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.     

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.     

Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle.

1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.     

Regional variation in the sensitivity of longitudinal smooth muscle to histamine at H1-receptors in guinea-pig ileum and colon.

The sensitivity of the distal ileum, proximal colon, medial colon, and distal colon of the guinea-pig to histamine has been evaluated. The rank order of sensitivity was ileum greater than medial colon greater than proximal colon approximately equal to distal colon. The mean -logEC50 values at receptors in the ileum, medial, proximal, and distal colon were 6.74, 6.18, 5.79, and 5.72, respectively. The apparent dissociation constant for the interaction of histamine with receptors in the various regions was determined. The -log Kd values at receptors in the ileum, proximal colon, medial colon, and distal colon were 4.68, 4.65, 4.62, and 4.44, respectively. The mean apparent -log Kd values for the antagonism of histamine by mepyramine were 9.0, 9.0, 9.1, and 8.9 for receptors on the ileum, proximal, medial, and distal colon, respectively. The results of these experiments provide no evidence that histamine receptors in the colon are distinguishable from H1-receptors as characterized on the ileum. The differences in sensitivity to histamine in the various regions of the intestine may be due to differences in the density of H1-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.     

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.     

Electrophysiological and mechanical characteristics of histamine receptors in smooth muscle cells of the guinea-pig ileum

To elucidate the role of the histamine receptor in functions related to intestinal motility, we investigated the effects of histamine and its antagonists on electrical and mechanical activities in longitudinal and circular layers of the terminal region of the guinea-pig ileum. Histamine hyperpolarized the membrane in the circular smooth muscle cells by increasing the permeability of K+ and it transiently inhibited generation of the spike while resting tone was elevated. Cimetidine (CIM) inhibited the hyperpolarization and relaxation induced by histamine while mepyramine (MEP) inhibited the contraction but did not affect the histamine-induced hyperpolarization. In the presence of CIM, histamine did not depolarize the membrane but did lower the threshold potential required for generation of the spike potential, increased the appearance of the spike and enhanced the phasic contraction. Histamine, in 20 mM K+ solution, hyperpolarized the membrane and produced a biphasic response, an initial relaxation and a subsequently generated contraction, in a concentration-dependent manner. In the longitudinal smooth muscle cells, histamine depolarized the membrane, and enhanced both generation of the spike and the contraction. MEP (0.1 microM) but not CIM (1 microM) blocked the histamine-induced responses. CIM at a higher concentration (10 microM) enhanced the histamine-induced contraction, while histamine did not relax the tissue precontracted by 20 mM K+. These results indicate that the circular muscle cells have both H1 and H2 receptors while the longitudinal muscle cells have the H1 receptor. The excitatory responses induced by activation of the H1 receptor in smooth muscle cells differ in these layers.     

Evidence that histamine homologues discriminate between H3-receptors in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus.

1. The binding of the selective histamine H3-receptor agonist ([3H]-R-alpha-methylhistamine) to sites in guinea-pig cerebral cortex and ileum longitudinal muscle myenteric plexus has been characterized and a comparison made of the apparent affinities of a series of H3-receptor ligands. 2. Saturation analysis suggested that [3H]-R-alpha-methylhistamine labelled a homogeneous population of histamine H3-receptors in guinea-pig cerebral cortex (pKD=9.91+/-0. 07; nH=1.07+/-0.03; n=5) and ileum longitudinal muscle myenteric plexus (pKD=9.75+/-0.21; nH=0.97+/-0.02; n=5). There was no significant difference in the estimated affinity of [3H]-R-alpha-methylhistamine in the two tissues. The cerebral cortex had a significantly higher receptor density (3.91+/-0.37 fmol mg-1 tissue) than the ileum longitudinal muscle myenteric plexus (0. 39+/-0.11 fmol mg-1). 3. Overall, the apparent affinities of compounds, classified as H3-receptor ligands, in cerebral cortex and ileum longitudinal muscle myenteric plexus were well correlated (r=0. 91, P<0.0001) and consistent with the cerebral cortex and ileum longitudinal muscle myenteric plexus expressing histamine H3-receptor population(s) that are pharmacologically indistinguishable by the majority of histamine H3-receptor ligands. However, it was evident that the homologues of histamine within this group of compounds could discriminate between the receptor populations in the two tissues. Thus, the estimated affinity of five imidazole unbranched alkylamines (histamine, homohistamine, VUF4701, VUF4732 and impentamine) were significantly higher in the guinea-pig cerebral cortex than in the ileum longitudinal muscle myenteric plexus assay.     

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.     

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.     

An inhibitory action of histamine on the guinea-pig ileum

1. In atropinized, plexus-containing preparations of the longitudinal muscle from the guinea-pig ileum, in which histamine contractions were abolished by mepyramine or diphenhydramine, an inhibitory action of histamine was revealed on the "tetanic spasms" produced by field stimulation.2. The inhibitory action of histamine on the atropine-resistant tetanic spasms, which are due to the excitation of non-cholinergic neurones in Auerbach's plexus (Ambache & Freeman, 1968a, b), was reversible. It is specific for the tetanic spasms, because histamine did not reduce contractions elicited by bradykinin, 5-hydroxytryptamine, prostaglandin E(2), nicotine or dimethylphenylpiperazinium.3. L-Histidinol and 2-mercaptohistamine exerted a considerably weaker inhibitory effect upon the tetanic spasms than histamine. Four other imidazoles tested, L-histidine, murexine, dihydromurexine and imidazolecarboxylcholine, were ineffective; so was the pyrazole ring isomer of histamine, betazole.4. The inhibitory action of histamine persisted after adrenoceptor blockade by phentolamine and pronethalol and after prior reserpinization of the guineapigs.5. The inhibitory action of histamine was also obtained after ganglionic paralysis by hexamethonium or dimethylphenylpiperazinium but was antagonized specifically by nicotine.6. On atropinized preparations of the longitudinal muscle from the guinea-pig descending colon histamine exerted, at most, an insignificant inhibitory effect on the tetanic spasms.     

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.

     

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.     

Temperature-dependence of desensitization induced by acetylcholine and histamine in guinea-pig ileal longitudinal muscle.

1. The effects of temperature on the time course of desensitization induced by acetylcholine and histamine, and on the recovery from desensitization were studied in the longitudinal muscle of the guinea-pig ileum. 2. Self- and cross-desensitization produced by acetylcholine (10(-5) M) occurred rapidly in the first 10 min of exposure to the agonist, with the same time course and the same degree of desensitization over the temperature range of 11 degrees C to 31 degrees C. 3. Self-desensitization produced by histamine (10(-5) M) also occurred rapidly in the first 10 min of exposure to the agonist, and showed great temperature-dependence, especially at 11 degrees C and 21 degrees C, but scarcely occurred at 6 degrees C. 4. Cross-desensitization produced by histamine developed gradually with time and showed a moderate temperature-dependence between 11 degrees C and 31 degrees C, but scarcely occurred at 6 degrees C. 5. The recovery processes from desensitization showed marked temperature-dependence. Recovery was halted completely at 11 degrees C. 6. These studies suggest that acetylcholine-induced desensitization may be attributed to a single non-specific mechanism. Histamine-induced desensitization may be due to at least two mechanisms: it occurs in both a specific and non-specific manner. Each of these desensitizations can be characterized by its unique temperature-dependence.