Characterization of the P1-purinoceptors mediating contraction of the rat colon muscularis mucosae.

1. Previous studies had shown that adenosine and adenine nucleotides including adenosine 5'-triphosphate (ATP) caused contraction of the rat colon muscularis mucosae via P1 and P2Y-purinoceptors respectively, and that the stable ATP analogue adenylyl 5'-(beta,gama- methylene)diphosphonate (AMPPCP) had an unexpected direct action on the P1-purinoceptors. The P1-purinoceptors have now therefore been further characterized by use of the adenosine analogues 5'-N-ethylcarboxamidoadenosine (NECA) and N6-cyclopropyladenosine (CPA) and the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), which is selective for the A1 subtype. The P2-purinoceptor antagonist suramin was also used, to investigate the selectivity of the P2 agonists. 2. The order of potency of P1 agonists for contraction was CPA greater than NECA greater than AMPPCP greater than or equal to adenosine, and DPCPX (1 nM) caused greater than two fold shifts to the right of the log concentration-response curves for each of these agonists, although the shifts were not always parallel and Schild analysis of the inhibition of the effect of adenosine resulted in a plot with a slope greater than unity. These results indicate that the P1-purinoceptor mediating contraction is of the A1 subtype, as has been found in other tissues in which adenosine causes contraction. 3. The P2-purinoceptor antagonist suramin (300 microM) had no effect on the responses to adenosine or to AMPPCP, but abolished contractions induced by the related stable ATP analogue adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of purinoceptors mediating depolarization of rat isolated vagus nerve.

1. As part of a broader study to characterize neuronal purinoceptors, the effects of adenosine 5'-triphosphate (ATP) and a range of ATP analogues were investigated on the extracellularly recorded membrane potential of the rat isolated vagus nerve, using a 'grease-gap' technique. 2. ATP evoked depolarization of the rat vagus nerve. The concentration-effect curve to ATP was not monophasic: at the lower concentrations (1 x 10(-5)-1 x 10(-3) M) the curve was shallow (< 50% of the near maximal response to 5-hydroxytryptamine (5-HT)) whilst at higher concentrations the relationship between concentration and amplitude of depolarization was steeper (> 135% of the response to 5-HT at the highest concentration tested, 1 x 10(-2) M). On washout of the high drug concentrations large after-hyperpolarizations were often observed. 3. alpha,beta-methylene ATP (1 x 10(-6)-3 x 10(-4) M), beta,gamma-methylene ATP (1 x 10(-6)-1 x 10(-3) M), and 5'-adenylylimidodiphosphate (beta,gamma-imido ATP; 1 x 10(-6)-1 x 10(-3) M) were all more potent than ATP and produced large depolarizations of the rat vagus nerve at the highest concentrations tested (> 150% of the response to 5-HT). The overall rank order of potency was alpha,beta-methylene ATP > beta,gamma-methylene ATP = beta,gamma-imido ATP > ATP. 4. In contrast, 2-methylthio ATP (1 x 10(-6)-1 x 10(-3) M) produced relatively small depolarizations (< 100% of the response to 5-HT). As was the case with low concentrations of ATP, the concentration-effect curve to 2-methylthio ATP was very shallow.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study on P2X purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens.

1. We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5'-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2. Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential-70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5'-triphosphate (2-MeSATP) and alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with alpha,beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha,beta-meATP >> ATP = 2-MeSATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae.

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.     

Characterization of postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle

The present study was designed to characterize the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle by means of a series of muscarinic agonists and subtype-preferring key muscarinic antagonists. Cumulative addition of muscarinic agonists elicited concentration-dependent contractions with the following rank order of potency (pD₂ values): (+)-muscarine (6.36) ≥ oxotremorine M (6.21) ≥ arecaidine propargyl ester (APE) (6.18) > carbachol (5.68)=(±)-methacholine (5.65) > 4-(4-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (4-Cl-McN-A-343) (4.28) > 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) (3.89). (+)-Muscarine, oxotremorine M, carbachol and (±)-methacholine behaved as full agonists, whereas APE, 4-Cl-McN-A-343 and McN-A-343 displayed partial agonism. The contractile responses of the rat anococcygeus muscle to (±)-methacholine were competitively antagonized by pirenzepine (pA₂=6.92), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl] 5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AQ-RA 741; pA₂=6.75), himbacine (pA₂=7.11), (±)-p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; pA₂=7.68) and the (R)- and (S)-enantiomers of hexahydro-difenidol [(R)-HHD: pA₂=8.52; (S)-HHD: pA₂=6.06]. A comparison of the pA₂ values derived from studies of contraction in rat anococcygeus muscle with literature binding (pK_i values) and functional affinities (pA₂ values) obtained at native M₁-M₄ receptors strongly suggests that the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle are of the M₃ subtype. Received: 18 April / Accepted: 18 July 1997     

Selectivity of bradykinin analogues for receptors mediating contraction and relaxation of the rat duodenum.

1. Bradykinin produces a biphasic response in the rat duodenum that consists of a relaxation (pD2 = 8.44) followed by a contraction (pD2 = 6.91). 2. The B1 agonist des-Arg9-bradykinin produced a contraction (pD2 = 7.16) but no relaxation. Des-Arg9-[Leu8]-bradykinin, which is a B1 antagonist in other systems produced contraction (pD2 = 7.65) in the rat duodenum. 3. Four bradykinin analogues that are preferential B2 agonists in other tissues had a biphasic effect with pD2 values in the range 7.22-8.68 for relaxation and 6.26-6.91 for contraction. 4. [Thi5,8,D-Phe7]-bradykinin, which is a B2 antagonist in most other systems produced relaxation in the rat duodenum, with a pD2 of 7.49. 5. It is concluded that the contractile component of the response to bradykinin in rat duodenum may be mediated by a subtype of the B1 receptor and the relaxant component by a receptor of the B2 subtype.     

Investigation of the 5-hydroxytryptamine receptor mechanism mediating the short-circuit current response in rat colon.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3-morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone.     

Evidence for a functional alpha 1A- (alpha 1C-) adrenoceptor mediating contraction of the rat epididymal vas deferens and an alpha 1B-adrenoceptor mediating contraction of the rat spleen.

1. The alpha 1-adrenoceptor subtype mediating contraction of the rat epididymal vas deferens and rat spleen has been investigated by use of alpha 1-adrenoceptor antagonists that have shown selectivity between the different cloned receptor subtypes. 2. In the rat epididymal vas deferens the potency of noradrenaline and phenylephrine was increased in the presence of neuronal and extra-neuronal uptake blockers, cocaine and beta-oestradiol, but these did not alter that of methoxamine. The order of potency of the agonists in the presence or absence of uptake blockade was noradrenaline > phenylephrine > methoxamine. In the rat spleen the potency of these agonists was not altered in the presence of cocaine and beta-oestradiol, and their order of potency was the same as in the vas deferens. 3. The non subtype selective alpha 1-adrenoceptor antagonist prazosin (up to 1 x 10(-7) M) was found to antagonize contractions to noradrenaline in the vas deferens competitively (pA2 9.2), but only in a non competitive manner in the spleen. Contractions to phenylephrine in the spleen however were competitively antagonized by prazosin (up to 1 x 10(-7) M) with a pA2 of 9.2. This suggests that there is an alpha 1- and a non alpha 1-adrenoceptor response to noradrenaline in the rat spleen. 4. Pretreatment with chlorethylclonidine (10(-4) M for 30 min) did not alter the noradrenaline contractions in the vas deferens, but contractions to noradrenaline and phenylephrine in the spleen were shifted 30 and 300 fold to the right of the control curve, respectively. This suggests that only the contractions in the spleen were mediated by alpha 1B-adrenoceptors. 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity studies of analogues of blomhotin mediating contraction of rat fundus

Blomhotin is a novel peptide (pGlu1-Gly2-Arg3-Pro4-Pro5-Gly6-Pro7-Pro8-Ile9-Pro10-Arg11) which has been isolated from the venom of Agkistrodon halys blomhoffii and exhibits contractile activity on rat stomach fundus. We carried out a structure-activity study of blomhotin and its related peptides, and the findings suggested that the N-terminal portion of blomhotin is mainly responsible for affinity for the blomhotin receptor, whereas the C-terminal portion of blomhotin, Pro-Ile-Pro-Arg, is responsible for complete activation of the blomhotin receptor in the rat stomach fundus. In particular, the amino acids at positions 9 and 11 of blomhotin appear to be essential for binding and intrinsic activity. Using knowledge gained from this structure-activity analysis, we have identified photoactive blomhotin analogues that have sufficient biological activity to probe the target molecule of blomhotin.     

Endothelin receptors mediating contraction in goat cerebral arteries.

1. The aim of the present study was to identify the subtype of receptor mediating contraction to endothelin-1 and sarafotoxin S6b in goat isolated middle cerebral arteries. 2. Endothelin-1, endothelin-2 and endothelin-3 contracted cerebral arteries in a concentration-dependent manner. Although the three peptides were full agonists, the order of potency was endothelin-1 = endothelin-2 > endothelin-3, with a relative potency of endothelin-1 and endothelin-2 versus endothelin-3 of approximately 280. Sarafotoxin S6b induced concentration-dependent contractions with lower potency than endothelin-1/endothelin-2, higher potency than endothelin-3 and a higher maximum response than the three endothelins. 3. The selective ETA-receptor antagonist, BQ-123, did not induce changes in either the resting tension or in the active tone developed by depolarization. In contrast, BQ-123 produced concentration-dependent relaxations of endothelin-1-precontracted cerebral arteries, and to a greater extent of sarafotoxin S6b-precontracted arteries. 4. Concentration-response curves to endothelin-1 and sarafotoxin S6b were competitively antagonized by BQ-123 (pA2 of 7.43 +/- 0.12 and 8.41 +/- 0.09, respectively). In contrast, BQ-123 had no effect on 5-hydroxytryptamine-elicited contractions even at 10(-6) M. 5. It is concluded that both the order of potency of endothelin isopeptides and the antagonism of BQ-123 point to the existence of ETA receptors mediating vasoconstriction to endothelin-1 and sarafotoxin S6b in the goat middle cerebral artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysophosphatidic acids induce contraction of rat isolated colon by two different mechanisms.

Lysophosphatidic acids (1-linoleoyl-, 1-linolenoyl, 1-arachidonoyl- and 1-O-hexadecyl-sn-glycero-3-phosphate) induced rapid contraction of rat isolated colon which was dependent on external Ca2+, 1-linolenoyl-lysophosphatidic acid having the greatest effect. The contraction induced by 1-linolenoyl-lysophosphatidic acid was reduced considerably by nifedipine and verapamil, but not by atropine or indomethacin. Phosphatidic acids with two short-chain acyl groups induced a small, atropine-sensitive contraction at 100 microM, but phosphatidic acids with two long-chain acyl groups were inactive. These results suggest that unlike phosphatidic acids, lysophosphatidic acids act directly on extracellular sites of the plasma membrane of smooth muscle cells in rat colon, mainly through activation of voltage-sensitive Ca2+ channels.     

Evidence that A2 purinoceptors are involved in endothelium-dependent relaxation of the rat thoracic aorta.

1. The effect of adenosine and some adenosine analogues on the isolated thoracic aorta from rats was compared with the effect of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP). 2. Both ATP and adenosine analogues caused relaxation of the noradrenaline (30 nM)-contracted thoracic aorta. 3. The order of potency for adenosine analogues was 5'-(N-ethyl) carboxamidoadenosine (NECA) greater than L-N6-phenylisopropyladenosine (L-PIA), adenosine 5'-monophosphate (AMP), adenosine indicating the presence of adenosine A2 receptors. 4. Removal of the endothelium or prior treatment with haemoglobin (10 microM) attenuated relaxant responses to both ATP and NECA, attenuation being greater for ATP than NECA. 5. 8-Phenyltheophylline (10 microM) reduced relaxant responses to NECA but not to ATP in the intact tissue. 6. These results provide evidence that there are two components to relaxation of the rat thoracic aorta induced by purinoceptor agonists. The first is an endothelium-dependent mechanism involving release of endothelium-derived relaxant factor (EDRF) and the second is due to a direct effect on smooth muscle.     

The ontogeny of purinoceptors in rat urinary bladder and duodenum.

1. The ontogeny of responses to purines and analogues of smooth muscle preparations was studied in rat duodenum and rat urinary bladder. 2. Responses to adenosine and to adenosine 5'-triphosphate (ATP) mediated by P1- and P2-purinoceptors respectively were present as early as postnatal day 2, the earliest day studied. 3. In rat bladder, adenosine was inhibitory and ATP and adenosine 5'-(beta, gamma-methylene) triphosphonate (AMP-PCP) were excitatory, acting on the P2X subtype of P2-purinoceptors. Adenosine was more potent in the neonate than in the adult, while the potency of the nucleotides initially increased with age but then declined, being highest between postnatal days 10 and 25. 4. In rat duodenum also, adenosine was inhibitory, its potency being less than the adult before day 15. 5. ATP at low concentrations was inhibitory in rat duodenum at every age studied and its potency increased with age, but higher concentrations of ATP (3 microM and above) were excitatory until day 15. Both relaxations and contractions were mediated by the P2Y subtype of P2-purinoceptors. These ATP-induced contractions were not inhibited by indomethacin (25 microM) or by tetrodotoxin (1 microM) and are therefore not due to prostaglandin synthesis or to ATP-induced release of transmitter substances from nerves. 6. These results show that responses to adenosine and to adenine nucleotides are present from birth and vary with age, and that the changes seen indicate a differential development for P1-, P2X- and P2Y-purinoceptors.     

Characterization of 5-HT receptors mediating contraction and relaxation of the longitudinal muscle of guinea-pig distal colon in vitro

A range of agonists and antagonists were used to characterize the receptors through which 5-hydroxytryptamine (5-HT) contracts and relaxes the longitudinal muscle of segments of guinea-pig distal colon, in vitro. 5-HT contracted the longitudinal muscle over the concentration range 10^–9 to 10^–4 mol/l. The 5-HT3 receptor agonist, 2-methyl-5-HT, produced concentration dependent contractions over the range 10^–6 to 10^–4 mol/l. 5-methoxytryptamine, an agonist at 5-HT4 receptors, caused contractions over a concentration range of 10^–8 to 10^–4 mol/l. The 5-HT4 antagonist, SDZ 205-557 (5 × 10^–7 mol/l) substantially suppressed the responses to low concentrations of 5-HT and to 5-methoxytryptamine, but had no effect on the responses to higher concentrations of 5-HT. In contrast, the 5-HT3 antagonist, granisetron (10^–6 mol/l), blocked the effect of 2-methyl-5-HT and substantially depressed responses to high concentrations of 5-HT, but had no effect on lower concentrations of 5-HT. Granisetron produced a small reduction in the response to 5-methoxytryptamine. Tetrodotoxin (TTX) (3 × 10^–7 mol/l) almost abolished the response to 5-methoxytryptamine and markedly suppressed the response to 2-methyl-5-HT, but the responses to 5-HT were only partially reduced. The 5-HT, antagonist, methiothepin 10^–6 mol/l. depressed the response to 5-HT 10^–7 to 10^–4 mol/l. and blocked its TTX insensitive component. The 5-HT₂ antagonist, ketanserin, in concentrations up to 10^–5 mol/l, had no effect on the contractions evoked by 5-HT.The response to 5-HT was substantially depressed by hyoscine (3 × 10^–6 mol/l. The tachykinin antagonist, spantide 10^–5 mol/l. depressed the response to 5-HT but to a lesser extent than hyoscine. Spantide and hyoscine combined completely blocked the contractile responses to 5-HT Responses to 2-methyl-5-HT were partially suppressed by hyoscine (3 x 10^–6 mol/l. and spantide (10^–5 mol/l) and completely blocked when both byoscine and spantide were present. Contractions evoked by 5-methoxytryptamine were partially blocked by hyoscine (3 × 10^–6 mol/l) and were unaffected by spantide (10^–5 mol/l), but a combination of hyoscine and spantide completely blocked such responses.When the excitatory transmission was blocked with hyoscine (3 × 10^–6 mol/l) and spantide 10^–5 mol/l) and the tone of the muscle raised, an inhibitory response to 5-HT was revealed that had a threshold concentration between 10^–7 mol/l) and 3 × 10^–7 mol/l, and a maximum effect at 10^–4 mol/l. It was blocked by TTX (3 × 10^–7 mol/l) and granisetron 10^–6 mol/l. while N-nitro-l-arginine (NOLA) (10^–4 mol/l) and SDZ 205-557 (5 × 10^–7 mol/l) had no effect. Apamin A 10^–6 mol/l. partially suppressed this response.It is concluded that 5-HT3, 5-HT4 and 5-HT₁-like receptors mediate contraction of the longitudinal muscle of the distal colon. The 5-HT3 and 5-HT4 receptors are located on the excitatory motor neurons innervating the longitudinal muscle and the 5-HT₁-like receptor is located on the muscle. 5-HT3 receptors are also found on inhibitory neurons to the muscle. Correspondence to: D. J. Woollard at the above address     

Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat

1. We examined the endothelin (ET) receptors mediating contractions to ET-1, ET-3 and sarafotoxin S6c (SX6c) in rat pulmonary resistance arteries by use of peptide and non-peptide ET receptor antagonists. Changes induced by pulmonary hypertension were examined in the chronically hypoxic rat. The effect of the mixed ET_A/ET_B receptor antagonist SB 209670 on endothelin-mediated contraction was also examined in human pulmonary resistance arteries.
2. In rat vessels, the order of potency for the endothelin agonists was SX6c=ET-3>ET-1 (pEC₅₀ values in control rats: 9.12±0.10, 8.76±0.14 and 8.12±0.04, respectively). Maximum contractions induced by ET-3 and ET-1 were increased in vessels from chronically hypoxic rats.
3. The ET_A receptor antagonist FR 139317 (1 μM) had no effect on the potency of ET-1 in any vessel studied but abolished the increased response to ET-1 in the chronically hypoxic vessels. The ET_A receptor antagonist BMS 182874 (1 μM) increased the potency of ET-1 in control rat vessels without effecting potency in the pulmonary hypertensive rat vessels.
4. Bosentan (non-peptide mixed ET_A/ET_B receptor antagonist) increased the potency of ET-1 in control rat vessels but was without effect in the pulmonary hypertensive rat vessels. Bosentan (1 μM) inhibited responses to SX6c in control and chronically hypoxic rat vessels with pK_b values of 5.84 and 6.11, respectively. The ET_B receptor antagonist BQ-788 (1 μM) did not inhibit responses to ET-1 in any vessel tested but did inhibit responses to both SX6c and ET-3 (pK_b values in control and chronically hypoxic rat vessels respectively: SX6c 7.15 and 7.22; ET-3: 6.68 and 6.89). BQ-788 (1 μM) added with BMS 182874 (10 μM) did not inhibit responses to ET-1 in control vessels but caused a significant inhibition of responses to ET-1 in chronically hypoxic preparations.
5. SB 209670 inhibited responses to ET-1 in both control and chronically hypoxic vessels with pK_b values of 7.36 and 7.39, respectively. SB 209670 (0.1 and 1 μM) virtually abolished responses to ET-1 in the human pulmonary resistance artery.
6. In conclusion, in rat pulmonary resistance arteries, vasoconstrictions induced by ET-1, SX6c and ET-3 are mediated predominantly by activation of an ET_B–like receptor. However, lack of effect of some antagonists on ET-1 induced vasoconstriction suggests that ET-1 stimulates an atypical ET_B receptor. The increase in potency of ET-1 in the presence of some antagonists suggests the presence of an inhibitory ET_A-like receptor. The influence of this is reduced, or absent, in the chronically hypoxic rats. Increased responses to ET-1 are observed in the chronically hypoxic rat and may be mediated by increased activation of ET_A receptors. SB 209670 is unique in its potency against responses to ET-1 in both control and chronically hypoxic rats, as well as human, isolated pulmonary resistance arteries.

     

Characterization of alpha-adrenoceptors mediating contraction in isolated ovine umbilical vein.

Responses to alpha-adrenoceptor agonists were studied in isolated umbilical vessels obtained from fetal lambs within 2 weeks of term (term = 145 days). Norepinephrine produced concentration-dependent contractions of the umbilical vein, but not in the umbilical artery. The contraction elicited by norepinephrine in the umbilical vein was not potentiated by cocaine (10(-5) M). Epinephrine also produced contractions of the umbilical vein with a potency similar to that of norepinephrine. Phenylephrine was less potent than norepinephrine while clonidine and xylazine were inactive in producing contractions of the umbilical vein. The dissociation constant (KA) of norepinephrine in the umbilical vein was 172 +/- 41 nM. There was little, if any, alpha-adrenoceptor reserve in the umbilical vein. Contraction produced to norepinephrine in the umbilical vein was effectively blocked by prazosin and phentolamine. Yohimbine was 500 times less potent than prazosin in blocking norepinephrine-induced contraction in the umbilical vein. Idazoxan only minimally antagonized the contractions elicited to norepinephrine. We conclude that alpha 1-adrenoceptors are present and mediate vasoconstriction in the ovine umbilical vein and that alpha 2-adrenoceptors are not present in this tissue.     

Cross talk between receptors mediating contraction and relaxation in the arterioles but not the dilator muscle of the rat iris.

1. Sympathetic nerve stimulation causes contraction of the dilator muscle and the large arterioles of the iris via the activation of alpha 1B-adrenoceptors. We have investigated whether increases in adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and the activation of receptors in these tissues can modulate these nerve-mediated contractions. 2. Increasing intracellular cyclic AMP with dibutyryl cyclic AMP (1 mM), forskolin (50 microM) or isobutylmethylxanthine (100 microM) produced relaxation of both the dilator and the arterioles, abolished the nerve-mediated constriction of the arterioles, but potentiated the nerve-mediated contraction of the iris dilator. 3. Pretreatment of the preparations with cholera toxin, to activate Gs permanently, caused a dilatation of the arterioles and abolished the nerve-mediated constriction but had no effect on the dilator muscle. 4. The beta-adrenoceptor agonist, isoprenaline (1 microM), the adenosine-A1,-A2 agonist, N-ethylcarboxamidoadenosine NECA (100 nM), in the presence of the A1-selective antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 10 nM), and calcitonin gene-related peptide (CGRP, 10 nM) all separately caused a dilatation of the arterioles and abolished the nerve-mediated constriction, while only isoprenaline (1 microM) produced an effect on the dilator, i.e. a relaxation but a potentiation of the nerve-mediated contraction. These results suggest the presence of at least 3 types of receptor linked to Gs and an increase in cyclic AMP in the arterioles, i.e. beta-adrenoceptor, adenosine-A2 and CGRP, but only 1 Gs-linked receptor, i.e. beta-adrenoceptors, on the dilator muscle cells.2+ '     

Pharmacological characterisation of neurokinin receptors mediating anion secretion in rat descending colon mucosa

Summary Substance P(SP), neurokinin A (NKA), neurokinin B (NKB), [Sar⁹, Met (O₂)¹¹]-SP (SMSP), senktide, [Ala⁸]-NKA(4–10) and neuropeptide (NP) all stimulate secretory responses in rat descending colon mucosa under voltage clamp conditions. Secretory responses (measured as short circuit current under voltage clamp conditions) were transient and those evoked by SP, SMSP, NKA and senktide were significantly reduced by pretreating tissues with the chloride channel blocker, diphenylamine carboxylate (DPC). Concentration-response curves showed varying degrees of sensitivity to tetrodotoxin (TTX). Senktide-induced secretion was virtually abolished by TTX, while NP and [Ala⁸]-NKA(4–10) were not significantly altered. Rightward shifts of concentration-response curves were observed for SMSP, NKA and SP in TTX treated preparations compared with controls. NKA response curves in the presence of TTX were further inhibited by MEN10,207 and CP-96,345. GR71251, GR82334 and CP-96,345 all inhibited SMSP secretory responses with pA₂ values of 5.8, 6.5 and 6.9 respectively. In conclusion three types of neurokinin receptor exist in preparations of rat colon mucosa and their relative location within neuronal and epithelial surfaces are discussed. Correspondence to H. M. Cox at the above addressK. G. and S. Y. were project students with H. M. C. in the Department of Pharmacology, University of Cambridge. S. Y. was also a Wellcome Trust Vacation Scholar     

Subtypes of purinoceptors in rat and dog urinary bladder smooth muscles.

1. The role of the adhesion glycoproteins CD18 and intercellular adhesion molecule-1 (ICAM-1) in inflammatory responses produced during a reversed passive Arthus (RPA) reaction and induced by zymosan and zymosan-activated plasma (ZAP) were studied in rabbit skin. 2. Oedema formation and haemorrhage were quantified by measuring accumulation of 125I-albumin and 111In-labelled red blood cells (111In-RBC) respectively. 3. Monoclonal antibody (mAb) R15.7 (anti-CD18), administered intravenously, abolished accumulation of 125I-albumin and 111In-RBC in dermal RPA reactions and in response to locally injected zymosan and ZAP. 4. When administered intravenously, the mAb RR1/1 (anti-ICAM-1) suppressed 125I-albumin and 111In-RBC accumulation in dermal RPA reactions and at sites treated with zymosan and ZAP. 5. Oedema formation in response to platelet-activating factor (PAF) and bradykinin (BK) either in the presence or absence of prostaglandin E2 (PGE2) were not affected by mAb R15.7 or by mAb RR1/1.1.1. 6. We conclude that oedema formation and haemorrhage associated with RPA reactions and in responses to zymosan and ZAP are completely CD18-dependent, and are mediated, at least in part, via ICAM-1. Responses to the neutrophil-independent oedema forming mediators, PAF and BK are not dependent upon CD18 or ICAM-1.