Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways

We have investigated whether prejunctional inhibitory muscarinic receptors ("autoreceptors") exist on cholinergic nerves in human airways in vitro and whether guinea pig trachea provides a good model for further pharmacological characterization of these receptors. Pilocarpine was used as a selective agonist and gallamine as a selective antagonist of these autoreceptors. Acetylcholine (ACh) release from postganglionic cholinergic nerves was elicited by electrical field stimulation (EFS) (40 V, 0.5 ms, 32 Hz). In human bronchi, pilocarpine inhibited the contractile response to EFS in a dose-related fashion; the dose inhibiting 50% of the control contraction was 2.2 +/- 0.4 x 10(-7) (SE) M (n = 22), and the inhibition was 96% at 3 x 10(-5) M. The inhibitory effects of pilocarpine were antagonized by gallamine in a dose-related fashion. The results were qualitatively the same in the guinea pig. Gallamine significantly enhanced the contractile response to EFS in the guinea pig, whereas pirenzepine failed to do so, which suggests that M2-receptors are involved. We conclude that prejunctional muscarinic receptors that inhibit ACh release are present on cholinergic nerves in human airways and that guinea pig trachea is a good model for further pharmacological characterization of these receptors, which appear to belong to the M2-subtype.     

Differential sensitivities of the sphincter of Oddi and gallbladder to cholecystokinin in the guinea pig: their role in transsphincteric bile flow.

Cholecystokinin (CCK) is considered to simply contract the gallbladder and relax the sphincter of Oddi with meals. In this study, we examined this hypothesis by investigating the action of CCK on the sphincter of Oddi and gallbladder of the guinea pig. The experimental design used an in vitro preparation of the sphincter of Oddi to measure contraction of the circular muscle. CCK increased tone in both the gallbladder and the sphincter of Oddi in a concentration-dependent manner. The normalized concentration-response curves for CCK, however, revealed that the gallbladder had a greater sensitivity to CCK (ED50 7 nM) than the sphincter of Oddi (ED50 22 nM; p < 0.01). Conversely, the sphincter was more sensitive to bethanechol than was the gallbladder. When the sphincter of Oddi was stimulated maximally with CCK in the presence of atropine (10(-6) M) or tetrodotoxin (10(-6) M), the contractile response was significantly reduced (p < 0.05) although not abolished. Conversely, atropine completely abolished the responses to bethanechol (10(-3) M) and transmural field stimulation (70 V, 10 Hz, 1 ms, for 20 s). Transmural field stimulation of the sphincter that had been precontracted with CCK (26 nM) caused a transient, initial relaxation followed by contraction. Pretreatment with atropine augmented the duration of this relaxation, which could be completely abolished by tetrodotoxin. Thus, CCK contracts the sphincter of Oddi in the guinea pig by a direct (myogenic) and a neural (likely cholinergic) mechanism. Relaxation of the sphincter of Oddi also occurs in the guinea pig via noncholinergic inhibitory nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of cholinergic neurotransmission in human airways by opioids.

Opioids reduce the cholinergic responses to electrical field stimulation (EFS) in guinea pig and canine airways by a prejunctional effect. We determined whether a similar effect operates in human airways in vitro. [D-Ala2-NMePhe4-Gly-ol5]enkephalin (DAMGO) (10(-8)-10(-6) M), a selective mu-opioid receptor agonist, inhibited the response to EFS in a dose- and frequency-dependent manner. DAMGO (10(-6) M) produced 86% inhibition at 0.5 Hz and 38% inhibition at 4 Hz, but at 32 Hz there was no significant inhibition. Another selective mu-opioid receptor agonist H-Tyr-D-Arg-Gly-Phe(4-NO2)-Pro-NH2 diacetate (BW 443C) also inhibited responses to EFS, producing 57.7% inhibition at 4 Hz at a concentration of 10(-6) M. The inhibitory effect on EFS was blocked by the opioid receptor antagonist naloxone (10(-5) M), indicating that opioid receptors are involved. DAMGO (10(-6) M) had no effect on the contractile response to exogenous acetylcholine, indicating a prejunctional effect. We conclude that mu-opioid agonists inhibit cholinergic neurotransmission in human airways in vitro, and this could have therapeutic potential in the treatment of airway disease.     

Cholinergic neuromodulation by prostaglandin D2 in canine airway smooth muscle

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.     

The electrophysiological and mechanical effects of atrial natriuretic peptide and acetylcholine on guinea pig ventricular papillary muscle

The direct effects of atrial natriuretic factor (ANF) and acetylcholine (ACh) on isolated guinea pig ventricular papillary muscle were studied. ANF (3 x 10(-9) - 3 x 10(-7) M), a cardiogenic hormone, had no significant electrical or mechanical effects on guinea pig papillary muscle driven at a frequency of 60 beats/min in normal (4 mM) and high [K]0 (27 mM) Tyrode solutions. On the other hand, ACh (3 x 10(-8) - 3 x 10(-7) M) caused a significant shortening of action potential duration and the contractile force showed no change or a slight decrease. At high concentration (5 microM), ACh reduced action potential durations at 50% and 90% repolarization (APD50 and APD90) by 10.5 +/- 2.1% and 12.4 +/- 1.8%, respectively, but the contractile force was slightly increased by 9.8 +/- 1.2%. In eleven of twenty-six preparations, spontaneous activity occurred and intermingled with driven activity. The ectopic rhythms were suppressed by ACh (1-5 microM). The changes in electrical but not mechanic activity induced by ACh were suppressed in the presence of five micromolar atropine. These results reveal that, in guinea pig papillary muscle, ANF had no direct chronotropic or inotropic effect. ACh may reduce APD and spontaneous discharges through an activation of muscarinic receptors but enhance twitch tension through other mechanisms.     

Different responsiveness of excitatory and inhibitory enteric motor neurons in the human esophagus to electrical field stimulation and to nicotine

To compare electrical field stimulation (EFS) with nicotine in the stimulation of excitatory and inhibitory enteric motoneurons (EMN) in the human esophagus, circular lower esophageal sphincter (LES), and circular and longitudinal esophageal body (EB) strips from 20 humans were studied in organ baths. Responses to EFS or nicotine (100 microM) were compared in basal conditions, after N(G)-nitro-l-arginine (l-NNA; 100 microM), and after l-NNA and apamin (1 microM). LES strips developed myogenic tone enhanced by TTX (5 microM) or l-NNA. EFS-LES relaxation was abolished by TTX, unaffected by hexamethonium (100 microM), and enhanced by atropine (3 microM). Nicotine-LES relaxation was higher than EFS relaxation, reduced by TTX or atropine, and blocked by hexamethonium. After l-NNA, EFS elicited a strong cholinergic contraction in circular LES and EB, and nicotine elicited a small relaxation in LES and no contractile effect in EB. After l-NNA and apamin, EFS elicited a strong cholinergic contraction in LES and EB, and nicotine elicited a weak contraction amounting to 6.64 +/- 3.19 and 9.20 +/- 5.51% of that induced by EFS. EFS elicited a contraction in longitudinal strips; after l-NNA and apamin, nicotine did not induce any response. Inhibitory EMN tonically inhibit myogenic LES tone and are efficiently stimulated both by EFS and nicotinic acetylcholine receptors (nAChRs) located in somatodendritic regions and nerve terminals, releasing nitric oxide and an apamin-sensitive neurotransmitter. In contrast, although esophageal excitatory EMN are efficiently stimulated by EFS, their stimulation through nAChRs is difficult and causes weak responses, suggesting the participation of nonnicotinic mechanisms in neurotransmission to excitatory EMN in human esophagus.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.     

Neural control of relaxation in cat airways smooth muscle

Functional innervation of cat airways smooth muscle was examined in isolated segments of trachea and bronchi using electrical field stimulation (EFS) techniques. Field stimulation caused contraction in tissues at resting tone and biphasic responses (contraction followed by relaxation) in tissues precontracted with 5-hydroxytryptamine (5-HT). Contractions were abolished by 10(-6) M atropine. Inhibitory responses were dependent on impulse voltage, duration, and frequency. At low voltages (less than or equal to 10 V) and pulse durations (less than or equal to 0.3 ms), EFS induced relaxations were abolished by 3 X 10(-6) M tetrodotoxin (TTX). Greater stimulus parameters elicited TTX-resistant relaxations. Pretreatment of the tissues with 10(-6) M propranolol and 10(-5) M guanethidine caused rightward shifts in relaxation frequency-response curves. These findings indicate that cat airways are innervated by excitatory cholinergic, inhibitory adrenergic, and inhibitory nonadrenergic noncholinergic (NANC) nerves. Pretreatment of the tissues with hexamethonium, cimetidine, indomethacin, or nordihydroguaiaretic acid did not affect NANC relaxation responses. It is concluded that NANC inhibitory responses in cat airway smooth muscle are mediated through intrinsic postganglionic nerve fibers and occur independently of histamine H2-receptor activation and without involvement of cyclooxygenase or lipoxygenase products of arachidonic acid metabolism.     

Effect of quinidine and tetrodotoxin on the activation of non-adrenergic nerves in guinea-pig trachealis muscle

Activation of non-adrenergic neurones in guinea-pig trachealis muscle was accomplished by electrical field stimulation and by the neurotoxin aconitine, in the presence of atropine and propranolol. Aconitine (10(-5) M) activated non-adrenergic neurones more slowly, but was as efficacious as supramaximal field stimulation (70 V, 1 msec, 1-100 Hz), producing 70-80% of the maximal relaxation to forskolin or theophylline. Quinidine (3 X 10(-5) M-3 X 10(-4) M) and tetrodotoxin (5 X 10(-9) M-3 X 10(-6) M) blocked relaxations to aconitine and field stimulation, without affecting smooth muscle relaxant responses to forskolin. The results suggest that the non-adrenergic inhibitory effects of quinidine are related to its presynaptic local anaesthetic actions, rather than to postsynaptic receptor blockade of the non-adrenergic inhibitory neurotransmitter.     

Influence of maturation on constrictive response to stimulation of C-fiber afferents in isolated guinea pig airways

We investigated whether the airway constrictive response to stimulation of bronchopulmonary C-fiber afferents is altered during the maturation process. Isometric tension was measured in airway rings isolated from three tracheobronchial locations (intrathoracic trachea and main and hilar bronchi) and compared in mature [M, 407 +/- 10 (SE) g body wt, n = 36] and immature (IM, 161 +/- 5 g body wt, n = 35) guinea pigs. Our results showed no difference in the ACh (10(-5) M)- or KCl (40 mM)-induced contraction between M and IM groups, regardless of the airway location. In sharp contrast, the concentration-response curves of 10(-8)-10(-6) M capsaicin were distinctly lower in IM hilar bronchi; for example, response to the same concentration of capsaicin (10(-6) M) was 89.2 +/- 15.3% of the response to 10(-5) M ACh in IM and 284.7 +/- 43.2% in M animals. Similar, but smaller, differences in the bronchoconstrictive response to capsaicin between IM and M groups were also observed in the trachea and main bronchus. Electrical field stimulation induced airway constriction in all three locations in M and IM groups. However, after administration of 10(-6) M atropine and 10(-6) M propranolol, electrical field stimulation-induced contraction was significantly smaller in the hilar bronchus of IM than M animals, and this difference was not prevented by pretreatment with 5 x 10(-5) M indomethacin. Although radioimmunoassay showed no difference in the tissue content of substance P between M and IM airways, the constrictive responses to exogenous substance P and neurokinin A were markedly greater in M airways at all three locations. In conclusion, the constriction of isolated airways evoked by C-fiber stimulation was significantly weaker in the IM guinea pigs, probably because of a less potent effect of tachykinins on the airway smooth muscle.     

Noncholinergic contractile response to nicotine in the guinea pig isolated tracheal smooth muscle

The existence of substance P immunoreactive nerves in the trachea of guinea pig is known. In this study, capsaicin induced a long-lasting and marked contraction in the guinea pig trachea and nicotine-induced contraction was partially reduced in the capsaicin-treated muscle. Furthermore, the contractile response to nicotine (10(-5) M) in the presence of atropine (10(-7) M) was abolished by a substance P antagonist, [D-Arg1, D-Pro2, D-Trp7,9 Leu11]substance P (10(-5) M). These findings suggest that noncholinergic contractile response to nicotine may be due to the release of material(s) resembling substance P in the isolated tracheal smooth muscle preparation of guinea pig.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Airway neutral endopeptidase-like enzyme modulates tachykinin-induced bronchoconstriction in vivo

To determine whether neutral endopeptidase (NEP), also called enkephalinase (EC 3.4.24.11), modulates the effects of exogenous and endogenous tachykinins in vivo, we studied the effects of aerosolized phosphoramidon, a specific NEP inhibitor, on the responses to aerosolized substance P (SP) and on the atropine-resistant response to vagus nerve stimulation (10 V, 5 ms for 20 s) in guinea pigs. SP alone (10(-7) to 10(-4) M; each concentration, 7 breaths) caused no change in total pulmonary resistance (RL, P greater than 0.5). Phosphoramidon (10(-4) M, 90 breaths) caused no change either in base-line RL (P greater than 0.5) or in the response to aerosolized acetylcholine (P greater than 0.5). However, in the presence of phosphoramidon, SP (7 breaths) produced a concentration-dependent increase in RL at concentrations greater than or equal to 10(-5) M (P less than 0.001). Phosphoramidon (10(-7) to 10(-4) M; each concentration, 90 breaths) induced a concentration-dependent potentiation of SP-induced bronchoconstriction (10(-4) M, 7 breaths; P less than 0.01). Vagus nerve stimulation (0.5-3 Hz), in the presence of atropine, induced a frequency-dependent increase in RL (P less than 0.001). Phosphoramidon potentiated the atropine-resistant responses to vagus nerve stimulation (P less than 0.001) at frequencies greater than 0.5 Hz. The tachykinin antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-substance P abolished the effects of phosphoramidon on the atropine-resistant response to vagus nerve stimulation (2 Hz, P less than 0.005). NEP-like activity in tracheal homogenates of guinea pig was inhibited by phosphoramidon with a concentration producing 50% inhibition of 5.3 +/- 0.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of substance P on neurally mediated contraction of rabbit airway smooth muscle

The neuromodulatory action of substance P (SP) was investigated in isolated rabbit tracheal smooth muscle (TSM) segments contracted with electrical field stimulation (ES). The tissues were placed in organ baths containing modified Krebs-Ringer solution and stimulated at a constant voltage (8 V; 24.5 mA) and pulse duration (2 ms) with ES frequencies ranging from 1 to 100 Hz. In the presence of SP, there occurred a dose-dependent augmentation of the TSM contractile response to any given ES, with the maximal effect of SP obtained at a dose of 10(-7) M. Accordingly, with the administration of 10(-7) M SP, the ES frequency-response relationship was altered so that 1) the mean (+/- SE) maximal tension (Tmax) induced by ES significantly increased (P less than 0.02) from a base-line value of 273 +/- 53 to 402 +/- 45 g/g TSM; and 2) the mean (+/- SE) log ES frequency producing 50% of Tmax (ES50) significantly decreased from a base-line value of 1.278 +/- 0.069 to 1.102 +/- 0.070 Hz (P less than 0.01). In contrast to these effects on ES-induced contraction, SP administration did not affect the TSM contractile response to administered methacholine chloride (10(-8) to 10(-3) M). On the other hand, the effects of SP on ES-induced contraction were independently blocked by the cholinergic antagonist, atropine (10(-6) M); the neurotoxin, tetrodotoxin (10(-6) g/ml); and the SP antagonist, D-Arg1,D-Pro2,D-Trp7,9,Leu11-SP (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin potentiates cholinergic neurotransmission in ferret trachea

We studied the effect of somatostatin on contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. Somatostatin (up to 10(-5) M) did not change resting tension, but it potentiated the contractile response to EFS dose dependently, with a maximum effect at 10(-6) M. Thus, at a concentration of 10(-6) M, somatostatin significantly decreased the mean log of EFS frequency producing 50% of maximum contraction from a control value of 0.52 +/- 0.07 to 0.24 +/- 0.06 (SE) Hz (P less than 0.01). The potentiating effect of somatostatin (10(-6) M) was not inhibited by hexamethonium, indomethacin, BW755C, pyrilamine, methysergide, or D,Pro2,D,Trp7,9-SP, but it was inhibited by atropine or by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by somatostatin at a concentration of 10(-6) M. These results suggest that somatostatin potentiates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific somatostatin receptor.     

Activation-dependent descending reflex evacuation motority of anal canal in rat model

The evacuative motor responses of the anal canal and recto-anal reflexes during defecation were studied in an isolated rat recto-anal model preparation using (i) partitioned organ bath, (ii) electrical stimulation, (iii) balloon distension and (iv) morphological techniques. Electrical field stimulation applied to the anal canal or to the distal part of the rectum elicited tetrodotoxin (10(-7) M)-sensitive frequency-dependent local or descending contractions of the anal canal and the local responses were bigger in amplitude (14.9 ± 1.35 mN) than the descending contractions (5.3 ± 0.7 mN at frequency of 5 Hz, p < 0.05). The balloon-induced distension of the distal rectum evoked descending responses of the anal canal consisting of a short contraction (1.50 ± 0.18 mN) followed by deep relaxation (3.12 ± 0.34 mN). In the presence of atropine (3 x 10(-7) M) the electrically-elicited (5 Hz) local or descending contractions of the anal canal were suppressed and a relaxation revealed. The initial contraction component of the distension-induced response was decreased while the relaxation was not changed. During atropine treatment, spantide (10(-7) M) lowered even more the contractile component of the anal canal response. NG-nitro-L-arginine (5 x 10(-4) M) enhanced the contraction, prevented the atropine-dependent relaxation of the electrically-elicited response and inhibited the distension-induced relaxation. L-Arginine (5 x 10(-4) M) suppressed the contraction and extended the relaxation. ChAT-, substance P- and NADPH-diaphorase-positive perikarya and nerve fibers were observed in myenteric ganglia of the anal canal. The results suggest activation-dependent descending reflex motority of the anal canal involving electrical stimulation-displayed cholinergic and tachykininergic and distension manifested nitrergic neuro-muscular communications.     

Modulation of cholinergic neurotransmission by vasoactive intestinal peptide in ferret trachea

We studied the effect of vasoactive intestinal peptide (VIP) on the contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. VIP did not change resting tension up to 2 X 10(-7) M, but it showed a biphasic effect on the responses to EFS. In concentrations up to 10(-9) M, VIP potentiated the response; at higher concentrations VIP reduced responses. Thus, at a concentration of 10(-9) M, VIP decreased the mean (+/- SE) log EFS frequency, producing 50% of maximum contraction significantly from a control value of 0.476 +/- 0.062 to 0.214 +/- 0.057 Hz (P less than 0.01); at a concentration of 2 X 10(-7) M VIP increased the half-maximal frequency from a control value of 0.513 +/- 0.086 to 0.752 +/- 0.053 Hz (P less than 0.05). The potentiating effect of VIP (10(-9) M) was not inhibited by hexamethonium, indomethacin, pyrilamine, methysergide, or [D-Pro2,D-Trp7,9] substance P. The inhibitory effect of VIP (2 X 10(-7) M) was also not inhibited by hexamethonium, indomethacin, or naloxone. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by VIP at concentrations of 10(-9) and 2 X 10(-7) M. These results suggest that VIP modulates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific VIP receptor.     

Studies of the innervation of rabbit myometrium and cervix

We characterized the innervation of isolated circular and longitudinal-oriented muscle strips from the nulliparous rabbit uterus and cervix by field stimulation (FS). FS with increasing frequency (2.5-50 pps) and voltage (2.5-70 V) caused graded increases in isometric contraction with no relaxation or inhibition of spontaneous activity. Tetrodotoxin (TTX, 3.1 X 10(-6) M) significantly reduced the FS response by 75% in all strips at higher stimulus frequencies. Contractile responses to FS were also significantly inhibited by atropine (3.5 X 10(-6) M) in circular uterus and in longitudinal cervix. Guanethidine (5 X 10(-6) M) reduced the response in all strips, as did phentolamine (3.6 X 10(-6) M) in longitudinal uterus and circular cervix. Propranolol (3.9 X 10(-6) M) did not significantly change the response in longitudinal uterus or circular cervix. In longitudinal uterus, combined guanethidine and atropine produced significant inhibition, but not statistically different from either drug alone. Similar results were seen in circular uterus. Electron microscopy and glyoxylic acid histofluorescence indicate that both blood vessels and smooth muscle in rabbit uterus are supplied with adrenergic nerves. The results suggest the presence of TTX-sensitive adrenergic and cholinergic excitatory innervation of rabbit uterus and cervix.     

Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 microM) or atropine (1 microM) and concentration-dependently (10 nM-1 microM) inhibited by N/OFQ (Emax, 11.5+/-1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7+/-2.9% inhibition, P<0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 microM) while prevented by the selective NOP receptor antagonist UFP-101 (10 microM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 microM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.