Differential Relaxant Responses of Guinea-pig Lung Strips and Bronchial Rings to Sodium Nitroprusside: A Mechanism Independent of cGMP Formation

The biochemical mechanism subserving smooth muscle relaxant effects of sodium nitroprusside was examined on U46619, 9, 11-dideoxy-9α, 11α-methanoepoxy PGF_2α, precontracted guinea-pig lung strips and hilar bronchial rings. Lung strips were resistant to the relaxant action of sodium nitroprusside or sodium nitrite (NaNO₂), whereas they markedly relaxed to 8-bromo-cyclic GMP (8-Br-cGMP), a membrane permeable analogue of cGMP. Precontracted bronchial rings completely relaxed to sodium nitroprusside, NaNO₂, or 8-Br-cGMP in a concentration-dependent manner. Sodium nitroprusside (10 μm ) substantially raised tissue cGMP level in lung strips. Conversely, sodium nitroprusside had no detectable effect on cGMP levels in bronchial rings. In the presence of 10 μm dipyridamole, an agent which preferentially inhibits cGMP-specific phosphodiesterase, cGMP levels in lung strips treated with sodium nitroprusside was significantly enhanced, but sodium nitroprusside demonstrated no relaxant effect on the preparations. However, dipyridamole potentiated sodium nitroprusside-induced precontracted bronchial ring relaxation without affecting the bronchial tissue cGMP level. In the presence of 10 μm LY83583 (6-anilino-5,8-quinoline-dione), a specific cGMP concentration-lowering agent, sodium nitroprusside-mediated elevation of cGMP level in lung strips was significantly reduced with no effect on the functional response. LY83583 demonstrated no inhibitory effect on either relaxation or cGMP level in bronchial rings treated with sodium nitroprusside. Our results suggest that precontracted smooth muscle in lung strips and in hilar bronchi respond distinctly to sodium nitroprusside. Furthermore, sodium nitroprusside mediates bronchial smooth muscle relaxation by mechanisms unrelated to cGMP.     

Inhibition of ATP-Induced Contraction in the Guinea-pig Urinary Bladder in Vitro and in Vivo

Abstract Contractions were elicited by adenosine 5′-triphosphate (ATP) in the guinea-pig urinary bladder in vitro and in vivo. In isolated detrusor strips, tetrodotoxin (3.1 × 10^-6-4.4 × 10^-5M) did not affect contractions induced by a submaximum concentration (10^-3M) of ATP, nor did atropine (1.7 × 10^-6 - 2.1 × 10^-4M), or the anticholinergic agent PR 197 within the concentration range 2.6 × 10^-8 - 2.6 × 10^-5M. In higher concentrations (5.2 × 10^-5-2.6 × 10^-4M), PR 197 inhibited the ATP-response by 60-70% in a way that was not clearly concentration-related. Isoprenaline (10^-7-2.0 × 10^-5M) and noradrenaline (2.5 × 10^-6-10^-4M) reduced the ATP-induced contractions by up to 79%. The effects of the amines were abolished by propranolol (5.2 × 10^-6-3.8 × 10^-5M). Adenosine, 1.0-2.0 × 10^-2M, reduced the ATP-response by about 50%; in lower concentrations, it had no effect. Nifedipine, 7.8 × 10^-7-1.2 × 10^-5M, reduced the responses by 15-795%. Indomethacin (≤2.0 × 10^-4M), and theophylline (2.0 × 10^-4M) had no consistent effects on ATP-induced contractions. Exposure of the preparations to a calcium-free medium reduced and abolished the ATP-response within 60 min. Intravenous injection of ATP (1 -20 mg/kg) caused a rapid and transient increase in intravesical pressure in the anaesthetized guinea-pig. The effect of ATP (3mg/kg) was reduced by atropine(5-10 mg/kg) by approximately 35%. PR 197 (2.5-5 mg/kg) abolished the ATP-response. Isoprenaline (5-100 pg/kg) caused a 53-95% inhibition that could be blocked by propranolol (1 mg/kg). The inhibiting effect of noradrenaline (10-100,μg/kg) could not be blocked by propranolol (1 mg/kg). Adenosine (1.5-3.0 mg/kg) given immediately before ATP completely inhibited the ATP-response. Nifedipine, 0.1-0.2 mg/kg, reduced the ATP-induced contraction by 34 to 100%. It is concluded that the ATP-induced contraction is elicited by a direct effect on the smooth muscle cell. It can be inhibited non-specifically by drugs with different modes of action.     

Effect of Mn2+ on the Development of Tension Induced in Guinea-pig Taenia Coli by Bay K 8644

We have studied the effects of Mn²⁺ on the contractile response induced by Bay K 8644, a dihydropyridine Ca²⁺ agonist, on guinea-pig taenia coli. Mn²⁺ (5 mm ) completely inhibited Bay K 8644 (10^?6 m )-induced rhythmic contraction and contracture to baseline values in normal Ca²⁺ medium; thereafter, the contraction progressively increased to about 90% of the K+ (60 mm )-induced tonic response. In Ca²⁺-free medium in the presence of Bay K 8644 Mn²⁺ also evoked contraction and a concomitant increase in Mn²⁺ influx into the cytoplasm. These results suggest that during the opening of voltage-dependent Ca²⁺ channels activated by Bay K 8644, Mn²⁺ can enter cytoplasm through the channels and induce contraction in taenia coli.     

Pharmacology: Pharmacological Modulation of Endothelial Function by Insulin in the Rat Aorta

Nitric oxide (NO)-mediated vasodilation induced by hyperinsulinaemia might involve an indirect action which promotes agonist-stimulated endothelial function. Our aim was to attempt to demonstrate such modulation of endothelium-dependent vasodilation by insulin in the rat isolated aorta. We found that vasodilation in response to acetylcholine, but not to adenosine diphosphate (ADP), histamine or the calcium ionophore A23187, was modestly enhanced after 20-min pretreatment with human insulin (100 nM) whereas endothelium-independent responses to the NO donor sodium nitroprusside were not significantly affected. Human insulin thus has the acute pharmacological action of selectively enhancing muscarinic receptor-mediated endothelial function in rat aortic vascular smooth muscle in-vitro.     

Prostaglandin D2 Induced Potentiation of the Anticonvulsant Actions of Phenobarbitone and Phenytoin in Rats. Role of Serotonin

Prostaglandin D₂ (PGD₂) produced a dose-related potentiation of the anticonvulsant actions of sub-effective doses of phenobarbitone and phenytoin against maximal electroshock-induced seizures in rats. PDG₂-induced potentiation of phenobarbitone and phenytoin was significantly attenuated following pretreatment with centrally administered 5,6-dihydroxytryptamine, a selective neurotoxin for serotonergic neurones, p-chlorophenylalanine, a specific inhibitor of serotonin biosynthesis, and methysergide, a serotonin receptor antagonist, indicating that the potentiation was serotonin-mediated.     

Effect of an Histaminergic H3 Agonist on the Non-adrenergic Non-cholinergic Contraction in Guinea-pig Perfused Bronchioles

Abstract— From the bronchioles of guinea-pigs, preparations were isolated for registration of perfused pressure on electrical field stimulation (EFS) and by application of drugs. The perfused bronchioles contracted when EFS was applied in the presence of atropine and phentolamine suggesting a non-adrenergic non-cholinergic (NANC) response. (R)-α-Methylhistamine (methylhistamine), a selective H₃ agonist, reduced the NANC bronchoconstrictor response in a concentration-dependent manner. β-Adrenoceptors, muscarinic and histamine (H₁) and H₂ receptor) antagonists, epithelial removal and cyclooxygenase inhibition had no effect on this inhibitory action of methylhistamine whereas the H₃ antagonist, thioperamide, reduced the inhibitory effect of methylhistamine with a K_i value of 2·98 × 10^?9 m . Methylhistamine had no effect on the concentration-dependent contraction induced by exogenous substance P and neurokinin A, demonstrating that an H₃ receptor might inhibit the release of transmitter from NANC nerves on guinea-pig perfused bronchioles in-vitro.     

Biochemical Pharmacology: Polyamine-mediated Heart Hypertrophy Induced by Clenbuterol in the Mouse

The use of β-agonists as growth-promoting agents in cattle could lead to toxic side-effects in man. One such effect is the accumulation of polyamines which seem to be implicated in muscle and heart hypertrophy. We have studied the induction of cardiac hypertrophy after treatment with clenbuterol and the role of polyamines in this effect. Treatment of mice with repeated doses of clenbuterol, a specific β-adrenergic agonist, resulted in a marked increase in heart muscle weight whereas total body weight did not change significantly. Clenbuterol-linked cardiac hypertrophy could be prevented by coadministration of either the non-specific β-adrenergic antagonist, propranolol, or the irreversible inhibitor of ornithine decarboxylase, α-difluoromethylornithine. The clenbuterol-induced cardiac hypertrophy was associated with a corresponding increase in the level of the polyamines putrescine, spermidine and spermine. These observations are indicative of the role of polyamines in cardiac hypertrophy induced by clenbuterol.     

白英乙醇提取物诱导人肺癌SPC-A-1细胞凋亡的实验研究

目的:探讨白英乙醇提取物对人肺癌SPC-A-1细胞凋亡及凋亡相关基因fas和caspase-3表达的影响。方法:采用体外培养人肺癌SPC-A-1细胞,随机分为正常对照组、白英乙醇提取物处理组(浓度分别为2.5、5、10mg.L-1)和阳性对照组(顺铂);药物处理48h后,用MTT法检测细胞增殖抑制率,转移酶介导脱氧尿苷三磷酸缺口末端标记法检测细胞凋亡率,半定量反转录-聚合酶链反应检测fas和caspase-3mRNA表达水平。结果:与正常对照组比较,白英各浓度组细胞抑制率显著上升(P<0.001),细胞凋亡率明显升高(P<0.05或P<0.01),fas和caspase-3mRNA表达显著上升(P<0.05或P<0.01)。结论:白英乙醇提取物可能通过上调fas和caspase-3基因表达,诱导细胞凋亡,从而抑制SPC-A-1细胞增殖。     

Analysis of Neurogenic Contractions Induced by ML-1035 and Other Benzamides in the Guinea-pig Non-stimulated Isolated Ileum

Abstract— 4-Amino-5-chloro-substituted benzamides have been shown to increase gastric motility in-vivo and enhance field-stimulated and peristaltic contractions in-vitro. The present experiments examined the contractile response to a series of benzamides in the guinea-pig non-stimulated ileum. Four benzamides elicited contractions in the isolated ileum which were expressed as a percentage of the contraction induced by 1 μm acetylcholine (% acetylcholine response = 12 ± 2, 19 ± 3, 26 ± 2, 51 ± 3, n= 13, 8, 17, and 21, with EC50 values of 0·85, 1·8, 5·7, and 14·2 μm for cisapride, zacopride, metocloprarmde, and ML-1035 (4-amino-5-chloro-2-((2-methylsulphinyl)-ethoxy)-N-(2-(diethylamino)-ethyl)-benzamide hydrochloride), respectively). ML-1035 contractions were completely blocked by atropine and tetrodotoxin, while ganglionic blockade with hexamethonium was ineffective. Metoclopramide has been reported to sensitize postjunctional muscarinic receptors, however, ML-1035 did not enhance acetylcholine-induced contractions. Tropisetron (ICS 205–930, 1 μm ), caused a parallel rightward shift in the concentration-response curve for both ML-1035 and zacopride (EC50 = 14·2 ± 1·3 and 1·8 ± 0·8 μm in the absence, and 26 ± 2·7 and 6·9 ± 2·3 μm in the presence of tropisetron for ML-1035 and zacopride, respectively) with apparent pK_B values of 5·9 and 6·0 for the respective compounds. 5-Hydroxytryptaminergic receptor desensitization by 2-methyl-5-hydroxytryptamine (5-HT₃) and 5-methoxytryptamine (5-HT₄), attenuated the response to ML-1035. We also examined the effect of the benzamides on [³H]acetylcholine release from longitudinal muscle myenteric plexus preparations; however, these compounds had little effect on basal [³H]acetylcholine release. Thus, the pharmacological data indicate that the benzamides can elicit neurogenic contractions in the non-stimulated ileum by activating postganglionic, cholinergic neurons which is independent of an effect on smooth muscle.     

Prostaglandin D2-induced catalepsy in rats: role of 5-hydroxytryptamine

Prostaglandin D2 (PGD2), the major PG in the rat brain, induced a dose-related catalepsy in rats on intracerebroventricular (i.c.v.) administration. This cataleptic response was significantly attenuated following the i.c.v. administration of pharmacological agents that decrease rat brain 5-hydroxytryptamine (5-HT) activity. PGE1 synergized but PGF2 alpha antagonized the catalepsy induced by PGD2. PGD2 and PGE1 have previously been shown to augment rat brain 5-HT activity, whereas PGF alpha inhibited it. It is therefore likely that the observed effects of these PGs on catalepsy involve a central 5-HT mechanism.     

Enhancement of alpha2-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic U46619 in the porcine isolated ear artery: role of the ERK-MAP kinase signal transduction cascade

1. Alpha(2)-adrenoceptor-mediated contractions in porcine blood vessels can be enhanced in the presence of the thromboxane-mimetic U46619, and forskolin. The aim of this study was to determine the role of U46619 in the enhanced contractions, and to determine whether signalling through the ERK-MAP kinase pathway is involved. 2. Responses to the alpha(2)-adrenoceptor agonist UK14304 (1 micro M) were increased from 22+/-3% of the response to 60 mM KCl to 68+/-12% (n=8, mean+/-s.e.m.) in the presence of a low concentration of U46619 (< 20% of the 60 mM KCl response). 3. Both the direct and the U46619-enhanced UK14304 responses were inhibited by 50 microM PD98059, an inhibitor of the ERK-MAP kinase pathway. UK14304-induced contractions were associated with an increase in ERK2 phosphorylation, indicating an increased activity. In the presence of U46619, there was an enhanced phosphorylation of ERK2. U46619 on its own had no effect on ERK phosphorylation. 4. Both the direct and enhanced UK14304 contractions were inhibited in the absence of extracellular calcium. These conditions also prevented the increase in ERK2 phosphorylation. This indicates a role for calcium influx in the enhanced contractions. 5. In conclusion, this study demonstrates that precontraction with the thromboxane-mimetic U46619 enhances alpha(2)-adrenoceptor-mediated vasoconstriction through the enhancement of the ERK-MAP kinase pathway, and influx of extracellular calcium.     

Antagonism of cocaine self-administration by selective dopamine D(1) and D(2) antagonists

Effects of the dopamine D(1) antagonist SCH 39166 were compared with those of the D(2) antagonist eticlopride in squirrel monkeys responding under a second-order fixed-interval schedule of i.v. self-administration of cocaine. Dose-response curves were determined for a range of doses of self-administered cocaine (0.01-1.7 mg/kg/injection) alone and after pretreatment with SCH 39166 (0.01-0.1 mg/kg) or eticlopride (0.001-0.006 mg/kg). Cocaine maintained self-administration behavior in a dose-related manner; as the dose of cocaine was increased, rates of responding first increased and then either decreased or leveled off. Optimum doses (0.03-0.3 mg/kg) maintained high rates of responding (0.7-1.7 responses per second) among the different monkeys, and patterns of responding that were characteristic for second-order schedules. Pretreatment with either SCH 39166 or eticlopride altered self-administration behavior in all monkeys. In most cases, dose-response curves for cocaine were shifted to the right, indicative of surmountable antagonism, and a 3 to 6-fold increase in dose of cocaine was necessary to restore optimal performances. In some instances, dose-response curves were shifted either downward or downward and to the right, indicating that the antagonistic effects of SCH 39166 and eticlopride were not always fully surmountable. These results show that self-administration of cocaine can be comparably modified by drugs that selectively block dopamine D(1) or D(2) receptors.     

Role of cytosolic phospholipase A2 in the enhancement of alpha2-adrenoceptor-mediated vasoconstriction by the thromboxane-mimetic U46619 in the porcine isolated ear artery: comparison with vasopressin-enhanced responses

Pre-contraction with the thromboxane-mimetic U46619 enhances the subsequent alpha(2)-adrenoceptor-mediated vasoconstriction in the porcine ear artery through an enhanced activation of ERK-MAP kinase. In this study we determined the role of cPLA(2) in this enhanced response, and determined whether vasopressin is also able to enhance alpha(2)-adrenoceptor-mediated vasoconstriction through the same pathway. The cPLA(2) inhibitors AACOCF3 (50 microM) and MAFP (50 microM) both inhibited the U46619-enhanced alpha(2)-adrenoceptor response, but had no effect on the direct alpha(2)-adrenoceptor response. AACOCF3 also inhibited the enhanced ERK activation associated with the enhanced alpha(2)-adrenoceptor-mediated vasoconstriction. Pre-contraction with arachidonic acid mimicked the effect of U46619 by enhancing the contractile response to the alpha(2)-adrenoceptor agonist UK14304 (1 microM) and enhancing the alpha(2)-adrenoceptor-mediated ERK activation. Pre-contraction with vasopressin also enhanced the contractile response to UK14304, but neither PD98059 (50 microM) nor AACOCF3 (50 microM) had any effect this vasopressin-enhanced response, indicating that neither the ERK pathway, nor cPLA(2) are involved in vasopressin-enhanced responses. The alpha(2)-adrenceptor-stimulated activation of ERK was also unaffected by pre-contraction with vasopressin. On the other hand, inhibition of PKCzeta inhibited the enhanced alpha(2)-adrenoceptor contraction after pre-contraction with both U46619 and vasopressin. This study demonstrates that alpha(2)-adrenoceptor-mediated vasoconstriction can be enhanced through two different pathways-one dependent upon the enhanced activation of ERK-MAP kinase through activation of cPLA(2), and the other through a different, ERK/cPLA(2)-independent pathway.     

Effect of TMB-8 on the pulmonary vasoconstrictor action of prostaglandin F2 alpha and the thromboxane mimic,U 46619.

1 TMB-8 (8-(N,N-diethylamino)octyl-3,4,5 trimethoxybenzoate HCl), an intracellular calcium antagonist, had no direct action on the pulmonary vasculature of the perfused canine lung lobe preparation. 2 The pulmonary pressor response to the thromboxane mimic, U46619, was not affected by TMB-8. 3 The vasopressor response to prostaglandin F2 alpha (PGF 2 alpha) was significantly attenuated but not completely blocked by TMB-8. 4 We conclude that the pulmonary pressor response to PGF 2 alpha is dependent on both intracellular and extracellular calcium pools for contraction and that U46619 facilitates either solely extracellular calcium influx or mobilizes an intracellular calcium pool not inhibited by TMB-8.     

Effects of Noradrenaline and Prostaglandin F2α on Angiotensin-Induced Contraction and Tachyphylaxis in Rat Aortic Rings

Abstract: The aim of this study was to examine the effects of noradrenaline (NA) and prostaglandin F_2α (PGF_2α) on angiotensin II (AII)-induced contraction and tachyphylaxis in aortic rings of the rat. Neither NA (10^–9 M) nor PGF_2α (10^–7 M) had significant effect on the response of the rings to the spasmogenic concentrations (10^–10 to 10^–7 M) of AII, but lowered significantly the threshold response of the aortic rings to AII (from 10^–9 to 10^–12 M). In rings that were tachyphylatic to AII, both NA and PGF_2α attenuated significantly the tachyphylaxis of the rings to AII at the concentrations of 10^–10 and 10^–7 of the octapeptide; and also lowered the threshold of the tachyphylatic rings to AII (from 10^–9 to 10^–11 M for NA, and from 10^–9 to 10^–10 M for PGF_2α). The specific properties of noradrenaline and PGF_2α were not shared by the non-specific potassium chloride. Because the lowering of threshold and attenuation of tachyphylaxis occurred at the physiological levels of AII and NA, it is possible that the in vivo actions of AII are under constant modulation by circulating and localised (higher than circulating) levels (e.g. of PGF_2α) of spasmogens. The results also call into question the physiological significance of angiotensin tachyphylaxis and may suggest that it is only an in vitro phenomenon occurring in the absence of endogeneous spasmogens.