptake was depressed and the % inhibition of ¹¹C uptake in the gray matter of the brain at 30 min after [¹¹C]Ro15-1788 injection was 15.3–23.5% (mean, n=6) following 30 g/kg CZP when compared with that in the control experiment without any previous treatment. The ¹¹C uptake in the cerebral cortex in the subject who received both doses decreased in a dose-related manner after 30 g/kg and 50 g/kg CZP. The P300 latency was prolonged significantly by 30 g/kg CZP [31.6±16.3 ms (mean±SD, n=6), P<0.05]. The P300 latency in the same subject was prolonged in a dose-related manner by 30 g/kg and 50 g/kg CZP. The technique using [¹¹C]Ro15-1788 and PET permits comparison of the pharmacological effects with the percentage of receptor sites which benzodiazepines occupy in the human brain. P300 also seems to be useful to investigate the pharmacological effects of benzodiazepines.     

5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well.Antagonists to block the contractile response to 5-HT via 5-HT₂, 5-HT₃ or 5-HT₄ receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) strongly inhibited the relaxations to ATP and VIP. l-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of l-NNA (100 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70–100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -Chymotrypsin (0.015 mg·ml^–1) and trypsin (0.005 mg·ml^–1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-d-Phe⁶, Leu¹⁷]VIP (1 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.     

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.     

Cardiovascular effects in rats of alpha1 and alpha2 adrenergic agents injected into the nucleus tractus solitarii

Summary The cardiovascular effects of selective alpha₁ and alpha₂ agonists and antagonists injected into the nucleus tractus solitarii (NTS) were studied in urethane-anesthetized rats. Methoxamine (0.3–3 g) injected bilaterally into the NTS caused a dose-dependent increase in blood pressure and heart rate. Phenylephrine (6 g) and an imidazolidine derivative St 587 (3 g) similarly injected also produced an increase in blood pressure, whereas a-methylnoradrenaline and an azepine derivative B-HT 920 (1 and 3 g) caused a decrease in blood pressure and heart rate. The pressor response to methoxamine (1 g) was markedly inhibited by prazosin (0.3 pg) injected into the same sites or hexamethionum (25 mg/kg, i. v.). Prazosin (0.3 g) alone injected bilaterally into the NTS did not affect the blood pressure, while yohimbine (0.1 g) similarly injected increased the pressure. These results suggest that in the rat NTS there exist alpha₁ adrenoceptors responsible for an increase in arterial pressure. The NTS alpha2 adrenoceptors seem to be involved in the tonic regulation of arterial pressure. Send offprint requests to T. Kubo at the above address     

Effects of intracerebroventricular administration of prostaglandin D2 on behaviour,blood pressure and body temperature as compared to prostaglandins E2 and F2α

The present work examined some central nervous actions of prostaglandin D₂ (PGD₂), which is the most prevalent prostaglandin in rodentorain. The effects of PGD₂ were compared with those of PGE₂ and PGF₂. The prostaglandins were administered intracerebroventricularly (ICV) to conscious rats using the method of Herman (1970). All three prostaglandins studied produced depressive behavioral effects, causing obvious sedation at doses of 2.0 g and 20.0 g ICV. PGD₂ and PGE₂ significantly reduced spontaneous motor activity at doses of 2.0 g and 20.0 g ICV. PGF₂ was less effective; only 20.0 g significantly inhibited motor activity. At a dose of 20.0 g ICV all three compounds were shown to block convulsions induced by pentylenetetrazol. PGD₂, the most effective prostaglandin in this respect, was still slightly anticonvulsive at a dose of 2.0 g ICV. PGF₂ hat the weakest anticonvulsive potency. PGE₂ and PGF₂ (2.0 g and 20.0 g ICV) caused a marked hypertensive effect, whereas PGD₂ at the same dose levels only produced a small increase in blood pressure. PGE₂ and PGF₂ (2.0 g and 20.0 g) also exerted marked pyrogenic actions. The effects of PGD₂ on body temperature were variable. When given at a dose of 20.0 g ICV, it caused slight hyperthermia whereas a lower dose (2.0 g ICV) induced a moderate fall in body temperature. These findings suggest a relationship between the actions of the different prostaglandins on blood pressure and body temperature.A preliminary report was given at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, March 1983 (Förstermann and Heldt, 1983)     

GABAergic modulation of D-1 dopamine receptor-mediated 3H-acetylcholine release from rabbit retina

Summary Dopamine evokes calcium-dependent release of ³H-acetylcholine from superfused rabbit retina labeled in vitro with ³H-choline, through activation of a D-1 dopamine receptor. This study investigates the activation of this receptor by endogenous dopamine and the modulation of the spontaneous and dopamine-evoked release of ³H-acetylcholine from rabbit retina labeled with ³H-choline by GABAergic agonists and antagonists. Endogenous dopamine, released from dopaminergic amacrine neurons by the indirect amines tyramine or D-amphetamine evoked the calcium-dependent release of ³H-acetylcholine from rabbit retina. The release of ³H-acetylcholine elicited by tyramine (10 M) or D-amphetamine (10 M) was attenuated by the selective D-1 antagonist SCH 23390 (0.1 M) and by the dopamine uptake inhibitor nomifensine (3 M). At concentrations of 1 mM and 1 M respectively, GABA and muscimol inhibited the spontaneous release of tritium from rabbit retina labeled in vitro with ³H-choline. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium. GABA and the GABA agonist muscimol (0.01–100 M) inhibited in a concentration-dependent manner the release of ³H-acetylcholine elicited by 100 M dopamine with IC₅₀ values of 4.5 M and 0.02 M respectively. The inhibition of dopamine-evoked ³H-acetylcholine release by GABA (10 M) and muscimol (0.1 M) was antagonized by the GABA antagonists bicuculline and picrotoxin. Picrotoxin and bicuculline (10 M) increased the spontaneous release of tritium, and potentiated the release of ³H-acetylcholine evoked by 100 M dopamine consistant with a tonic, inhibitory GABAergic input to the cholinergic amacrine neurons in rabbit retina. Dopamine-evoked acetylcholine release in rabbit retina may be of physiological importance as D-1 dopamine receptor-mediated increases in ³H-acetylcholine release from rabbit retina can be elicited by endogenous dopamine. In addition, activation of GABA receptor sites modulates the spontaneous and dopamine-evoked acetylcholine release from rabbit retina. Send offprint requests to M. L. Dubocovich at the above address     

The novel cardioprotective agent BMS-180448 activates a potassium conductance in cardiac and vascular smooth muscle

The goal of the present study was to further characterize the effects of the novel cardioprotective agent BMS-180448 on potassium fluxes in cardiac and vascular smooth muscle. Exposure of voltage-clamped guinea pig ventricular myocytes to BMS-180448 (300 M) produced an inhibition of IK followed by the delayed (5.5 ± 0.5 min) activation of a large time-independent potassium current. At 100 M, BMS-180448 produced only inhibition of I_K. The BMS-180448 activated current was refractory to block by 30 M, glyburide but was largely inhibited by 100 M alinidine (84 ± 6% inhibition at + 40 mV). Cromakalim (100 M)-activated currents were fully inhibited by 3 M, glyburide and 79 ± 4% blocked by 100 M alinidine. The current responses to BMS-180448 were unaffected by the inclusion of 10 mM UDP (100 M, ATP) in the pipette. BMS-180448 also produced a concentrationdependent increase in ⁸⁶Rb efflux from aortic strips; efflux responses were increased in low calcium medium and fully antagonized by 3 M, glyburide. Thus, BMS-180448 activates a potassium conductance in both cardiac and smooth muscle. The glyburide sensitivity of the BMS-180448-induced increase in ⁸⁶Rb efflux from the aortic preparations suggests that this drug activates I_KATP in vascular smooth muscle. Moreover, the observation that BMS-180448 (100 M) partially inhibits the effects of cromakalim in ventricular muscle cells suggests that these drugs interact, directly or indirectly, with a common site in cardiac muscle.     

The relaxant action of osthole isolated from Angelica pubescens in guinea-pig trachea

The effect of osthole, isolated from Angelica pubescens, on the contraction of guinea-pig trachea was studied. Osthole (25–100 mol/l), theophylline (10–1000 mol/l) and higher concentrations of nifedipine (0.1–100 mol/l) suppressed the contraction response curves of tracheal smooth muscle caused by carbachol, prostaglandin F₂ (PGF₂), U46619 (thromboxane A₂ analogue) and leukotriene C₄ (LTC₄) in a concentration-dependent manner. The contraction caused by high K⁺ (120 mmol/1) and cumulative concentrations of CaCl₂ (0.03–3 mmol/1) was also inhibited concentration-dependently by osthole (25–100 mol/l), theophyl line(10–1000 mol/l) and lower concentrations of nifedipine (0.01–0.1 mol/l). The relaxant actions of osthole were not affected by propranolol (1 mol/l), glibenclamide (10 mol/l) or removal of tracheal epithelium. Osthole (100 mol/l) was still effective in causing tracheal relaxation in the presence of nifedipine (1 mol/l). In Ca²⁺-free- and EGTA (0.2 mmol/1)-containing medium, the relaxing effect of osthole was more potent than in normal Krebs solution. Osthole (25 and 50 mol/l) caused 2.9 and 6.5, or 3.0 and 5.6 fold, respectively, increase in potency of forskolin or sodium nitroprusside in causing tracheal relaxation but did not affect that by cromakalim. Osthole (50 mol/l) enhanced the increase in tissue cAMP and cGMP levels induced by forskolin and sodium nitroprusside, respectively, and in higher concentrations (100 and 250 mol/l), itself increased markedly tissue cAMP and cGMP contents. Osthole (10–250 mol/l) inhibited the activity of cAMP and cGMP phosphodiesterases in a concentration-dependent manner. It is concluded that osthole exerts a nonspecific relaxant effect on the trachealis by inhibiting the cAMP and cGMP phosphodiesterases. Correspondence to: C. M. Teng at the above address