Potentiation of neostigmine and pyridostigmine by 4-aminopyridine in the rat

The interaction between 4-aminopyridine and neostigmine or pyridostigmine was studied in vivo in the rat sciatic nerve-anterior tibialis preparation using the constant infusion of pancuronium technique. The ED50 (dose of drug which produced a 50% antagonism) of neostigmine, pyridostigmine and 4-aminopyridine were 18, 49 and 440 μg kg^?1 respectively. The addition of 100 μg kg^?1 of 4-aminopyridine, which produced no antagonism by itself, decreased the neostigmine ED50 to 7·4 μg kg^?1. The addition of 200 μg kg^?1 of 4-aminopyridine, which produced a 30% antagonism by itself, decreased the ED50 of pyridostigmine to 11 μg kg^?1. We conclude that both neostigmine and pyridostigmine interact with 4-amino-pyrine synergistically.     

Neural Involvement in 5-Hydroxytryptamine-induced Net Electrogenic Ion Secretion in the Rat Intestine In-vivo

5-Hydroxytryptamine (5-HT) induces active electrogenic anion secretion by both the small intestine and the colon, responses that can be detected from measurements of transmural electrical activity. This approach was adopted to examine the involvement of neural mechanisms in 5-HT-induced secretion in rat proximal jejunum, distal ileum and proximal colon in-vivo. Under control conditions, 5-HT caused maximum rises in transintestinal potential difference of 4.7 ± 0.3, 3.8 ± 0.4 and 7.6 ± 0.3 mV, respectively, with corresponding ED50 values of 28 ± 3, 38 ± 4 and 41 ± 4 nmol kg^?1 (n = 12). In each region examined a neural component in the secretory response to 5-HT was identified. Hexamethonium (22 μmol kg^?1) reduced the 5-HT response in each region; in the jejunum and colon, it also attenuated the responses to the 5-HT₃ agonist, phenylbiguanide and to 5-methoxytryptamine (5-MeOT), an agonist at all 5-HT receptors except 5-HT₃, indicating that in these regions the nicotinic pathway can be activated by more than one 5-HT receptor subtype. Atropine (0.27 and 2.7 μmol kg^?1) was found to have regional effects on the intestinal responses to 5-HT receptor agonists. In the jejunum, evidence for a pro-secretory muscarinic pathway which could be activated by more than one 5-HT receptor subtype was found. In the ileum and colon no muscarinic pro-secretory pathway was identified, indeed in the colon, an anti-secretory pathway may be present. This muscarinic anti-secretory pathway was observed with phenylbiguanide and 5-MeOT, but not 5-HT. Substance P release does not appear to be involved in mediating the intestinal secretory response to 5-HT. 5-HT-induced intestinal anion secretion may involve a direct secretory action on the enterocyte which can be modified by neurally-mediated pro-secretory and anti-secretory pathways, the balance between these processes varying down the length of the gut.     

The character of the antagonism by polyphloretin phosphate of contractions to prostaglandins E1 and F2α in guinea-pig ileum

Polyphloretin phosphate (PPP) produced a dose-dependent decrease in the tone and reduction of the spontaneous phasic contractions of the longitudinal muscle of guinea-pig isolated ileum. PPP (100 μg ml^?1) after a 2 min contact with the ileum decreased the contractile effects of PGE₁ 0·1 μM by 40·6 ± 7·4%, of PGE₁ 0·01 μM by 86·7 ± 3·3% and of PGF_2α 0·1 μM by 62·2 ± 8·6%. After 10 min contact of PPP the contractile effect of PGE₁ 0·1 μM was decreased by 47·7 ± 4·7% and that of PGF_2α0·1 μM by 89·6 ± 1·7%. When the contact was longer, PPP showed a pronounced after-effect in respect to the effects of PGE₁ and particularly of PGF_2α. PPP significantly reduced contractions to 5-HT and BaCl₂, but not to acetylcholine, histamine or substance P. The type of antagonism of PGE₁ by PPP was examined using cumulative concentration-effect curves for PGE₁ in the presence of increasing concentrations of PPP. We conclude that on guinea-pig ileum PPP acts as a non-competitive antagonist of PGE₁ and PGF_2α.     

Effect of Cholecystokinin-B/Gastrin Receptor Blockade on Gastric Acid Secretion in Conscious Rats

Abstract: Gastrin, histamine and acetylcholine are physiological stimuli of gastric acid secretion. The cholecystokinin-B/gastrin receptor antagonists YM022 and RP73870 were used to study the effect of gastrin receptor blockade on acid secretion. Gastrin, histamine, insulin or bethanechol were administered to conscious gastric fistula rats with or without the concomitant intravenous infusion of YM022 or RP73870. Other rats were subjected to pylorus ligation. YM022 and RP73870 inhibited the gastrin-induced acid secretion in a dose- and time- dependent manner; maximal inhibition was observed at a dose of 0.3 μmol·kg^?1 · hr^?1 for both YM022 and RP73870, the ID₅₀ values being 0.02 μmol · kg^?1 · hr^?1 and 0.05 μmol · kg^?1 · hr^?1 for YM022 and RP73870, respectively. At a dose of 0.3 μmol · kg^?1 · hr^?1 YM022 and RP73870 failed to inhibit basal and histamine-, bethanechol-, and insulin-evoked secretion. They also failed to affect the secretion evoked by infusion of a cocktail of maximally effective doses of gastrin-17, histamine and bethanechol. YM022 and RP73870, finally, were without effect on the acid response to pylorus ligation. We suggest that endogenous gastrin in the conscious rat does not contribute to the basal acid secretion and does not participate in the acid response to histamine or to vagus stimulation.     

Tetrodotoxin inhibits directly acting stimulants of intestinal fluid secretion

Intravenous injection of tetrodotoxin (20 μg kg^?1) to anaesthetized rats blocks PGE₁- and inhibits VIP-induced fluid secretion from the mucosa of the jejunum without affecting the physiological rates of net water and glucose absorption. It is suggested that the toxin might release an endogenous inhibitor of secretion or that it might possess direct antisecretory activity on the epithelial cells of the jejunum. It is further suggested that results where tetrodotoxin has been used to investigate secretagogue action should be treated with caution.     

Biochemical pharmacology and toxicology of 8-azaadenosine alone and in combination with 2′-deoxycoformycin(pentostatin)

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2′-deoxycoformycin (dCF). The LD₁₀ (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. × 5) schedule was 30mg·kg^?1·day^?1. When the animals were pretreated with 0.1 mg·kg^?1·day^?1 of dCF, the ld₁₀ dose was reduced to 10 mg·kg^?1·day^?1× 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the ld₁₀ dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 μmoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 μmoles/g of cells when 8-azaAdo was used in combination with dCF (1 μg/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.     

Characterization of the Prejunctional Adenosine Receptors in the Rat Anococcygeus Muscle

Abstract— Adenosine receptor agonists inhibited electrically-evoked contractions of the rat isolated anococcygeus muscle. The compounds tested were: N⁶-cyclopentyladenosine (CPA), N((S, trans)-2-hydroxycyclopentyl)adenosine (GR79236), the R- and S-isomers of phenylisopropyladenosine (PIA), 5′-N-ethylcarboxamidoadenosine (NECA), ((2-(4-(2-carboxyethyl)phenyl)ethyl)amino)-N-ethylcarbox-amidoadenosine (CGS 21680) and N-((2-methylphenyl)methyl)adenosine (metrifudil). The rank order of agonist potency was: CPA = R-PIA = GR79236 = NECA » S-PIA > metrifudil > CGS 21680, which is consistent with an effect mediated by adenosine A₁ receptors. A similar rank order of potency was obtained for inhibition of electrically-evoked contractions of the guinea-pig ileum. However, there may be a lower receptor reserve in rat anococcygeus compared with the guinea-pig ileum, since higher concentrations of agonists were necessary to produce effects in the anococcygeus than in the guinea-pig ileum and S-PIA behaved as a partial agonist. The effect of NECA was antagonized in rat anococcygeus and guinea-pig ileum by the mixed A₁/A₂ receptor antagonist, 8-phenyltheophylline (pA₂ values of 6·8 and 6·9, respectively). The selective A₁-receptor antagonist, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX), also blocked the inhibitory response to NECA in both tissues. Here, however, the pA₂ values (9·6 and 8·6, respectively) were slightly but significantly different. These values confirm that the prejunctional adenosine receptors of the rat anococcygeus are of the A₁ type, and suggest that they are similar but not necessarily identical to those of the guinea-pig ileum. The differing potencies of DPCPX as an antagonist of NECA between the preparations may reflect a tissue-dependent variation in sensitivity to this antagonist.     

Opiate and opiate antidiarrhoeal drug action on rat isolated intestine

1 Stimulation of segments of rat jejunum (2.5, 5, 10, 20 and 40 Hz for 8 s) and ileum (10 Hz) resulted in a fast atropine-sensitive contraction during stimulation and a non-cholinergic after-contraction. Stimulation of segments with single pulses at 0.1 Hz had no effect. The colon (10 Hz) usually responded only with single large atropine-sensitive contractions. 2 The responses of the jejunum (2.5–40 Hz, 8 s) were unaffected by the μ-receptor ligands morphine (0.3 μM) and RX 783006 (0.3 μM) or the k-receptor ligand, ethylketocyclazocine (0.3 μM). The cholinergic contraction of the colon was unaffected by ethylketocycazocine (0.3 μM), but was reduced slightly by morphine at high concentrations only. 3 The prototype δ-opiate receptor ligand D-Ala²-D-Leu⁵-enkephalin (DADLE; 30 nM) reduced the contraction during stimulation (2.5, 5, 10 and 20 Hz for 8 s) of the rat jejunum. The IC₅₀ value for inhibition of the contraction elicited by stimulation at 10 Hz was 3.2 in the jejunum and was 12 nM in the ileum. Contractions of the colon were only slightly inhibited at a high concentration of DADLE (1 μM). 4 DADLE had no effect on contractions elicited by acetylcholine, at a concentration of up to 3 μM in segments of jejunum. Naloxone (1 μM) abolished the inhibitory effect of DADLE on the cholinergic contraction of the electrically stimulated jejunum. In addition, naloxone caused a large enhancement of control responses to stimulation. 5 Only high concentrations of the antidiarrhoeals loperamide and diphenoxylate inhibited the contraction elicited at 10 Hz in the jejunum (IC₅₀, 1.3 and 7.1 μM, respectively) and ileum (loperamide IC₅₀, 0.5 μM). The same concentrations of these drugs also inhibited the effect of exogenously added acetylcholine. A similar pattern of findings was made in the colon. 6 The results show marked differences in responses to transmural stimulation and to opiate drugs compared to those previously obtained from guinea-pig ileum. Furthermore, it appears from the results that the cholinergic nerves of the rat jejunum and ileum possess non-μ, non-k, enkephalin-sensitive opiate receptors, which upon stimulation reduce the amount of depolarization-induced acetylcholine release. Finally, the opiate antidiarrhoeals appear to act postsynaptically.     

Buspirone and 1-(2-pyrimidinyl)-piperazine attenuate xylazine-induced antinociception in the mouse

Abstract— The effects of subcutaneous pretreatment with buspirone and its major metabolite 1-(2-pyrimidinyl)-piperazine (1-PP) on the antinociceptive effect of xylazine were examined using the mouse acetic acid assay. Both buspirone and 1-PP dose-dependently attenuated the antinociceptive action of subcutaneously administered xylazine (0·8 mg kg^?1), with ED50 values of 7·3 mg kg^?l for buspirone and 3·4 mg kg^?1 for 1-PP. Pretreatment with either buspirone (8 mg kg^?1) or 1-PP (4 mg kg^?1) increased the antinociceptive ED50 of xylazine 3–4-fold. These data support the involvement of α₂-adrenoceptor and 1-PP in the pharmacological activity of buspirone.     

Rolipram inhibits airway microvascular leakage induced by platelet-activating factor,histamine and bradykinin in guinea-pigs

Abstract— Rolipram (0·1–1000 μg kg^?1, i.v.) reduced the increase in microvascular permeability induced by platelet-activating factor (PAF; 50 ng kg^?1, i.v.) at different sites of the guinea-pig airways. Rolipram (1–100μg kg^?1, i.v.) inhibited histamine (30μg kg^?1, i.v.)-and bradykinin (0·3 μg kg, i.v.)-induced airway microvascular leakage. These effects of rolipram were obtained at doses which inhibit histamine (7–20 μg kg^?1 min^?1)-induced bronchoconstriction (IC50 = 3 ± 1 μg kg, i.v.) without depressing arterial blood pressure in the guinea-pig. Aminophylline (50 mg kg^?1) did not change the effect of PAF. The anti-exudative effect of rolipram is of potential therapeutic value in asthma.     

Antagonism by SB 204070 of 5-HT-evoked Contractions in the Dog Stomach: an In-vivo Model of 5-HT4 Receptor Function

The ability of 5-hydroxytryptamine (5-HT) to evoke contractile activity in the gastric Heidenhain pouch was measured in conscious dogs using a method in which 5-HT₄ receptor-antagonist activity can be measured in-vivo. At doses of 5-HT which evoked short-lived measurable responses (5 or 10 μg kg^?1, i.v.), it was found that this activity was greatly reduced by atropine (100 μg kg^?1, i.v.), but was unaffected by methysergide, methiothepin, ketanserin (each at 100 μg kg^?1, i.v.) or granisetron (10 or 100 μg kg^?1, i.v.). At best SDZ 205–557 2-diethylaminoethyl-[2-methoxy-4-amino-5-chloro] benzoate; 100 μg kg^?1, i.v.) reduced the action of 5-HT in 4/5 animals and increased it in the other but its effects were variable in magnitude and not consistently maintained. However, the more potent and selective 5-HT₄-receptor antagonist SB 204070 (1-butyl-4-piperidinylmethyl 8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride) dose-dependently antagonized the 5-HT-evoked contractions in all dogs tested. This action was reversible, but long-lasting with an effective half-life of 18·0 h when administered at 1 μg kg^?1. The estimated ID50 value was 0·55 μg kg^?1.     

Ontogeny of mammalian myocardial beta-adrenergic receptors.

Dogs with gastric fistulae and denervated gastric pouches received graded doses of pentagastrin with and without a background infusion of somatostatin (1 μg kg^?1 h^?1). Similarly, graded doses of somatostatin (0.25, 0.5, 1, 2 and 4 μg kg^?1 h^?4) were infused after a steady state gastric secretion had been achieved with pentagastrin (1.5 μg kg^?1 h^?1), about twice the dose required to produce half maximal (D₅₀) response. Somatostatin inhibited pentagastrin-stimulated gastric acid secretion with competitive inhibition kinetics, but its precise site of action remains uncertain. The minimum effective dose of somatostatin on a twice D₅₀ dose of pentagastrin was 0.25 μg kg^?1 h^?1.     

Catecholamine uptake blockade in anaesthetized dogs: influence on cardiovascular responses

The effects of differential and combined catecholamine uptake antagonism on cardiovascular responses of anaesthetized dogs to isoprenaline, noradrenaline, and electrical stimulation of the left ansa subclavia nerve have been studied. Uptake 1 inhibition by cocaine HCl (5 mg kg^?1 and 1 mg kg^?1 every 45 min) enhanced responses to noradrenaline (0·1 to 2·0 μg kg^?1 i.v.) and sympathetic nerve stimulation (1 to 20 Hz), but did not affect those to isoprenaline. Uptake 2 inhibition by metanephrine (40 μg kg^?1 min^?1) enhanced cardiac responses to isoprenaline (0·05 to 1·0 μg kg^?1 i.v.), but did not significantly alter those to noradrenaline or nerve stimulation. Responses to all agonist interventions were increased by the combined administration of cocaine and metanephrine. Cocaine preferentially enhanced the positive chronotropic cardiac response to noradrenaline, but metanephrine did not differentiate between heart rate and contractility. These results have been discussed in the light of the mechanism of drug action involved.     

Amelioration of Cisplatin Nephrotoxicity with Glycine: Dose Dependency in Rats

The effects of glycine (0·1-1·0 g μg kg^?1, i.v.) on the acute changes in renal haemodynamics and nephrotoxicity produced by cisplatin (6·0 mg g kg^?1, i.v.) were investigated in the rat. Cisplatin produced decreases of 50% in the clearance of [³H] inulin (C_IN) and renal blood flow (RBF), 110 min following its injection. Glycine at a dose of 0·1 g kg^?1 produced no attenuation of the cisplatin-induced decrease in C_IN or RBF. Furthermore, this dose of glycine provided no significant protection of renal function over a 7-day period following cisplatin injection. By contrast, glycine at a dose of either 0·5 or 1·0 g kg^?1 markedly attenuated cisplatin-induced falls in C_IN and RBF, with the highest dose completely preventing any falls in these indices during the course of the experiment. Treatment with these higher doses of glycine produced prominent protection from the nephrotoxic actions of cisplatin, as evidenced by improvements in a range of indices of renal function which included plasma urea and creatinine concentrations, urine output, sodium excretion, C_IN and the clearance of [¹⁴C]P-aminohippurate. The results of experiments with an intermediate dose of 0·25 g kg^?1 glycine revealed some degree of amelioration of acute renal haemodynamic effects of cisplatin, particularly with regard to C_IN; whilst in the nephrotoxicity study, 0·25 g kg^?1 glycine produced a modest but significant reduction in cisplatin-induced acute renal dysfunction. The results have revealed a clear association between the acute renal haemodynamic effects produced by glycine in cisplatin-injected rats with the longer-term renal protective effects of glycine in cisplatin nephrotoxicity. The findings indicate that glycine's ability to prevent the falls in RBF and glomerular filtration rate produced by cisplatin plays an important role in the protective effect of glycine in cisplatin-induced nephrotoxicity.     

The Action of 1,2,3,5,6,11b-Hexahydro-[1]benzothieno [3,2-g]indolizine Hydrochloride (ADT 16) on Peripheral and Central Responses Mediated by 5-Hydroxytryptaminergic and Adrenergic Systems of the Rat

Abstract— Some acute pharmacological effects have been examined of racemic ADT 16 (1,2,3,5,6,11b-hexahydro[1]benzothieno[3,2-g]indolizine hydrochloride), on peripheral and central responses mediated by 5-HT and adrenergic systems in the rat. In-vitro, ADT 16(10–1000 Nm ), similarly to mianserin, antagonized the inhibitory responses to B-HT 920 of the electrically-stimulated rat isolated prostatic vas deferens. High concentrations of ADT 16 (10 μm ), also resembled those of mianserin by potentiating twitch responses to electrical stimulation of the tissue. Contractile responses to phenylephrine of rat isolated epididymal vas deferens were antagonized by ADT 16 (0·3–1 μm ). In the rat stomach fundus strip, ADT 16 (1–3 μm ) antagonized contractions due to 5-HT. ADT 16 (0·1–1 μm ) had no effect on responses to acetylcholine of the guinea-pig isolated ileum. In-vivo, in spinalized, decerebrated rats, fenfluramine- or clonidine-induced facilitation of flexor reflex activity of the anterior tibialis muscle was attenuated by ADT 16 (3 and 10 mg kg^?1, i.v., and 3 mg kg^?1, i.v. respectively). In the anaesthetized rat, l -3,4-dihydroxyphenylalanine (l -dopa)- or l -5-hydroxytryptophan (l -5-HTP)-induced increases in the frequency of spontaneous twitches of the anterior digastricus muscle were attenuated by ADT 16 (1 and 3 mg kg^?1, i.v.; n = 4). It is concluded that ADT 16, similarly to mianserin, is a novel peripherally and centrally active antagonist of 5-HT and adrenergic responses in the rat.     

The effect of number of stimuli and rate of stimulation on the inhibition by PGE1 of adrenergic transmission

An inverse correlation was found between the number of pulses and sympathetic blockade by PGE₁ (10^?8g/ml) in guinea-pig isolated field stimulated atria and vas deferens. The higher the number of pulses applied at a given frequency the smaller was the inhibition by PGE₁ of neuroeffector transmission. At 100 shocks using 10 Hz stimulation, PGE₁ no longer decreased the height of twitches of the guinea-pig vas deferens, however, the velocity of contractions was decreased. Sympathetic transmission in the rabbit jejunum was blocked by a somewhat higher dose of PGE₁ (3 × 10^?8g/ml). In this organ an increase in pulse number produced no detectable change in PGE₁-induced blockad. At a temperature of 37°C the vas deferens of the guinea-pig was highly sensitive to PGE₁, whereas that of the rat was virtually insensitive. On cooling the guinea-pig vas deferens to a temperature of 20°C the sensitivity to PGE₁ decreased and the shape of the shock number-effect curve became similar to that observed in that rat vas deferens. It is suggested, that the different shape of the curve may be caused by reduced release and/or effect of an inhibitory substance, probably prostaglandin. The biological responsesobtained with racemic PGE₁ were qualitatively identical with those elicited by natural PGE₁ the latter being about twice as potent.     

Acetylcholine release from the myenteric plexus of guinea-pig ileum by prostaglandin E1

Release of acetylcholine (ACh) by prostaglandin E₁ from the nerve terminals of the guinea-pig longitudinal muscle strip was studied in order to reveal the effect of PGE₁ on myenteric plexus activity. The ACh released was collected in the presence of physostigmine (2·1 μg ml^?1) and choline (0·1 μg ml^?1) at 38° C. Five to 100 ng ml^?1 PGE₁ enhanced the release dose-dependently. The effect was maintained during the presence of PGE₁ in the organ bath, while rapid tachyphylaxis was observed with the ACh-releasing action of nicotine. Tetrodotoxin or morphine almost completely inhibited the effect of PGE₁ on ACh release. Hexamethonium, in a concentration which completely blocked the effect of nicotine, partially inhibited the effect of PGE₁. In the late phase of nicotine action, the tissue was still sensitive to PGE₁ despite the continued exposure to nicotine. These data suggest the presence in the myenteric plexus of PG receptors which can increase ACh release.     

Protective Effects of Arginine-vasopressin on Aspirin-induced Gastric Mucosal Damage in Anaesthetized Dogs

Abstract— The protective effects of graded doses of arginine-vasopressin (AVP) on acidified acetylsalicylic acid (ASA) solution-induced changes in gastric prostaglandin E₂ (PGE₂) secretion, mucus production, acid back-diffusion and mucosal damage were studied in the bilateral truncal vagotomized stomach of anaesthetized dogs. After 1–4 h intragastric irrigation of the stomach with AVP (1–100 ng kg^?1) plus 20 min acidified ASA solution, a significant (P < 0·05) inhibition in gastric mucosal lesions and acid back-diffusion produced by acidified ASA solution was observed. The reduction in the gastric PGE₂ secretion and in mucus production provoked by the same dose of acidified ASA solution was also diminished. Furthermore, a correlation (r = 0·883; P < 0·01) between AVP-induced inhibition in ASA-provoked reduction in gastric PGE₂ secretion and in mucus production was found. During the experiment, the heart rate, the peripheral arterial blood pressure and the gastric arterial blood pressure were not altered by AVP (1–100 ng kg^?1). Thus, intragastric AVP protects gastric mucosa against ASA-induced damage without producing cardiovascular side effects. The inhibitory effects of AVP (100 ng kg^?1) on acidified ASA-induced reduction in PGE₂ and mucus secretion, as well as on ASA-induced enhancement in acid back-diffusion and erosion production were dose-dependently reversed by a specific V₁ antagonist, 1-(β-mercapto-β,β-cyclopentamethylene-propionic acid), 2-(o-methyl)tyrosine-Arg⁸-vasopressin. From the above results, it is suggested that the protective effects of intragastric AVP on gastric mucosa against acidified ASA-induced damage is at least partly due to stimulation of the biosynthesis of gastric PGE₂, which may contribute to the increase in the gastric mucus secretion and to the decrease in acid back-diffusion. Furthermore, the endogenous PGE₂ stimulated by AVP may be mediated by V₁-receptor activation.     

Prejunctional stimulation of cholinergic nerves in rat airway smooth muscle by an adenosine analogue

The effect of 5′-N-ethylcarboxamidoadenosine (NECA) on rat bronchial smooth muscle was examined in vitro. Both the nerve mediated muscle contraction induced by electrical stimulation and the potassium evoked release of [³H]ACh were enhanced by NECA. The apparent affinity (EC₅₀) of NECA in the contraction experiments was 0.30 ± 0.06 μM. The adenosine (ADO) receptor antagonist, 8-phenyltheophylline (8-PT), inhibited the NECA induced potentiation of both the electrical induced contraction and the potassium evoked release of [³H]ACh. The EC₅₀ and intrinsic activity of exogenous ACh were not altered in the presence of NECA (1 μM) in experiments where smooth muscle contraction were measured, indicating that NECA has a prejunctional effect and not a postjunctional effect on muscarinic receptors. The new A₂ specific ADO receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) and ADO also enhanced the nerve-mediated contraction (EC₅₀ = 35 ± 8 μM and 69 ± 20 μM, respectively). 8-PT (10 μM) and enprofylline (ENPF) (10 μM) inhibited the electrically induced contraction by 55 ± 16% and 45 ± 5% respectively. The potassium evoked release, however, was stimulated 56 ± 6% and 39 ± 7% by 50 μM 8-PT and ENPF respectively. The results provide evidence for a NECA specific ADO receptor in rat bronchi that is most likely prejunctional. Stimulation of this receptor, which may be of an A₂ receptor subtype, enhances the nerve mediated release of ACh and thereby induce contraction of the bronchial smooth muscle.     

Cirsimarin and Cirsimaritin,Flavonoids of Microtea debilis (Phytolaccaceae) with Adenosine Antagonistic Properties in Rats: Leads for New Therapeutics in Acute Renal Failure

In traditional medicine Microtea debilis is used against proteinuria. In ligand-binding studies extracts of Microtea debilis have been shown to inhibit the binding of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) to adenosine-A₁ receptors in rat forebrain membranes. Subsequently, cirsimarin, a flavonoid, was isolated as the active component and was shown to function as adenosine antagonist at the adenosine-A₁ receptor in-vitro. In this study we have investigated the adenosine-A₂ receptor activity of cirsimarin the in-vivo inhibition of the effects of adenosine by cirsimarin in rats, the absorption of cirsimarin and the inhibition of the binding of [³H]DPCPX to the adenosine-A₁ receptor by urine samples obtained after oral administration of crude extract of Microtea debilis, cirsimarin or cirsimaritin to rats. Cirsimarin inhibited the binding of [³H]5′-N-ethylcarboxamidoadenosine ([³H]NECA) to adenosine-A₂ receptors in rat striatum with an inhibition constant, K_i, of 6.5 ± 0.3 μm . The decrease of heart rate and blood pressure induced by adenosine was significantly inhibited by cirsimarin. After oral administration of 8 and 80 mg kg^?1 cirsimarin, the compound could not be detected in either plasma or urine, but the presence of cirsimaritin was established. By use of β-glucuronidase, glucuronides of cirsimaritin were also detected in the urine. The concentrations of cirsimaritin in the plasma were 0.126 ± 0.04, 0.138 ± 0.015, and 0.120 ± 0.022 μm , respectively, 2, 5 and 12 h after administration of 8 mg kg^?1 cirsimarin. The concentrations of cirsimaritin in the urine at the same times after administration of the same dose were 205 ± 1.86, 5.05 ± 2.6 and 2.06 ± 0.09 μm , respectively. The inhibition of the binding of [³H]DPCPX to the adenosine-A₁ receptor by urine samples collected 2, 5 and 12 h after oral administration of 8 mg kg^?1 cirsimarin or a crude extract of Microtea debilis containing approximately 8 mg kg^?1 cirsimarin and 2.8 mg kg^?1 cirsimaritin, or 6.8 mg kg^?1 cirsimaritin, was not significantly different from that of urine samples collected from untreated rats, in contrast with urine samples collected 1 and 2 days after oral administration of 80 mg kg^?1 cirsimarin. Approximately 3% of the cirsimarin was excreted in the urine as cirsimaritin. The results indicate that in the kidney and urinary tract the concentrations of cirsimaritin produced after ingestion of more than 8 mg kg^?1 cirsimarin can be high enough to inhibit the interaction of adenosine with its receptors; this might explain the effectiveness of Microtea debilis preparations against proteinuria in traditional medicine.