Muscle Of Guinea-pig Ileum Research Articles

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors.

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle.

1. This study examined the effects of sodium rhein (0.03-30 microM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded. 2. Sodium rhein (0.3, 3 and 30 microM) significantly potentiated (52+/-11% at 30 microM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 microM) or omega-conotoxin GVIA (10 nM) sodium rhein (3 and 30 microM) did not enhance, but significantly reduced (49+/-10% and 44+/-8%, respectively, at 30 microM) acetylcholine-induced contractions. 3. Sodium rhein (0.3, 3 and 30 microM) significantly increased (65+/-11% at 30 microM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 microM), omega-conotoxin GVIA (10 nM) or atropine (0.1 microM), sodium rhein (3 and 30 microM) significantly reduced (50+/-10%, 55+/-8% and 46+/-10%, respectively, at 30 microM) substance P-induced contractions. 4. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 microM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30+/-6%)- or substance P (36+/-6%)-induced contractions. 5. Sodium rhein (30 microM) significantly potentiated (29+/-9%) the electrically-evoked contractions. L-NAME (100 microM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 microM) significantly increased (32+/-9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment. 6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca2+ entry through the N-type Ca2+ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

Potentiation by neurotensin of carbachol-induced tension development in beta-escin-skinned smooth muscle of guinea-pig ileum.

Effect of neurotensin (NT) on carbachol(CCh)-induced tension development due to Ca2+ release from intracellular stores was investigated in beta-escin-skinned smooth muscle of guinea-pig ileum. NT (10 nM) increased the tension development in response to CCh. NT also increased the tension response to caffeine, another store-Ca2+ releaser. NT did not exert such an effect in pertusis toxin (PTX)-treated preparations. Treatment with isoprenaline to elevate endogenous cyclic AMP levels or with dibutyryl cyclic AMP did not affect the effect of NT. A nonpeptide NT antagonist, SR 48692, failed to block the effect of NT. NT shifted the pCa-tension relationship in the lower direction of Ca2+ concentration. NT was incapable of releasing Ca2+ from intracellular stores. The results suggest that NT may cause an increase in Ca2+ sensitivity of contractile elements to potentiate the CCh-induced tension development due to release of stored Ca2+ and that the effect is mediated by SR 48692-insensitive NT receptors linked to a PTX-sensitive G protein which works with no relation to a change in cytosolic cyclic AMP levels.

O-261 - Transient resensitization inhibits progress of desensitization in muscarinic receptors and histamine H1-receptors at its early stage in longitudinal smooth muscle of guinea-pig ileum

O-261 - Transient resensitization inhibits progress of desensitization in muscarinic receptors and histamine H1-receptors at its early stage in longitudinal smooth muscle of guinea-pig ileum

Pharmacological properties of non-adrenergic, non-cholinergic inhibitory transmission in chicken gizzard.

1. Non-adrenergic, non-cholinergic (NANC) inhibitory transmission in chicken gizzard was studied by use of intracellular microelectrode techniques. Changes in membrane potential in response to NANC nerve stimulation were recorded in the gizzard smooth muscle pretreated with atropine (1 microM) and guanethidine (1 microM). 2. Field stimulation of the intramural nerves (FS) evoked inhibitory junction potentials (i.j.ps) which were abolished by tetrodotoxin (1 microM), but not inhibited at all by K+ channel blockers including apamin (0.5 microM), tetraethylammonium (TEA, 10 mM), charybdotoxin (0.2 microM) and glibenclamide (10 microM). 3. NG-nitro-L-arginine (3 mM), an inhibitor of nitric oxide (NO) synthase, inhibited i.j.ps. The effect was reversed by L-arginine (3 mM), but not by D-arginine (3 mM). 4. 8-Bromo cyclic GMP (100 microM), a membrane permeable analogue of cyclic GMP, produced a membrane hyperpolarization which was blocked by TEA (10 mM) or glibenclamide (10 microM). 5. NO at concentrations of up to 400 microM affected neither i.j.ps nor resting membrane potential. On the other hand, NO (80 microM) caused the membrane to hyperpolarize in the smooth muscle of guinea-pig ileum. 6. These results suggest that in the chicken gizzard, NANC i.j.ps may not arise from opening of conventional types of K+ channel and that NO seems unlikely to be involved in the generation of i.j.ps. A possible mechanism by which the inhibitory effect of NG-nitro-L-arginine on i.j.ps was brought about will be discussed.

Activation of M2 muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M3 muscarinic receptors.

In longitudinal muscle of guinea-pig ileum, activation of muscarinic receptors causes contraction antagonised by M3 receptor subtype antagonists despite a preponderance of M2 receptor subtype binding sites. Experiments on single smooth muscle cells under voltage-clamp described here show that the cationic current evoked by carbachol which normally causes depolarization of the muscle is inhibited competitively by M2 antagonists with affinities typical of antagonism at a M2 receptor. However, M3 antagonists strongly reduced the maximum cationic current which could be evoked by carbachol in a non-competitive manner with affinities typical for an action at M3 receptors. Thus cation channels are gated by M2 receptor activation but strongly modulated by activation of M3 receptors.

Effects of (-)-RO363 at human atrial beta-adrenoceptor subtypes, the human cloned beta 3-adrenoceptor and rodent intestinal beta 3-adrenoceptors.

1. Chronic treatment of patients with beta-blockers causes atrial inotropic hyperresponsiveness through beta 2-adrenoceptors, 5-HT4 receptors and H2-receptors but apparently not through beta 1-adrenoceptors despite data claiming an increased beta 1-adrenoceptor density from homogenate binding studies. We have addressed the question of beta 1-adrenoceptor sensitivity by determining the inotropic potency and intrinsic activity of the beta 1-adrenoceptor selective partial agonist (-)-RO363 and by carrying out both homogenate binding and quantitative beta-adrenoceptor autoradiography in atria obtained from patients treated or not treated with beta-blockers. In the course of the experiments it became apparent that (-)-RO363 also may cause agonistic effects through the third atrial beta-adrenoceptor. To assess whether (-)-RO363 also caused agonistic effects through beta 3-adrenoceptors we studied its relaxant effects in rat colon and guinea-pig ileum, as well as receptor binding and adenylyl cyclase stimulation of chinese hamster ovary (CHO) cells expressing human beta 3-adrenoceptors. 2. beta-Adrenoceptors were labelled with (-)-[125I]-cyanopindolol. The density of both beta 1- and beta 2-adrenoceptors was unchanged in the 2 groups, as assessed with both quantitative receptor autoradiography and homogenate binding. The affinities of (-)-RO363 for beta 1-adrenoceptors (pKi = 8.0-7.7) and beta 2-adrenoceptors (pKi = 6.1-5.8) were not significantly different in the two groups. 3. (-)-RO363 increased atrial force with a pEC50 of 8.2 (beta-blocker treated) and 8.0 (non-beta-blocker treated) and intrinsic activity with respect to (-)-isoprenaline of 0.80 (beta-blocker treated) and 0.54 (non-beta-blocker treated) (P < 0.001) and with respect to Ca2+ (7 mM) of 0.65 (beta-blocker treated) and 0.45 (non-beta-blocker treated) (P < 0.01). The effects of (-)-RO363 were resistant to antagonism by the beta 2-adrenoceptor antagonist, ICI 118,551 (50 nM). The effects of 0.3-10 nM (-)-RO363 were antagonized by 3-10 nM of the beta 1-adrenoceptor selective antagonist CGP 20712A. The effects of 20-1000 nM (-)-RO363 were partially resistant to antagonism by 30-300 nM CGP 20712A. 4. (-)-RO363 relaxed the rat colon, partially precontracted by 30 mM KCl, with an intrinsic activity of 0.97 compared to (-)-isoprenaline. The concentration-effect curve to (-)-RO363 revealed 2 components, one antagonized by (-)-propranolol (200 nM) with pEC50 = 8.5 and fraction 0.66, the other resistant to (-)-propranolol (200 nM) with pEC50 = 5.6 and fraction 0.34 of maximal relaxation. 5. (-)-RO363 relaxed the longitudinal muscle of guinea-pig ileum, precontracted by 0.5 microM histamine, with intrinsic activity of 1.0 compared to (-)-isoprenaline and through 2 components, one antagonized by (-)-propranolol (200 nM) with pEC50 = 8.7 and fraction 0.67, the other resistant to (-)-propranolol with pEC50 = 4.9 and fraction 0.33 of maximal relaxation. 6. (-)-RO363 stimulated the adenylyl cyclase of CHO cells expressing human beta 3-adrenoceptors with pEC50 = 5.5 and intrinsic activity 0.74 with respect to (-)-isoprenaline (pEC50 = 5.9). (-)-RO363 competed for binding with [125I]-cyanopindolol at human beta 3-adrenoceptors transfected into CHO cells with pKi = 4.5. (-)-Isoprenaline (pKi = 5.2) and (-)-CGP 12177A (pKi = 6.1) also competed for binding at human beta 3-adrenoceptors. 7. We conclude that under conditions used in this study, (-)-RO363 is a potent partial agonist for human beta 1- and beta 3-adrenoceptors and appears also to activate the third human atrial beta-adrenoceptor. (-)-RO363 relaxes mammalian gut through both beta 1- and beta 3-adrenoceptors. (-)-RO363, used as a beta 1-adrenoceptor selective tool, confirms previous findings with (-)-noradrenaline that beta 1-adrenoceptor-mediated atrial effects are only slightly enhanced by chronic treatment of patients with beta-blockers. Chronic treatment with

Endogenous NO inhibits NANC but not cholinergic neurotransmission to circular muscle of guinea pig ileum.

Nitric oxide (NO) mediates neurogenic relaxations of gastrointestinal (GI) smooth muscle. NO synthase (NOS) inhibitors also alter neurogenic contractions, suggesting NO modulates excitatory neurotransmitter release. In circular muscle-myenteric plexus preparations, guanethidine and either scopolamine or CP-96,345, a neurokinin-1 (NK1) receptor antagonist, were used to isolate nonadrenergic, noncholinergic (NANC) or cholinergic contractions, respectively. NOS inhibitors and hemoglobin potentiated neurogenic NANC but not cholinergic contractions and did not affect NK1 receptor agonist [substance P methyl ester (SPME)]-induced contractions. Sodium nitroprusside (SNP), a NO donor, attenuated NANC and cholinergic neurogenic contractions, but cholinergic contractions were less sensitive to SNP. SNP partially attenuated SPME-induced contractions, and apamin reduced inhibition of NANC contractions by SNP. Bethanechol responses were not affected by SNP. These data indicate NANC but not cholinergic contractions are inhibited by endogenous NO, suggesting differential regulation of release of tachykinins and acetylcholine from enteric nerves. NK1 receptor-but not muscarinic receptor-activated postjunctional pathways are also inhibited by NO. Therefore, prejunctional and postjunctional modulation of NANC contractions are mechanisms for inhibition of GI motility by endogenous NO.

Inhibition of Na+,K+‐ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Ouabain, an Na+K+ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca2+ -independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [3H]ACh. Release of [3H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the omega-conotoxins GVIA (selective N-type calcium channel blocker) and MVIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [3H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [3H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, omega-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [3H]ACh. Thus, extracellular Ca2+ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na+K+-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.

The longitudinal muscle of rat ileum as a sensitive monoreceptor assay for bradykinin B1 receptors.

1. Various bradykinin derivatives, acting preferentially at B1 or B2 receptors, were tested in the isolated longitudinal smooth muscle of rat ileum. Experiments were carried out in the presence of chlorpheniramine and atropine (both 1 microM), guanethidine and indomethacin (both 3 microM) and of the peptidase inhibitors (captopril, bestatin and thiorphan, all 1 microM). 2. The rank order of potency was (pD2 values +/- s.e.mean, n = 5 in parentheses, at 5 h from set-up): [des-Arg9]-BK (8.27 +/- 0.11) > or = [des-Arg10]-kallidin (7.67 +/- 0.24) > bradykinin (6.69 +/- 0.25). The B2 receptor selective agonist, [Hyp3,Tyr(Me)8]-BK, was approximately 10 fold less active than bradykinin. Contractile responses to all agonists increased with time. The maximal response to the B1 receptor agonist, [desArg9]-BK at 5 h (94 +/- 2%) was significantly (P < 0.05) greater than that measured at 2 h (74 +/- 2%). 3. The B2 receptor antagonist, D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK (Hoe 140, 0.1 microM) did not affect responses to the B1 receptor agonist [des-Arg9]-BK (0.1 nM--1 microM) nor those to the B2 receptor agonist, [Hyp3,Tyr(Me)8]-BK (1 nM--10 microM). In control experiments performed in the longitudinal smooth muscle of guinea-pig ileum and rat isolated urinary bladder as bioassays for B2 receptors, the B2 receptor antagonist Hoe 140 (0.1 microM) antagonized bradykinin-induced contractions. 4. In the rat isolated ileum the B1 receptor antagonist, D-Arg[Hyp3, Thi5, D-Tic7, Oic8, des-Arg9]-BK ([des-Arg10]-Hoe 140, 0.3 - 10 microM) competitively antagonized contractile responses to [des-Arg9]-BK with an estimated pKB of 6.74 +/- 0.08 (Schild plot slope with confidence limits 1.22, (0.70 - 1.73) n = 13). In control experiments in the guinea-pig isolated ileum and rat isolated urinary bladder, [des-Arg10]-Hoe 140 (1 - 10 microM) did not inhibit B2 receptor-mediated contractile responses. 5. The putative B1 receptor antagonist, [Leu8,des-Arg9]-BK, behaved as a partial agonist when responses were determined 2 h from set-up (pD2 6.43 +/- 0.21, n = 5; Emax 30% of that evoked by [des-Arg9]-BK); at 5 h from set-up it behaved as a full agonist (pD2 7.48 +/- 0.12, n = 5; Emax 90% of that evoked by [des-Arg9]-BK). At this time the response to [Leu8,des-Arg9]-BK was antagonized in a concentration-dependent manner by [des-Arg10]-Hoe 140, which at 1 microM and 10 microM, produced dose-ratios of 6.33 +/- 3.66 (n = 4) and 103 +/- 40 (n = 4). 6. In view of the rank order of potency of agonists, the antagonist activity by [des-Arg10]-Hoe 140 and the lack of antagonist activity of Hoe 140, we conclude that the longitudinal smooth muscle of rat ileum, after histamine, acetylcholine, noradrenaline, and prostanoid production blockade, is a sensitive monoreceptor assay for studying the pharmacology of bradykinin B1 receptors. Further the preparation can also be used as a sensitive bioassay to identify partial agonist activity of B1 receptor antagonists such as [Leu8,desArg9]-BK.

Phenoxybenzamine discriminates between two distinct populations of histamine H1-receptors in longitudinal muscle of guinea-pig ileum.

The longitudinal muscle of guinea-pig ileum was used as a test tissue. Progressive (up to 70 min) treatment with phenoxybenzamine (10(-6) M) inhibited progressively the response to histamine and progressively decreased the density of histamine H1-receptors. However, the 90 min treatment has no further significant inhibitory effect on the contractile response and did not decrease the density of the receptors. These results suggest that phenoxybenzamine discriminates between two distinct populations of H1-receptors. Furthermore, it was found that the presence of GTP gamma-S made some populations of H1-receptors resistant to phenoxybenzamine and facilitated an interaction of mepyramine with H1-receptors.

Pharmacological Characterization of Adrenoceptors Mediating Contractile and Relaxant Responses to Noradrenaline in the Longitudinal Muscle of Guinea-Pig Ileum.

Mechanical responses to noradrenaline (NA) were recorded in longitudinal muscle strips from the terminal and intermediate regions (3-10 cm and 30-40 cm from the ileo-caecal junction, respectively) of the guinea-pig ileum. NA(0.16-1,600 microM) produced a concentration-dependent contraction in the terminal ileum with the EC50 value of 11.9 +/- 4.3 microM (n = 5). In the intermediate ileum, NA produced relaxation at concentrations ranging from 0.016 microM to 1.6 microM. Responses to NA at 16 microM varied among preparations: no noticeable change in tension, a moderate relaxation and a small contraction. At higher concentrations, NA produced contractile responses and their peak tension increased in a concentration-dependent manner. The contractile effect of NA in the terminal ileum remained unaltered after treatment with propranolol (1.4 microM) or yohimbine (1.1 microM), or a combination of these drugs. In the intermediate ileum, the relaxant effect of NA was markedly reduced or abolished by propranolol (1.4 microM). Yohimbine (1.1 microM) had little effect on NA-induced relaxation. The contractile effect of NA in both the terminal ileum and intermediate ileum was inhibited by prazosin (1.1 microM), so that the contractions were converted to relaxation or markedly reduced. Propranolol abolished the relaxant response induced by NA at 0.16 microM, but did not inhibit those induced by NA at 1.6 microM. Methoxamine (0.16-1,600 microM) produced a concentration-dependent contraction in both the terminal ileum and the intermediate ileum. The EC50 was 93.5 +/- 28.5 microM (n = 8) in the terminal ileum and 83.3 +/- 27.7 microM (n = 10) in the intermediate ileum. There was no significant difference between the values. The results showed that NA is capable of producing contraction or relaxation in the longitudinal muscle layer of the terminal and intermediate regions of the guinea-pig ileum. The contraction, which is mediated by alpha 1-adrenoceptors, predominates in the terminal ileum, but relaxation, which is mediated by beta-adrenoceptors and uncharacterized adrenoceptors, predominates in the intermediate ileum.

Competitive and selective antagonistic action of NPC 17731 on bradykinin-induced relaxant and contractile responses of the guinea pig isolated ileum

1. 1. The aim of the present study was to evaluate, by means of functional studies, the effect of the newly developed bradykinin (BK) receptor antagonist NPC 17731, D-Arg 0, Arg 1-Pro 2-Hyp 3-Gly 4-Phe 5-Ser 6-[DHype (transpropyl) 7]-Oic 8-Arg 9, against BK-mediated biphasic response in the guinea pig ileum “in vitro” (circular and longitudinal layers). 2. 2. In the circular muscle, NPC 17731 (0.1–1000 nM) dose dependently and reversibly antagonized, with similar potency, both the contractile and relaxant responses caused by BK, as well as BK-induced contraction in longitudinal smooth muscle with IC 50 of 23, 29 and 37 nM, respectively. 3. 3. NPC 17731 (10–300 nM) also caused a concentration-dependent displacement to the right of BK-induced contraction and relaxant responses in the circular smooth muscle of guinea pig ileum, without changing BK maximal response. Schild plot analyses were linear (correlation close to 1), yielding pKb values of 8.89 ± 0.19 and 8.73 ± 0.18, respectively, and slopes not significantly different from unity, providing evidence that NPC 17731 acts as a pure B 2 competitive receptor antagonist against both BK responses. 4. 4. Similarly, NPC 17731 (3-100 nM) antagonized, in a graded and competitive manner, BK-induced contraction in longitudinal muscle from guinea pig ileum with a slope not different from unity, yielding a pKb value of 8.62 ± 0.13. 5. 5. These results indicate that the new B 2 BK receptor antagonist NPC 17731 antagonizes, with high affinity and with similar potency through simple competitive and selective mechanisms, BK receptor-mediating contraction or relaxant responses in circular and longitudinal smooth muscles from guinea pig ileum. In addition, these results also suggest that although BK-induced contraction or relaxation responses in guinea pig ileum are coupled to distinct second messengers, NPC 17731 interacts with a homogeneous population of B 2 receptors in both guinea pig ileum preparations.

Phosphorus‐containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo

1. We have examined several phosphorus-containing peptides as potential mixed inhibitors of two neurotensin-degrading zinc metallopeptidases, endopeptidase 3.4.24.15 and endopeptidase 3.4.24.16. 2. Among a series of 13 phosphonamide peptides, N-(2-(2-naphtyl)ethylphosphonyl-glycyl-prolyl-norleucine (phosphodiepryl 08) was found to inhibit potently the hydrolysis of neurotensin by purified endopeptidase 3.4.24.15 and 3.4.24.16 with an identical Ki value of 0.4 nM. 3. Phosphodiepryl 08 displayed a strong selectivity towards the two peptidases since it failed to inhibit several other zinc-containing peptidases such as endopeptidase 3.4.24.11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase and carboxypeptidases A and B. 4. The protective effect of phosphodiepryl 08 on neurotensin degradation was examined in vitro and in vivo in central and peripheral bioassays. 5. Phosphodiepryl 08 virtually abolished neurotensin degradation by 4-day-old plated pure cultured neurones from mouse embryos and greatly potentiated neurotensin-induced antinociception in the mouse hot plate test. 6. In the periphery, phosphodiepryl 08 inhibited neurotensin degradation by membranes prepared from isolated longitudinal smooth muscle of guinea-pig ileum and greatly potentiated the neurotensin-induced contraction of the same longitudinal smooth muscle preparation. 7. Our study indicates that phosphodiepryl 08 behaves as a potent and selective mixed inhibitor of endopeptidase 3.4.24.15 and 3.4.24.16 and can be used as a powerful agent to prevent neurotensin degradation, in vitro and in vivo, in central and peripheral assays.

Caffeine and carbachol act on common Ca 2+ stores to release Ca 2+ in guinea-pig ileal smooth muscle

To characterize intracellular Ca 2+ stores, the Ca 2+-releasing effects of caffeine, carbachol and inositol 1,4,5-trisphosphate (IP 3) were compared by measuring the drug-induced tension development in β-escin-skinned longitudinal smooth muscle of guinea-pig ileum. Caffeine (20 mM), carbachol (10 or 100 μM) or IP 3 (40 μM), applied after loading Ca 2+ within intracellular stores, produced a transient rise in tension in a Ca 2+-free solution. This change in tension occurred in response to release of Ca 2+ from the stores. The effect of either caffeine or carbachol was markedly reduced or abolished after preceding application of the other drug. IP 3 was without effect when applied subsequently to caffeine. The effects of carbachol and IP 3 were abolished after combined treatment with ryanodine (30 μM) and caffeine (20 mM) which causes functional removal of caffeine-releasable Ca 2+ stores, but not after combined treatment with ryanodine (30 μM) and carbachol (10 μM). The results suggest that caffeine, carbachol and IP 3 all act on common Ca 2+ stores to release Ca 2+.

Possible Involvement of a Small G-Protein Sensitive to Exoenzyme C3 of Clostridium botulinum in the Regulation of Myofilament Ca2+ Sensitivity in β-Escin Skinned Smooth Muscle of Guinea Pig Ileum

The effects of exoenzyme C3 of Clostridium botulinum on Ca(2+)- and drug-induced tension developments were investigated in beta-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca2+ sensitivity. C3 is known to ADP-ribosylate the rho p21 family of small G-proteins. Treatment with C3 (0.35 microgram/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca2+ concentration axis, indicating a decrease in Ca2+ sensitivity of the contractile elements. The inhibitory effect of C3 was not preserved after treatment with GDP beta S (1 mM), an antagonist of GTP for the binding to G-proteins. Stimulation of muscarinic receptors with carbachol (CCh, 100 microM) shifted the pCa-tension curve leftward, indicating Ca2+ sensitization of tension development. The Ca(2+)-sensitizing effect of CCh was not observed after C3 treatment. When GTP gamma S (10 microM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca2+, further increase in tension was elicited and the effect of GTP gamma S was inhibited by C3 treatment. The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca2+ sensitivity of smooth muscle myofilaments.

Effect of Phorbol Ester, 12-Deoxyphorbol 13-Isobutylate (DPB), on Muscle Tension and Cytosolic Ca2+ in Rat Anococcygeus Muscle

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10-6M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or β-escin in the anococcygeus muscle. 3) In the β-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca2+-developed tension. Moreover, the enhancement was inhibited by H-7 (3 × 10-5 M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.

Effects of calcitonin gene-related peptide on intracellular calcium concentration in longitudinal muscle of guinea pig ileum

The effect of rat calcitonin gene-related peptide (rCGRP) on intracellular free calcium concentration ([Ca 2+] i) and its relationship with muscle relaxation were examined in plexus-free longitudinal muscle (LM) of the guinea pig ileum using a [Ca 2+] i-tension simultaneous recording technique. Tissue was stimulated with either histamine (0.5 μM) or KCl (30 m M). rCGRP at a concentration of 263 n M (which displayed maximal relaxation of the LM) caused a small and brief but significant decrease in the [Ca 2+] i in histamine-treated tissue. On the other hand, the same concentration of rCGRP relaxed the muscle without affecting the [Ca 2+] i in KCl-treated tissue. rCGRP caused a dissociation between the changes in [Ca 2+] i and tension. The effects of forskolin (0.5 μ M) and nifedipine (10 n M) on both [Ca 2−] i and tension were examined in comparison with rCGRP. Forskolin lowered the tension to greater extent than the [Ca 2+] i whereas nifedipine (10 n M) diminished both the [Ca 2+] i and tension in a parallel manner in histamine-treated tissues. These results suggest that rCGRP may not reduce [Ca 2+] i as its primary mechanism of relaxation and it may change the calcium sensitivity of the contractile elements of the smooth muscle.

Spontaneous Tone in Different Types of Longitudinal Muscle Preparations of Guinea Pig Ileum

Spontaneous tone of longitudinal muscle in guinea pig ileum was investigated in three types of preparations, intact segment preparation, segment preparation deprived of the mucosal layer and longitudinal strip preparation. Tone was defined as the sustained contraction that was lost by 10−7 M atropine in the isotonically recorded manner. The magnitudes of tone were constant for at least 3 hr in the two types of segment preparations. Contractions in response to 10−6 M acetylcholine, which were induced 9 times with an interval of 20 min between each induction, were almost identical throughout the period. In the longitudinal strip preparation, on the other hand, the tone gradually decayed and was eventually lost, while the amplitudes of acetylcholine-induced contractions were reciprocally increased. The tone in the intact segment preparation was reduced to 19% of the control by tetrodotoxin (3 × 10−7 M), to 51% by indomethacin (3 × 10−6 M) and to 26% by N6-cyclopentyladenosine (10−7 M), but was not affected by AA-861 (3 × 10−6 M) or CP-96,345 (3 × 10−7 M). In the three types of preparations, the dose-response curves for acetylcholine were alike with similar EC50s. These results suggest that the tone of longitudinal muscle was mainly induced due to neural activity in the myenteric plexus of guinea pig ileum and that sensitivity to acetylcholine was not affected by the neural activity.

Effects of calcitonin gene-related peptide on cyclic AMP production and relaxation of longitudinal muscle of guinea pig ileum

The effects of rat calcitonin gene-related peptide (rCGRP) on the content of cyclic nucleotides in the cells of plexus-free longitudinal muscle (LM) of guinea pig ileum relative to smooth muscle relaxation were investigated. In the absence of isobutyl-methyl-xanthine (IBMX), a nonspecific inhibitor of phosphodiesterases, rCGRP significantly increased cyclic 3′5′-adenosine monophosphate (cAMP) content that correlated temporally to the relaxation of the muscle. Cyclic 3′5′-guanosine monophosphate (cGMP) content was not affected by this peptide. IBMX potentiated both the increase in cAMP content and the muscle relaxation induced by rCGRP. rCGRP increased cAMP content in a concentration-dependent manner, with an ED 50 value of 7.5 × 10 −9 M. Both the rCGRP-stimulated increase in cAMP content and the rCGRP-induced muscle relaxation were blocked by hCGRP(8–37), a selective antagonist of CGRP receptors. The pA 2 value of hCGRP(8–37) for rCGRP-induced inhibition of tension was calculated to be 6.48. These results suggest that CGRP relaxes the LM through its specific receptors and involves the generation of cAMP, but not the generation of cGMP.