Contractile Action Research Articles

Actions of amphetamine derivatives and cathinone at the noradrenaline transporter

We have recently shown that methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), cathinone and methylenedioxyethylamphetamine (MDEA) have a cocaine-like action to potentiate the contractile actions of noradrenaline but not isoprenaline in the 1-Hz paced rat right ventricle. The purpose of this study was to directly test the actions of these compounds at the noradrenaline transporter. In rat left ventricular slices, potency (−log IC 50) values at inhibiting uptake of [ 3H]noradrenaline were: cocaine 6.16±0.15, cathinone 6.03±0.16, MDMA 6.05±0.07, MDA 5.68±0.06 and MDEA 5.56±0.08. MDEA and MDA were significantly less potent. In rat cerebral cortex membranes, MDMA was significantly less potent at displacing [ 3H]nisoxetine binding; −log EC 50 values: cocaine 5.04±0.08, cathinone 5.40±0.14, MDA 4.66±0.11, MDEA 4.99±0.15, MDMA 4.22±0.07. The noradrenaline uptake studies showed that MDEA was least potent: MDEA was also least potent functionally in the paced rat right ventricle. The [ 3H]nisoxetine displacement studies did not compare with the functional studies.

An affiliate journal of the International Society for Free Radical Research

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H2O2) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2•−) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or NG-nitro-l-arginine methyl ester (l-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2•−, an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac l-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH4 in endothelial cells and, as a consequence, disrupt the balance between NO and O2•− produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits.

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.

Actions of endothelin and corticotropin releasing factor in the guinea-pig ileum: no evidence for an interaction with capsaicin-sensitive neurons

Both endothelins and corticotropin releasing factor (CRF) appear in capsaicin-sensitive neurons. We have investigated the effects of human endothelin-1 (ET-1) and CRF in the guinea-pig ileum longitudinal and circular preparations and sought for ways of specific antagonism. With the aid of tachyphylaxis to capsaicin (i.e., rendering capsaicin-sensitive neurons functionally impaired) it was tested if these neurons played a mediating role in the effects of ET-1 or CRF. We also tried to find out whether endogenous endothelin or CRF plays a role in the excitatory and inhibitory effects of capsaicin in the ileum. In preparations at basal tone, both exogenous ET-1 (1–100 nM) and CRF (3–100 nM) caused contraction. These responses were not influenced by capsaicin tachyphylaxis. The contractile effect of ET-1 was not affected by tetrodotoxin (1 μM), atropine (1 μM), methysergide (100 nM), chloropyramine (100 nM) or SR140333 (100 nM) but was significantly inhibited or even abolished by the receptor antagonist BQ123 (3 μM) or BQ788 (3 μM). CRF caused contraction that was fully sensitive to tetrodotoxin (1 μM), tachyphylaxis to CRF or to atropine (1 μM) plus the tachykinin NK 1 receptor antagonist SR140333 (200 nM). Atropine alone had a weak inhibitory effect on the contractile action of CRF. Neither the antagonist BQ123 (3 μM) nor CRF tachyphylaxis inhibited the contractile action of capsaicin (2 μM), even in the presence of a mixture of GR82334 (3 μM) and SR142801 (100 nM), for blocking tachykinin NK 1 and NK 3 receptors, respectively – a treatment that by itself significantly reduced the effect of capsaicin. Exogenous ET-1 (0.3–5 nM), but not CRF (30–100 nM), caused relaxation of the atropine-treated, histamine-precontracted ileum. This effect of ET-1 was significantly inhibited or abolished by BQ123 (10 μM), or BQ788 (3 μM), but was not influenced by capsaicin tachyphylaxis. Likewise, relaxation of the atropine-treated, histamine-precontracted ileum in response to capsaicin was not influenced by the endothelin receptor antagonist BQ788 (3 μM) or BQ788 (3 μM) plus BQ123 (3 μM). Apamin (300 nM) was also without effect on the capsaicin-induced relaxation. In circular muscle strips ET-1 inhibited the indomethacin-induced spontaneous activity. This effect was abolished by BQ123 (3 μM) or BQ788 (3 μM). CRF caused a stimulation of the circular muscle. This stimulatory effect was not influenced by atropine (1 μM) alone, but was inhibited by atropine plus tachykinin NK 1 and NK 2 receptor antagonists (SR140333 (200 nM) and SR48968 (200 nM)) and also by tetrodotoxin (1 μM). It is concluded that capsaicin-sensitive neurons do not play a role in the effects of exogenous ET-1 or CRF in the guinea-pig ileum. ET-1 can both contract and relax the ileal longitudinal smooth muscle directly, probably via both ET A and ET B receptors. CRF acts by specifically stimulating excitatory (but not inhibitory) neurons of the myenteric plexus. Neither endogenous ET-1 nor CRF seems to play a role in the excitatory or inhibitory effects of capsaicin.

Deletion of the UT receptor gene results in the selective loss of urotensin-II contractile activity in aortae isolated from UT receptor knockout mice.

1 Urotensin-II (U-II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U-II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U-II-induced vasoconstriction. 2 This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU-II in wild-type (UT((+/+))) and UT receptor knockout (UT((-/-))) mice. 3 Basal left ventricular end-diastolic and end-systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rates, cardiac outputs and ejection fractions in UT((+/+)) mice and in UT((-/-)) mice were similar. 4 Relative to UT((+/+)) mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC(50)=8.26+/-0.08) that evoked relatively little vasoconstriction (17+/-2% 60 mM KCl), vessels isolated from UT((-/-)) mice did not respond to hU-II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU-II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin-1, KCl) and endothelium-dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. 5 The present study is the first to directly link hU-II-induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU-II contractile action with no 'nonspecific' alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro, the contribution that hU-II and its receptor make to basal systemic haemodynamics appears to be negligible in this species.

Functional Roles of the Rho/Rho Kinase Pathway and Protein Kinase C in the Regulation of Cerebrovascular Constriction Mediated by Hemoglobin

Although there is evidence that the Rho/Rho kinase pathway and protein kinase C (PKC) are involved in the development of cerebral vasospasm, the mechanism by which subarachnoid hemorrhage (SAH) activates these pathways is unclear. A large body of evidence points to oxyhemoglobin (OxyHb) as a major causative component of blood clot responsible for vasospasm. Therefore, the present studies were conducted to explore whether the Rho/Rho kinase and PKC may be involved in a sustained vasoconstriction induced by OxyHb in cerebral arteries. OxyHb evoked sustained vasoconstriction in the endothelium-denuded rabbit basilar arteries, which was reversed by the selective inhibitors of Rho kinase, Y-27632, and HA-1077, with the IC50 values of 0.26+/-0.02 and 0.74+/-0.1 micromol/L, respectively. In quiescent cerebrovascular smooth muscle (CVSM) cells, OxyHb induced Rho translocation, as assessed by immunoblotting, with a time course, which paralleled the contractile action of OxyHb. Rho translocation was also observed in intact arteries stimulated with OxyHb for 24 hours (219%) and 48 hours (160%). The increase in Rho translocation was fully inhibited by GGTI-297, an inhibitor of Rho prenylation. OxyHb also caused significant translocation of both PKCalpha and PKCepsilon (P<0.01), which was maximal at the time corresponding to maximal tension developed in response to OxyHb. Ro-32-0432, an inhibitor of PKC, attenuated vasoconstriction mediated by OxyHb in basilar artery. These results show, for the first time, that OxyHb-mediated signaling in CVSM utilizes the Rho/Rho kinase and PKC-based mechanisms.

먹장어 (Eptatretus burgeri)의 피부로부터 Bradykinin-Related Peptide의 정제

Hagfish bradykinin (BK)-related peptide가 먹장어 (E. burgeri)의 피부추출물로부터 분리, 정제되어졌다. 분자량이 875.8 Da인 hagfish BK는 <TEX>$C_{18}$</TEX> 역상 및 양이온교환 high performance liquid chromatography로 순수하게 정제되었다. 자동아미노산서열분석기와 MALDI-TOF mass spectroscopy에서 얻은 결과의 조합으로 hagfish BK의 일차구조는 Gly-Thr-Ala-Gly-Ile-Gly-Pro-Phe-Arg로 판명되었다. 이 아미노산서열을 mammalian BK와 비교하면 다섯 개의 아미노산의 치환을 <TEX>$(Arg^1{\rightarrow}Gly,\;Pro^2{\rightarrow}Thr,\;Pro^3{\rightarrow}Ala,\;Phe^5{\rightarrow}Ile,\;Ser^6{\rightarrow}Gly)$</TEX> 포함한다. Hagfish BK는 먹장어 장관에 대해 수축활성을 나타냈으며, 약 <TEX>$10^{-11}\;M$</TEX>의 농도에서 역치반응이 나타났다. A hagfish bradykinin(BK)-related peptide was isolated and characterized from the skin of hagfish, Eptatretus burgeri. The hagfish BK with a molecular mass of 875.8 Da was purified to a homogeneity using <TEX>$C_{18}$</TEX> reverse-phase and cation-exchange high performance liquid chromatography. The primary structure of the hagfish BK was determined as Gly-Thr-Ala-Gly-Ile-Gly-Pro-Phe-Arg by a combination of an automated amino acid sequencing and MALDI-TOF mass spectrometry. This amino acid sequence contains five substitutions <TEX>$(Arg^1{\rightarrow}Gly,\;Pro^2{\rightarrow}Thr,\;Pro^3{\rightarrow}Ala,\;Phe^5{\rightarrow}Ile,\;Ser^6{\rightarrow}Gly)$</TEX> compared with that of mammalian BK. The hagfish BK showed a contractile action on the intestine of hagfish, Eptatretus burgeri. The threshold concentration of hagfish BK was around <TEX>$10^{-11}\;M.$</TEX>

Contractile actions of imidazoline α-adrenoceptor agonists and effects of noncompetitive α 1-adrenoceptor antagonists in human vas deferens

The contractile actions of imidazoline α-adrenoceptor agonists were investigated in human vas deferens longitudinal and circular muscle. The effects of phenoxybenzamine were studied in comparison to dibenamine and SZL-49 (4-amino-6,7-dimethoxy-2-quinazolinyl-4-(2-bicyclo[2,2,2]octa-2,5-dienylcarbonyl-2-piperazine), an alkylating prazosin analogue that discriminates between α 1H- and α 1L-adrenoceptor subtypes. The imidazoline α-adrenoceptor agonist, A-61603 ( N-[5-(4,5-dihydro-1 H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide), was a potent agonist (p D 2; longitudinal muscle 6.9, circular muscle 6.4) and cirazoline a partial agonist (p D 2; longitudinal muscle 6.1, circular muscle 5.1). Oxymetazoline was less effective, indanidine and clonidine were ineffective. SZL-49 produced a differential inhibition of contractions evoked by A-61603 in circular (α 1H) compared to longitudinal (α 1L) muscle and phenoxybenzamine had the opposite effect. Dibenamine inhibited the contractions comparably in both muscle types and analyses of its partial alkylation of receptors yielded identical estimates of equilibrium dissociation constant (p K d) for A-61603 in longitudinal (5.82) and circular (5.84) muscle. Receptor occupancy–response relationships revealed that whilst the muscle types are not different in receptor reserves for A-61603, contraction to the potent imidazoline is more efficiently coupled in longitudinal than in circular muscle. This underlies the markedly different responsiveness of the muscle types to cirazoline or oxymetazoline (α-adrenoceptor agonists with lower efficacies relative to A-61603).The differential inhibitory actions of phenoxybenzamine and SZL-49 are discussed.

Increased relaxant action of forskolin and isoproterenol against muscarinic agonist-induced contractions in smooth muscle from M2 receptor knockout mice.

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M(2) muscarinic receptor knockout mice. Forskolin (5.0 micro M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M(2) muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 micro M). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M(2) receptor knockout mice were approximately 3- to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M(2) receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M(2) receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M(2) receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M(2) muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels.

Pharmacological characterization of SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide), a novel peptidic urotensin-II receptor antagonist.

1. Human urotensin-II (hU-II), a cyclic undecapeptide, is amongst the most potent mammalian vasoconstrictors identified, suggesting that hU-II and its G-protein-coupled receptor (UT) may regulate cardiovascular homeostasis. Such a hypothesis would benefit greatly from the development of selective UT antagonists. 2. Although the somatostatin (SST) antagonist SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide) is purported to block U-II-induced contractions in rat isolated aorta, little is known about its specific pharmacological properties. 3. SB-710411 (10 micro M) inhibited hU-II-induced contraction in rat isolated aorta causing a significant, parallel shift in the agonist concentration-response curve (pK(b) 6.28+/-0.11; n=8) with no suppression of the E(max). In contrast, SB-710411 did not alter the contractile actions of angiotensin-II, phenylephrine, or KCl. Paradoxically, however, SB-710411 potentiated the contractile response to endothelin-1 (pEC(50) 8.02+/-0.16 and 8.54+/-0.11, P<0.01; n=8). Rather than being specific toSB-710411, this phenomenon appears to be related to somatostatin receptor affinity and not intrinsic activity since the SST agonist somatostatin-14 and antagonist cyclo-somatostatin also potentiated endothelin-1-induced contraction. 4. SB-710411 (10 micro M) did not inhibit carbachol, sodium nitroprusside, IBMX, isoprenaline, and levcromakalim-induced reversal of tone established with noradrenaline. In contrast, however, SB-710411 significantly inhibited the reversal of tone established with endothelin-1 using the same vasorelaxants. 5. In summary, although SB-710411 inhibits the vasoconstrictor actions of hU-II in a competitive, surmountable manner, it also possesses additional pharmacological actions. Thus, whilst the present study is amongst the first to detail the properties of a functional U-II receptor antagonist, the data suggest caution be used when assessing data generated utilizing this moiety and other SST analogues.

Contractile response to a cannabimimetic eicosanoid, 2-arachidonoylglycerol, of longitudinal smooth muscle from the guinea-pig distal colon in vitro

The effect of 2-arachidonoylglycerol, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 μM) and N G-nitro- l-arginine (100 μM), 2-arachidonoylglycerol (10 nM–10 μM) produced concentration-dependent and tetrodotoxin (1 μM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.2 μM), and partially by hexamethonium (100 μM) pretreatment. The response to 2-arachidonoylglycerol was mimicked with N-arachidonoylethanolamine (anandamide, 0.1–30 μM), another cannabimimetic eicosanoid, but the cannabinoid CB 1/CB 2 receptor agonist, R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) (0.1–10 μM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10–30 μM), were without effect. The cannabinoid CB 1 receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 μM), the cannabinoid CB 2 receptor antagonist, [ N-[1 S]-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 μM), and the vanilloid receptor antagonist, capsazepine (10 μM), did not shift the concentration–response curve for 2-arachidonoylglycerol to the right. The contractile action of 2-arachidonoylglycerol was also partially attenuated in the presence of nordihydroguaiaretic acid (10 μM), a lipoxygenase inhibitor. These results indicate that 2-arachidonoylglycerol produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB 1/CB 2 receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of 2-arachidonoylglycerol.

Cisapride reverses the anticholinergic effect of disopyramide on the isolated guinea-pig urinary bladder.

The present investigation aims to examine whether the prokinetic agent cisapride is able to reverse disopyramide's anticholinergic effect on the isolated guinea-pig urinary bladder. Acetylcholine, at concentrations ranging from 10(-7) to 10(-3) M, produced a stimulatory effect on the urinary bladder (pEC(50) value=5.1). Disopyramide competitively antagonized the contractile effect of acetylcholine with an ID(50)=4.4 x 10(-6) M. Although cisapride by itself had either no intrinsic contractile action or a modest effect on the urinary bladder, at concentrations ranging from 3 x 10(-7) to 10(-6) M, it significantly reversed the above inhibitory effect of disopyramide, and produced a parallel leftward shift of the concentration-response curve for acetylcholine in the presence of disopyramide. The pEC(50) values for acetylcholine in the presence of 3 x 10(-6) M and 10(-5) M disopyramide were 4.7 and 4.2, respectively, while in the presence of 10(-5) M disopyramide, after pretreatment with 5 x 10(-7) M cisapride, the pEC(50) value for acetylcholine was 4.6. It is concluded that cisapride is effective in reversing the anticholinergic activity of disopyramide on the isolated guinea-pig urinary bladder, probably by facilitating cholinergic neurotransmission.

In vitro pharmacological characterization of the prostanoid receptor population in the non-pregnant porcine myometrium

In order to characterize prostanoid receptors present in the non-pregnant porcine uterus, the effects of naturally occurring prostaglandins (D 2, E 2, F 2α, I 2) and synthetic prostanoid receptor agonists on contractility of the longitudinal and circular muscles were examined in vitro. The potent contractile actions of prostaglandin F 2α and cloprostenol indicate the presence of excitatory FP receptors in the porcine uterus. The longitudinal muscle was more sensitive to FP receptor agonists than was the circular muscle. Prostaglandin D 2 produced an excitatory response in the longitudinal muscle but completely inhibited the spontaneous contraction of the circular muscle. BW-245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin, 1 nM–10 μM, a DP receptor agonist) inhibited the spontaneous contractions of both muscles, but the inhibition was conspicuously stronger in the circular muscle. Prostaglandin I 2 caused excitatory and inhibitory responses in the longitudinal and circular muscles, respectively, at relatively high concentrations (10–100 μM). Cicaprost, an IP receptor agonist caused inhibition of the contraction in the circular muscle but contracted the longitudinal muscle. Iloprost, an EP 1/IP receptor agonist, caused excitatory responses in both muscles at relative high concentrations. Prostaglandin E 2 caused excitatory responses at 1–100 nM and inhibitory responses at 100 nM–10 μM in both muscle layers. ONO-DI-004 ((17 S)-2,5-ethano-6-oxo-17,20-dimethyl prostaglandin E 1, an EP 1 receptor agonist) and ONO-AE-248 ((16 S)-9-deoxy-9β-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene-19,20-didehydro prostaglandin F 2, an EP 3 receptor agonist) contracted the longitudinal muscle but had little effect on the circular muscle. ONO-AE1-259 (11,15- O-dimethyl prostaglandin E 2, an EP 2 receptor agonist) inhibited the spontaneous contractions of both muscle layers to almost the same degree, but ONO-AE1-329 (16-(3-methoxymethyl)phenyl-ω-tetranor-3,7-dithia prostaglandin E 1, an EP 4 receptor agonist) did not inhibit the myometrial contraction. The present results indicate that contractile (FP, EP 1, EP 3) and relaxatory (DP, IP, EP 2) prostanoid receptors are present in the non-pregnant porcine uterus. There are marked muscle layer-related differences in the degree of responsiveness of prostanoid receptor agonists, and these differences suggest that there is a heterogeneous distribution of prostanoid receptors in the longitudinal and circular muscles (FP, EP 1 and EP 3, longitudinal muscle>circular muscle; DP, circular muscle>longitudinal muscle).

Galanin and its analogues: A structure-activity relationship studies in rat isolated gastric smooth muscles

Galanin (GAL), a 29-amino-acid-residue neuropeptide, modulatesgastric smooth muscles activity by interacting with specific receptors. However due to the lack of specific antagonists in thegastrointestinal (GI) tract the actual level of GAL involvement in GI motility remains largely unknown. In our studies we have performed structure-activity relationship studies of two porcinegalanin fragments, two chimeric galanin analogues and several 15-amino-acid-residue galanin analogues modified in positions 2, 3, 4, 6, 8 or 14, investigating their contractile action on rat isolated gastric fundus strips, employed as in vitro assay of peptides activity. Thus we intended to characterize the moleculardomains of GAL responsible for binding and activation of GAL receptors in rat gastric smooth muscle cells. The data acquired in the course of our structure-activity relationship studies suggest that both N- and C-terminal fragment of GAL molecule contribute towards the affinity and activity of GAL gastric smooth muscle cell receptors. Moreover, we concluded that positions 2, 3, 4, 6, 8 and 14 in the amino acid sequence of GAL may play important roles in binding and activation of GAL receptors in rat gastric smooth muscle cells.

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT 1 antagonist losartan. AT 2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1×10 −6 M) but were partially attenuated by the PKC inhibitor H-7 (1×10 −6 M), Ca 2+ channel blocker nicardipine (1×10 −5 M), Rho kinase inhibitor HA-1077 (1×10 −7 M) or p 44/42 MAP kinase inhibitor PD 98059 (5×10 −5 M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT 1 and AT 2 receptors. We conclude that Ang II-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT 1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca 2+, and PKC, Rho kinase and p 44/42 MAP kinase.

Interaction of xenin with the neurotensin receptor of guinea pig enteral smooth muscles

Xenin, a 25 aminoacid peptide, interacts with the neurotensin receptor subtype 1 of intestinal muscles of the guinea pig. Replacement of the C-terminal Lys -Arg peptide bond in xenin 6 by a reduced pseudo-peptide bond augmented binding affinity to isolated jejunal and colonic muscle membranes by factors of 7.7 and 21.0 respectively; the potency to contract the jejunum and to relax the colon was increased by factors of 3.2 and 1.3. The C-terminus Trp-Ile-Leu (WIL) of xenin, in contrast to the C-terminus Tyr-Ile-Leu (YIL) of neurotensin, bound competitively to the muscle membranes. WIL blocked the contractile action of xenin in the jejunum and was synergistic with the relaxing action in the colon. The Lys -Arg motif and Trp in the C-terminus of xenin are essential structures in the action of xenin on the enteral smooth muscle receptors.

Blocking actions of the sea anemone, Bunodosoma cavernata extract on Histamine and Acetylcholine induced contractile actions of the wild grasscutter and rabbit Ilea

Extract from the sea anemone, Bunodosoma cavernata was prepared by homogenizing 100 of the sea anemones in 300ml of 0.9% saline in a Moulinex blender. This was centrifuged at 4000 revolutions per minute for 10 minutes. The supernatant was re – centrifuged to obtain the crude extract. The extract (1ml/l) was found to elicit long lasting contractions on the ilea of wild rabbit and grass cutter. It was also found to block acetylcholine (ACh) – induced contractions of the ileum of grass cutter (90 + 6.2% inhibition + SEM) and the contractile action induced by ACh (0.2mg and 0.4mg) (89 + 5.8% inhibition + SEM) and histamine (0.075mg and 0.15mg) on the rabbit ileum (80 + 4.6% inhibition + SEM) . The antagonism by the extract was non – selective. (Journal of Applied Sciences & Environmental Management: 2002 6(2): 11-16)

Anti-ischemic effects of angiotensin-converting enzyme inhibitors: a future therapeutic perspective.

The renin-angiotensin system and angiotensin-converting enzyme (ACE) are increasingly being implicated in the pathogenesis of coronary artery disease and its sequelae and the potential of ACE inhibitors to protect the heart is a topic that has emerged recently as a matter for scientific discussion. Experimental and clinical studies have shown the beneficial effects of ACE inhibitors on the metabolism, function and structure of healthy and damaged hearts and these data support the concept of both primary and secondary cardioprotection with this class of drugs. Animal studies have demonstrated the potential beneficial effects of ACE inhibition at a variety of sites, including improvement of endothelial function, inhibition of platelet aggregation, prevention of atherosclerotic lesions and inhibition of myointimal proliferation, extending the concept to a more general definition of cardiovascular protection with ACE inhibitors involving both the heart and the vessels. ACE inhibitors prevent stimulation of smooth muscle cell angiotensin-II (A-II) receptors, thereby blocking both contractile and proliferative actions of A-II. In addition, ACE inhibition of kininase inhibits the breakdown of bradykinin, a direct stimulant of nitric oxide release from the intact endothelial cell. Thus, at the cellular level ACE inhibition shifts the balance of ongoing mechanisms in favour of those promoting vasodilatory, anti-aggregatory, antithrombotic and antiproliferative effects. Although these data have not all been validated in human studies, the reduction of ischemic events in studies of ACE inhibition in left ventricular dysfunction (LVD) and, more recently, also in patients without LVD, cannot be explained solely by improved hemodynamics, and it is possible that actions on the endothelium, the atherosclerotic process and platelets are at least in part responsible. So, the available data underlie the potential benefits of ACE inhibition in the field of ischemic heart disease and atherosclerosis; the results of ongoing studies in humans looking more directly at the influence of ACE inhibitors in this setting are awaited with interest.

A possible role of lipoxygenase in the activation of vanilloid receptors by anandamide in the guinea-pig bronchus.

1. In the absence of indomethacin, anandamide did not contract the guinea-pig bronchus at concentrations up to 100 microM. In the presence of indomethacin (10 microM), anandamide induced concentration-related contractions with a pEC(50) value of 5.18+/-0.11. It was significantly less potent than capsaicin (pEC(50) 7.01+/-0.1). The anandamide uptake inhibitor AM404, produced only a 14.1+/-3.22% contraction at 100 microM. All experiments were conducted in the presence of PMSF (20 microM). 2. The vanilloid receptor antagonist, capsazepine (10 microM), significantly attenuated the contractile effect of anandamide, the response to 100 microM anandamide being 40.53+/-7.04% in the presence of vehicle and 1.57+/-8.93% in the presence of 10 microM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan. 3. The log concentration-response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC(50) values for anandamide were 4.88+/-0.08 and 5.17+/-0.19 in the presence of vehicle and SR141716A (1 microM) respectively. 4. The lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 microM anandamide from 34.7+/-1.9% (vehicle) to 7.7+/-5% (ETYA, 10 microM) and from 41.85+/-4.25% (n=6) (vehicle) to 10.31+/-3.54 (n=6) (ETI, 20 microM). Neither inhibitor significantly affected contraction of the tissue by substance P. 5. This study provides evidence that anandamide acts on vanilloid receptors in the guinea-pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

Role of small-conductance Ca 2+-dependent K + channels in in vitro nitric oxide-mediated aortic hyporeactivity to α-adrenergic vasoconstriction in rats with cirrhosis

Background/Aims : In vitro studies have shown that cirrhotic aortas are hyporeactive to the contractile effect of vasoconstrictors because upregulated endothelial nitric oxide-synthase (NOS) overproduces nitric oxide (NO). Although stimulation of endothelial small-conductance Ca 2+ -dependent K + (SK Ca ) channels may elicit vasorelaxation in normal arteries, the role of these channels in cirrhosis-induced hyporeactivity is unknown. Thus, the aim of the present study was to investigate the role of endothelial SK Ca channels in cirrhosis-induced, NO-mediated, in vitro aortic hyporeactivity to α 1 -adrenergic vasoconstrictors. Methods : Isolated thoracic aortas from cirrhotic and normal rats were used. The effects of apamin, a selective SK Ca channel blocker, were measured on the vascular reactivity to phenylephrine. In addition, SK Ca channel protein expression was studied. The effects of iberiotoxin and charybdotoxin, blockers of other K Ca channels, were also studied in cirrhotic aortas. Results : Apamin suppressed cirrhosis-induced aortic hyporeactivity to phenylephrine in an endothelium-dependent, NOS-inhibitor-sensitive manner. SK Ca channel protein was overexpressed in cirrhotic aortic walls. Iberiotoxin abolished cirrhosis-induced aortic hyporeactivity to phenylephrine in an endothelium-dependent but NOS-inhibitor-resistant manner. Charybdotoxin did not induce any significant increase in phenylephrine-elicited contraction. Conclusions : In cirrhotic aortas, the overexpression and overactivity of endothelial SK Ca channels contributes to in vitro NO-mediated hyporeactivity to the contractile action of α 1 -adrenergic agonists.