Morphine-tolerant longitudinal muscle strip from guinea-pig ileum

1. Implantation of morphine pellets in guinea-pigs produced a high degree of tolerance and dependence within 3 days.2. The contractions of the longitudinal muscle induced by electrical stimulation of the myenteric plexus-longitudinal muscle preparations obtained from tolerant animals were less depressed by morphine than the contractions evoked in preparations from non-tolerant animals.3. Naloxone did not alter the size of the evoked twitch but antagonized the depressant action of morphine in tolerant and in non-tolerant animals. When given to tolerant guinea-pigs, naloxone caused an increase in intestinal activity in vivo.4. The contractile response of the longitudinal muscle to acetylcholine was the same in preparations obtained from tolerant and non-tolerant animals. Electrically evoked contractions of the myenteric plexus-longitudinal muscle preparations from tolerant animals showed reduced sensitivity to the depressant effects of adrenaline, isoprenaline, and particularly, dopamine.     

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.     

Histamine-induced inositol phospholipid breakdown in the longitudinal smooth muscle of guinea-pig ileum.

The characteristics of histamine-stimulated inositol phospholipid breakdown in slices of guinea-pig ileal smooth muscle and cerebellum have been investigated. In cerebellar slices the inhibition of the inositol phospholipid response to histamine by mepyramine was consistent with competitive antagonism of histamine H1-receptors. In slices of the longitudinal smooth muscle of guinea-pig ileum, mepyramine produced only a weak inhibition of the response to histamine, at concentrations up to 1 microM. This was in striking contrast to the potent competitive antagonism of the H1-mediated contractile responses obtained with mepyramine in this tissue. The H1-receptor antagonists (+)-chlorpheniramine and promethazine similarly had no effect on the EC50 value for histamine in guinea-pig ileum, while promethazine competitively antagonized the muscarinic receptor-mediated inositol phospholipid response in this tissue (Ka 3.6 X 10(7)M-1). Cimetidine, on its own, did not significantly inhibit the inositol phosphate accumulation elicited by histamine in ileum. In the presence of 0.2 microM mepyramine, cimetidine (0.1 mM) produced a small parallel shift of the histamine concentration-response curve (Ka 3 X 10(4) M-1). This inhibition, however, was not consistent with antagonism of an H2-receptor-mediated response. The effect of a range of histamine analogues on inositol phospholipid breakdown was determined. Dose-response curves were constructed and characterized in terms of the EC50, slope and maximal response attainable relative to histamine. The H1-agonists, N alpha,N alpha-dimethylhistamine, N alpha-methylhistamine, 2-pyridylethylamine and 2-thiazolyethylamine produced the largest accumulations of [3H]-inositol-1-phosphate. A very weak response was produced by the H2-selective agonist impromidine, while dimaprit (also H2-selective) was without significant effect. Mepyramine appeared to antagonize competitively the response to the H1-selective agonist 2-pyridylethylamine. This was in contrast to the data obtained with other H1-agonists, where mepyramine produced only a small dextral shift of the agonist curves at low agonist concentrations and an increase in the Hill coefficient. This was particularly striking in the case of 2-methylhistamine. The results suggest that an H1-receptor component in guinea-pig ileum, may coexist with a larger inositol phospholipid response to histamine which is independent of the activation of H1- or H2-receptors.     

The Schultz-Dale response of the longitudinal muscle strip preparation of guinea-pig ileum

1. The mast-cell distribution in the various layers of the ileum has been described.2. Histamine-content, anaphylactic histamine-release and the anaphylactic dose-response curve of the full-thickness ileum and of the longitudinal muscle strip have been measured and compared.3. Tetrodotoxin 10^-7 g/ml had no obvious effect on the anaphylactic dose-response curve of either preparation. This suggests that the plexus is not of any great importance in the Schultz-Dale reaction.4. Exposure of the longitudinal muscle strip to octylamine 10^-3 g/ml for 1 min reduced the mast cell content by 95-100%. After this treatment the dose-response curve to antigen was eliminated, although the muscle still responded to small doses of histamine and to anaphylactic mediators. Pretreatment of antibody with octylamine did not impair passive sensitization and subsequent response to antigenic challenge. This suggests that the classical Schultz-Dale reaction in the strip is mediated mainly by mast cells, and possibly other cells, and is probably not due to a direct effect of the antigen-antibody reaction on the smooth muscle.5. The typical three-phase anaphylactic response (quick contraction, quick relaxation, slow contraction) of full-thickness ileum is discussed and compared with the predominantly two-phase response of the longitudinal muscle strip. No evidence was found for the release of a relaxation-factor. It is suggested that the initial fast phase may be due to mediators released from mast cells among the longitudinal muscle fibres, and the sustained contraction to a second wave of mediators reaching the longitudinal muscle from deeper layers of the ileum.     

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum

The binding affinities of selective muscarinic antagonists were compared with their ability to block receptor-mediated inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum. When measured by competitive inhibition of the binding of N-[3H]methylscopolamine, the binding properties of selective muscarinic antagonists were consistent with a two-site model. Approximately 84% of the binding sites (major sites) had high affinity for the M2-selective antagonists methoctramine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one), whereas the remainder of the sites (minor sites) had high affinity for hexahydrosiladifenidol and its para-fluoro derivative. There was good agreement between the estimates of the dissociation constants of muscarinic antagonists for the major binding site and those measured by antagonism of the adenylate cyclase response. There was also good agreement between the dissociation constants of muscarinic antagonists for the minor binding site and those measured by antagonism of the phosphoinositide response and the contractile response. Our data indicate that there are at least two types of muscarinic receptors in the longitudinal muscle of the ileum, the more abundant being an M2 receptor, which mediates an inhibition of adenylate cyclase activity, and the less abundant being an M3 receptor, which triggers contraction and phosphoinositide hydrolysis.     

Mechanism of action of muscarine on the longitudinal muscle of the guinea-pig isolated ileum.

1 DL-Muscarine elicited a contraction of the ileal longitudinal muscle of the guinea-pig and the contraction was characterized by an after-response. 2 Physostigmine (20 x 10(8) M) potentiated the contraction of the longitudinal muscle elicited by DL-muscarine. 3 Hemicholinium-3 (HC-3) caused a rightward shift of the dose-response curve to DL-muscarine on the ileal longitudinal muscle of the guinea-pig ileum. 4 beta-Bungarotoxin (10 microgram/ml) significantly (P < 0.025) reduced the contraction elicited by DL-muscarine (2.5 X 10(-8) M) suggesting presynaptic release of acetylcholine as an indirect mechanism of action of DL-muscarine. 5 Morphine (1.0 x 10(-8) M) significantly (P < 0.05) reduced the contractions elicited by DL-muscarine (2.5 x 10(-8) M) further suggesting presynaptic release of acetylcholine as an indirect mechanism of action of DL-muscarine. 6 A subthreshold dose of DL-muscarine (2.0 x 10(-10) M) potentiated the effect of acetylcholine (2.5 x 10(-8) M) and the potentiation was blocked by beta-bungarotoxin.     

Neurokinin3-receptors are linked to inositol phospholipid hydrolysis in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation.

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of longitudinal muscle from guinea-pig ileum. 2. Substance P, neurokinin A and neurokinin B induced a concentration-dependent accumulation of total [3H]-inositol phosphates in the presence of 12 mM lithium with similar maximal responses and EC50 values. 3. The selective NK1-receptor agonist, substance P methyl ester, and the selective NK3-receptor agonist succ-[Asp6, MePhe8]-SP(6-11) (senktide) also stimulated [3H]-inositol phosphate formation with maximum responses of 50.69 +/- 0.96 and 45.64 +/- 1.17% relative to 10 microM substance P, respectively. Substance P methyl ester was approximately equipotent with substance P, whereas senktide was approximately 100 times more potent. 4. When added together, maximally effective concentrations of substance P methyl ester and senktide gave responses that were fully additive. In contrast, responses to substance P and neurokinin B were not additive. 5. The stimulation of [3H]-inositol phosphate formation by substance P, neurokinin B and senktide was not affected by atropine (2 microM) or tetrodotoxin (TTX, 0.3 microM). 6. The contractile effect of senktide was inhibited completely by TTX and partially blocked by atropine. Contractions induced by substance P methyl ester were not changed in the presence of TTX or atropine. 7. [D-Pro4, D-Trp7,9,10]-SP(4-11) competitively antagonized the action of substance P methyl ester on inositol phospholipid hydrolysis and contraction, but had no significant effect on senktide-induced inositol phospholipid breakdown or contraction. 8. These results suggest that NK3-receptors in the guinea-pig ileum are coupled to inositol phospholipid hydrolysis.     

Post-tetanic potentiation at the nerve-muscle junction in the longitudinal muscle of the guinea-pig ileum

The effect of short tetanic stimulation (30 Hz for 25 s) on the following twitch responses of the myenteric plexus-longitudinal muscle preparation of guinea-pig ileum to electric stimulation (0.1 Hz) was investigated in the presence of naloxone and indomethacin. Post-tetanic potentiation (PTP) of the twitches observed in control experiments was abolished in preparations desensitized by substance P but it was not affected in preparations desensitized by serotonin or pretreated with methysergide. Immediately after 5 min tetanic stimulation a decreased sensitivity to substance P but unchanged sensitivity to serotonin were observed. Electromyogram (EMG) of the longitudinal muscle layer was picked up 4 and 10 mm aborally from the stimulation site in response to 1 to 16 impulse trains delivered at 100 Hz. In control conditions only the longer trains triggered this neurogenic response at the distal recording site. In the presence of substance P but not serotonin facilitation occurred so that the distal site was frequently recruited to respond with an EMG even to single impulses. A substance P-like compound rather than serotonin may be a candidate for the neuromodulator or neurotransmitter substance involved in PTP and changes in the response topography of muscarinic transmission.     

Histamine-induced hydrolysis of polyphosphoinositides in guinea-pig ileum and brain

The effect of histamine and the H1-selective agonist, 2-pyridylethylamine, on the accumulation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3) has been examined in lithium-treated slices of guinea-pig cerebellum and ileal smooth muscle. Following 45 min incubation, histamine produced a large accumulation of [3H]InsP and a smaller accumulation of [3H]InsP2 and [3H]InsP3 in both tissues. In cerebellar slices all three responses to histamine were potently and competitively inhibited by the selective H1-receptor antagonist, mepyramine. In contrast, incubation of ileal slices with mepyramine (0.1 microM) produced only a small reduction (circa 20%) in the maximal accumulation elicited by histamine of each [3H]inositol phosphate with no significant effect on the EC50 or Hill coefficient. However, when 2-pyridylethylamine, instead of histamine, was used to stimulate inositol phospholipid hydrolysis in ileal smooth muscle, the agonist-induced responses appeared to be competitively antagonised by mepyramine. The results presented indicate that there is an apparent dissociation between histamine-induced InsP3 accumulation and H1-receptor-mediated contractile activity in ileal smooth muscle and suggest that agonist-induced inositol phospholipid hydrolysis in this tissue may be involved in other cellular events separate from those involving calcium.     

Tachykinin receptors in the circular muscle of the guinea-pig ileum.

1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandin E1 and indomethacin on responses of longitudinal muscle of guinea-pig ileum to cholecystokinin.

The effect of prostaglandin E1 (PGE1) and indomethacin (IND) cholecystokinin (CCK)-induced contractions of guinea-pig isolated ileum longitudinal muscle were studied. PGE1 (2.8--28 nM) consistently and dose dependently increased the contractions evoked by CCK (indirect muscle stimulation) or by ACh. IND (2.7 microM) decreased the contractions to both compounds and this was reversed with 2.8--7 nM PGE1. Pretreatment of the preparations with phentolamine (2.6 microM) or pretreatment of the animals with reserpine (2 mg/kg i.p. 24 h before killing) did not affect PGE1 potentiation or IND inhibition of CCK-induced contractions. The results indicated that PGE1 potentiated CCK-induced contractions of the longitudinal muscle of guinea-pig ileum by increasing the response to released ACh. Experiments with IND suggested that endogenous PGs may modulate the effect of CCK or related gastrointestinal hormones.     

Relationship between stimulated inositol lipid hydrolysis and contractility in guinea-pig visceral longitudinal smooth muscle

Carbamylcholine caused a marked, concentration-dependent stimulation of [3H]Ins P, [3H] InsP2 and to a lesser extent [3H]InsP3 production in guinea-pig longitudinal smooth muscle prelabelled with myo-[3H]inositol. Accumulation of these three inositol phosphates showed differential sensitivity to LiCl. Muscle contraction was apparent at lower concentrations of carbamylcholine. Both responses were mediated via muscarinic-type receptors. An association of inositol phosphate production and contractility was also observed in response to substance P, histamine and noradrenaline, the latter via an alpha-adrenergic mechanism. The Ca2+-channel agonist CGP 28392 failed to stimulate inositol phosphate production despite inducing a contractile response. Carbamylcholine -induced inositol phosphate production persisted in the presence of D600 or Mn2+ despite loss of contractile activity. However, both responses showed a similar, marked dependence on the presence of Ca2+ in the extracellular medium. Mn2+ could restore basal and stimulated inositol phosphate production in low Ca2+ solutions but could not substitute for Ca2+ in restoring contractility. The results suggest that stimulated inositol lipid hydrolysis in longitudinal smooth muscle does not result from Ca2+ entry into the tissue, although the response does depend on the concentration of divalent cations in the extracellular medium. This dependency may be related to the maintenance of membrane potential and possibly phospholipid conformation.     

Some pharmacological properties of the circular and longitudinal muscle strips from the guinea-pig isolated ileum

A circular and a longitudinal muscle strip were prepared from adjacent parts of a guinea-pig ileum and a direct pharmacological comparison made under identical conditions. The longitudinal preparation was sensitive to acetylcholine, methacholine, carbachol, 5-hydroxytryptamine, histamine and nicotine, while the circular preparation was insensitive to 5-hydroxytryptamine, histamine and nicotine, and responded to the choline esters only in high concentrations. Incubation of the preparations with the anticholinesterase, mipafox (NN-diisopropylphosphodiamidic fluoride), sensitized both preparations to the action of acetylcholine; potentiation of the contraction of the longitudinal muscle was 16-times; that of the circular one 4,000-times. The longitudinal muscle was more sensitive than the circular muscle to acetylcholine whether both were treated with mipafox or not. Bradykinin and substance P both stimulated the longitudinal but not the circular muscle, an effect not modified after mipafox. Hyoscine antagonized the responses of the circular muscle strip, treated with mipafox, to acetylcholine and to histamine, but on the longitudinal muscle strip the response to histamine was not affected, the response to acetylcholine being competitively antagonized. Morphine, in the same concentrations on both circular and longitudinal muscle strips, antagonized the stimulant actions of nicotine and to a lesser extent of 5-hydroxytryptamine, but the responses to histamine on the longitudinal muscle strip were not antagonized by morphine which was in contrast to its action on the circular muscle strip. These observations showed that the main differences in the responses of the circular and longitudinal muscle of the guinea-pig ileum to drugs were in the intrinsic properties of the smooth muscle cells. In addition cholinesterase may protect the circular muscle cells. Finally the circular muscle strip preparation proved to be a useful tool to study the action of drugs on the nervous plexuses of the ileum of the guinea-pig.     

1,4-Dithiothreitol-induced changes in histamine H1-agonist efficacy and affinity in the longitudinal smooth muscle of guinea-pig ileum.

The effect of 1,4-dithiothreitol (DTT) on histamine H1-receptor agonist affinity and efficacy has been investigated in longitudinal muscle strips of guinea-pig ileum. Exposure of ileal smooth muscle to DTT significantly increased the maximal responses to the partial agonists SKF71473 and DE-2PEA, indicative of an increase in agonist efficacy. This effect was paralleled by a small decrease in EC50 values. In contrast, DTT produced a parallel displacement of the concentration-response curve to the full agonist histamine in the same muscle strips. Studies in which phenoxybenzamine and benzilylcholine mustard were used to reduce the maximum response to histamine suggested that DTT altered both agonist affinity and efficacy. The affinity constant for histamine, calculated by the method of Furchgott & Bursztyn (1967), increased by 2.7 fold in the presence of DTT. Furthermore, agonist efficacy also appeared to increase in the presence of DTT since the maximum response to histamine following phenoxybenzamine treatment increased on application of DTT. [3H]-mepyramine binding studies confirmed that DTT increased agonist affinity. DTT produced a significant parallel shift to the left of the displacement curves for histamine, 2-methylhistamine, 2-pyridylethylamine and 2-thiazolylethylamine. The results of this study therefore suggest that DTT potentiates H1-receptor-mediated contractile activity in guinea-pig ileum by increasing both agonist efficacy and affinity.     

Homogeneous and non-homogeneous distribution of inhibitory and excitatory adrenoceptors in the longitudinal muscle of the guinea-pig ileum

1 The effects of adrenoceptor agonists and antagonists on spontaneous and evoked membrane activities of longitudinal muscle cells from different parts of the guinea-pig ileum were observed, using microelectrode methods.

2 Isoprenaline inhibited the generation of spikes in cells in the terminal (0-3 cm from the ileocaecal valve) and proximal (more than 50 cm from the ileocaecal valve) regions of the ileum, with no change on the membrane potential and ionic conductance of the membrane. These actions of isoprenaline were abolished by propranolol.

3 Noradrenaline and phenylephrine depolarized the membrane and increased both the spike frequency and ionic conductance of cell membranes of the terminal ileum, whereas noradrenaline and clonidine hyperpolarized the membrane, increased the ionic conductance of the membrane and inhibited the spontaneously generated spikes from cells of the proximal ileum. The excitatory effect of phenylephrine in the cells of the proximal ileum and the inhibitory effect of clonidine on cells of the terminal ileum were less pronounced.

4 The excitatory actions of noradrenaline or phenylephrine were antagonized by prazosin and phentolamine, but not by yohimbine, whereas the inhibitory actions of noradrenaline or clonidine were antagonized by yohimbine and phentolamine but not by prazosin.

5 The cholinergic e.j.ps evoked by field stimulation to the tissue were not affected by isoprenaline or phenyleprine but were inhibited by noradrenaline and clonidine, in both the terminal and proximal regions of the ileum. These actions of noradrenaline and clonidine were antagonized by yohimbine but not by prazosin.

6 The results indicate that in the myenteric plexus and longitudinal muscle tissues of the guinea-pig ileum there are prejunctional inhibitory (α₂), postjunctional inhibitory (α₂ and β) and postjunctional excitatory (α₁) adrenoceptors. The homogeneous distributions of prejunctional α₂- and postjunctional β-adrenoceptors in the ileum are responsible for inhibitions of cholinergic excitatory junction potentials (e.j.ps) and spontaneous spike activities, respectively. The density of distribution of the postjunctional α₁-adrenoceptors is higher in the terminal than in the proximal regions, and these distributions are reversed in the case of the postjunctional α₂-adrenoceptors. The postjunctional α₁-adrenoceptors are probably responsible for the membrane depolarization and α₂-adrenoceptors for the hyperpolarization induced by catecholamines.