Tetrodotoxin-resistant Contraction Research Articles

Multiple Motor Effects of ATP and their Inhibition by P2 Purinoceptor Antagonist, Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic Acid in the Small Intestine of the Guinea‐Pig

Adenosine 5'-triphosphate (ATP) may be an important neurotransmitter in the gastrointestinal tract. The present study examined the motor effects of exogenous ATP on longitudinally-oriented preparations of the guinea-pig isolated ileum and the influence of drugs on the ATP-induced responses. High micromolar concentrations of ATP caused two types of contraction, a phasic, cholinergic response and a tonic, tetrodotoxin-resistant contraction. The phasic contraction was reduced by hexamethonium (5x10(-5) M), but left uninfluenced by capsaicin tachyphylaxis or tachyphylaxis to alpha,beta-methylene ATP. The tonic response was resistant to atropine, hexamethonium, capsaicin, omega-conotoxin GVIA, or pretreatment with alpha,beta-methylene ATP. Both types of ATP-induced contraction were diminished or abolished by the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 3x10(-6) and 3x10(-5) M, respectively). In the precontracted, atropine-treated ileum ATP (10(-6)-10(-4) M) caused guanethidine-resistant relaxation. This response was not influenced by tetrodotoxin, omega-conotoxin GVIA, or NG-nitro-L-arginine, but was abolished by apamin (10(-7) M), and inhibited by PPADS (3x10(-5) M) or reactive blue 2 (10(-5) M), in a surmountable manner. A high degree of tachyphylaxis was observed with the relaxant effect of ATP (10(-5)-10(-4) M). A high concentration (3x10(-4) M) of PPADS failed to influence ileum contractions to exogenous acetylcholine or histamine. It is concluded that, in addition to its direct contractile action in the guinea-pig ileum, ATP can activate (partly preganglionic) cholinergic neurones, an effect whose mechanism is largely different from that of alpha,beta-methylene ATP. ATP also causes relaxation by a direct, probably P2Y-receptor-mediated effect on the smooth muscle. All motor effects of ATP are inhibited by the antagonist PPADS.

Thromboxane A 2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity

Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP 1, EP 2, EP 3, DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9α, 11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM–10 μM), a stable thromboxane A 2 mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC 50 value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4( Z)-6-(2- o-Chlorophenyl-4- o-hydroxyphenyl-1,3-dioxan- cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1 S-[1α,2α( Z),3α,4α]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [ 3H]SQ29548 binding site with similar K d values (30 nM), but B max in the circular muscle (90.9±8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2±8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with p K i values in parentheses) was [1 S-[1,2( Z),3(1 E,3 S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2 S-[2α( Z),3β(1 E,3 R*)]]-5-heptenoic acid (CTA 2, 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1 S-[1α,2β( Z),3α(1 E,3 R*),5α]]-5-heptenoic acid (PTA 2, 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1 S-[1α,4α,5α(1 E,3 R*),6β( Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N- tert-butyl- N¢-[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 μM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP 1, EP 2 and EP 3) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A 2-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.

Tachykinin NK1receptor-mediated inhibitory responses in the guinea-pig small intestine

We used in vivo, in vitro studies and immunohistochemistry to elucidate the mechanisms activated by tachykinin NK1receptors in evoking inhibitory motor response in the guinea-pig small intestine. In vivo, the selective NK1receptor agonist GR 73,632 produced a dose-dependent suppression of the distension-induced duodenal contractions, and a decrease of basal tone. These effects were reduced by pretreatment with the NK1receptor antagonist SR 140,333. In L-Nω-nitro-L-arginine methylesther hydrochloride-pretreated animals, the suppressant effect of GR 73,632 on duodenal contractions was reduced, whereas the relaxation of the basal tone was unaffected. In vitro, GR 73,632 evoked a biphasic response consisting of a transient, tetrodotoxin-sensitive inhibitory effect followed by tetrodotoxin-resistant contractions. SR 140,333 blocked both inhibitory and excitatory motor responses induced by GR 73,632. NK1immunoreactivity was localized to myenteric and submucosal neurons and to interstitial cells of Cajal in the deep muscular plexus of the small intestine. NK1receptor-expressing neurons had Dogiel type I morphology and many of them were β-nicotinamide adenine phosphate dinucleotide-diaphorase-positive, indicating they are inhibitory neurons. In conclusion, in the guinea-pig small intestine, NK1receptor stimulation evokes a myogenic excitatory motor response and a neurogenic inhibitory motor response that involves, at least in part, a nitrinergic pathway.

Binding and functional characterization of alpha-2 adrenoceptors in isolated swine myometrium.

1. Alpha-adrenoceptors in the uterus of cyclic mature pigs were investigated for their binding characteristics as shown by a radioligand study employing [3H]-prazosin and [3H]-rauwolscine, and the alpha-adrenoceptors were studied for their functional roles on motility as shown mechanically in vitro. 2. Noradrenaline (10 nM-1 microM) caused a tetrodotoxin-resistant contraction in the cornual longitudinal muscle. The response was decreased by yohimbine (10 nM-1 microM) but not by prazosin (1 microM). 3. Clonidine, xylazine, phenylephrine and methoxamine also elicited a concentration-dependent contraction of the cornual longitudinal muscle. The ranking according to the potency was clonidine > xylazine >> phenylephrine > or = methoxamine. On the other hand, these alpha-adrenoceptor agonists did not elicit contraction of the cornual circular muscle even at 100 microM. 4. Noradrenergic contraction induced by transmural stimulation was significantly decreased by yohimbine (10-100 nM) but not by prazosin (1 microM). 5. The swine myometrium contained both alpha-1 (13%) and alpha-2 (87%) adrenoceptors. The distribution of alpha-adrenoceptors was muscle layer-specific, i.e. the number of both types of adrenoceptors in the longitudinal muscle was four times higher than that in the circular muscle. 6. These results show that although swine myometrium contains both alpha-1 and alpha-2 adrenoceptors, the alpha-2 type is predominant and largely responsible for mediating the excitatory response to both exogenous and endogenous noradrenaline in the cornual longitudinal muscle.

Role of nitric oxide in mediating non-adrenergic non-cholinergic relaxation of the cat ileocecal sphincter

The participation of nitric oxide (NO) in field stimulation- or nicotine-evoked non-adrenergic non-cholinergic (NANC) relaxation of cat ileocecal sphincter was studied in vitro. During a 30 μM noradrenaline-induced contraction, both the application of electrical field stimulation (2–20 Hz, 0.2 ms, supramaximal current intensity, 10 s duration) and (−)-nicotine (10–500 μM) produced a tetrodotoxin-sensitive relaxation. The maximal relaxation was observed at 10 Hz or 100 μM (−)-nicotine. In 12 out of 19 strips the pretreatment with N ω- nitro- L - arginine (100 μM) decreased the amplitude of the field stimulation-evoked relaxation, while in the remaining strips the relaxation was transformed into a contraction. By increasing the concentration of N ω- nitro- L - arginine up to 1 mM all strips responded to field stimulation with a frequency-dependent tetrodotoxin-resistant contraction. N ω- Nitro- L - arginine (100 μM) completely inhibited the nicotine-induced relaxation. L-Arginine (1 mM) restored the amplitude of both field stimulation- and nicotine-evoked relaxations. These data indicate that NO appears to be involved in both field stimulation- and nicotine-evoked NANC relaxations. Evidence has been obtained for the existence of tetrodotoxin-resistant NANC contraction in cat ileocecal sphincter.

Specific receptor for vasoactive intestinal contractor in myenteric cholinergic neurones

The receptor for vasoactive intestinal contractor (VIC) involved in acetylcholine release was examined in the guinea-pig ileum. VIC (10 −9 to 10 −7 M) evoked tritium outflow ileal strips preloaded with [ 3H]choline, in a concentration-dependent manner. The evoked tritium outflow was Ca 2+-dependent and tetrodotoxin-sensitive, suggesting that VIC acts on somato-dendritic regions of the cholinergic neurones to stimulate acetylcholine release. The response to VIC did not change after desensitization to either endothelin-1, endothelin-2 or endothelin-3, hence there is probably no cross-desensitization in evoked tritium outflow between VIC and endothelins. VIC-induced contractions consisted tetrodotoxin-sensitive and -resistant contractions, and the tetrodotoxin-resistant contraction was abolished after desensitization to either endothelin-1, endothelin-2 or endothelin-3. These results indicate that the VIC-specific receptor is located on cholinergic neurones, but not on the smooth muscle cells of guinea-pig ileum.

Non-adrenergic, non-cholinergic regulation of guinea-pig airway smooth muscle-indomethacin-induced changes and segmental differences

Several lines of evidence suggest a regulatory role for the non-adrenergic, non-cholinergic (NANC) nervous system in the airways of the guinea-pig and man. We examined NANC frequency-response characteristics (0.2–90 Hz) with respect to segmental differences and effects of cyclooxygenase-inhibition (indomethacin 10 μM). Furthermore, the neurogenic contribution to the contractile and the relaxatory NANC response was quantified with tetrodotoxin (1–10 μM). Frontally opened guinea-pig airway rings (n = 3–7) were used. NANC responses were obtained by electrical field stimulation (0.5 ms; 1200 mA; 240 s). A relaxatory NANC response was demonstrated in the proximal and the distal trachea. A contractile NANC response was demonstrated in the distal trachea and the main bronchus. Indomethacin lowered the baseline tension, decreased the relaxatory and increased the contractile NANC response (p < 0.05; at 0.8 Hz; n = 4). A relationship between the baseline tension and the NANC response is suggested. The relaxatory NANC response was tetrodotoxin-sensitive at 3 Hz stimulation frequency (p < 0.05; n = 3). At this frequency, the contractile NANC response was also mainly tetrodotoxin-sensitive (p < 0.05; n = 6) whereas at 60 Hz a dominant, tetrodotoxin-resistant contraction was demonstrated. The pronounced frequency-response relationship as well as the tetrodotoxin sensitivity at very low frequencies (0.2–1.6 Hz) indicates that tetrodotoxin-sensitive NANC nerves are stimulated optimally at low impulse frequencies.

4-Aminopyridine induces the release of neuropeptide Y (NPY) to produce an atropine- and tetrodotoxin-resistant contraction in rabbit isolated jejunum

4-Aminopyridine (4-AP) induced an atropine- and tetrodotoxin (TTX)-insensitive contraction (resistant contraction), in a concentration-dependent manner, in the isolated jejunum of rabbits. The failure of specific antagonists of histamine, serotonin and substance P to affect this resistant contraction ruled out the participation of histamine, serotonin and/or substance P. Antiserum against neuropeptide Y (NPY) reduced this resistant contraction in a concentration-dependent manner and inhibited the action of 4-AP totally at a high concentration (1.25% dilution) whereas normal serum lacked this ability. This suggested that the release of NPY was involved in this 4-AP-induced resistant contraction. Radioimmunoassay of NPY-like immunoreactivity in isolated synaptosomal preparations indicated that 4-AP possessed the ability to induce the release of NPY. However, guanethidine did not affect the actions of 4-AP, indicating that NPY is released mainly from non-adrenergic nerves. Our results indicate that 4-AP induces the release of NPY from non-adrenergic nerves to produce an atropine- and TTX-resistant contraction in the isolated jejunum of rabbits.

Further studies on the motor response of the human isolated urinary bladder to tachykinins, capsaicin and electrical field stimulation

1. 1. Muscle strips from the dome of the human urinary bladder responded to field stimulation with contractions which were atropine- (3 μM) and tetrodotoxin- (1 μM) sensitive. These contractions were sensitive to omega conotoxin (CTX, 0.1 μM). The atropine- and tetrodotoxin-resistant contractions produced by field stimulation were totally unaffected by CTX. 2. 2. DMPP (30–100 μM), a nicotinic agonist, produced transient bladder contractions which were hexamethonium- and atropine-sensitive. 3. 3. Tachykinins produced a contraction of the human bladder. Among several synthetic tachykinin analogs only those having activity at the NK-2 receptor produced a consistent contractile response. 4. 4. Either capsaicin (1 μM) or calcitonin gene-related peptide (10 nM−0.1 μM) had no motor effect. At 10 μM, capsaicin exerted a depressant effect on nerve-mediated contractions but this effect did not exhibit desensitization. 5. 5. These findings provide evidence that NK-2 receptors are the main if not the sole mediators of the contractile response of the muscle from the dome of the human isolated bladder to tachykinins. 6. 6. No evidence was found for a tachykininergic component in the excitatory response to field stimulation nor for motor responses mediated by capsaicin-sensitive nerves. 7. 7. CTX-sensitive calcium channels are probably present on cholinergic nerve terminals in the human bladder muscle.

The effect of omega conotoxin GVIA, a peptide modulator of the N-type voltage sensitive calcium channels, on motor responses produced by activation of efferent and sensory nerves in mammalian smooth muscle

1. The effect of omega-conotoxin (CTX) GVIA, a peptide which blocks neuronal calcium channels, were investigated on nerve-mediated motor responses in a variety of isolated smooth muscle preparations from rats and guinea-pigs. 2. In the rat or guinea-pig isolated vas deferens CTX (1 nM-1 microM) produced a concentration and time-related inhibition of the response to field stimulation, while the responses to KCl, noradrenaline or adenosine triphosphate were unaffected. In the presence of CTX a series of tetrodotoxin-resistant contractions could be elicited by field stimulation by increasing pulse width and/or voltage. 3. In the rat or guinea-pig isolated urinary bladder, CTX produced a concentration and time-dependent inhibition of twitch responses to field stimulation without affecting the response to exogenous acetylcholine. In the rat bladder the maximal effect did not exceed 25% inhibition while a much larger fraction of the response (about 70%) was inhibited in the guinea-pig bladder. The CTX-resistant response was abolished, in both tissues, by tetrodotoxin. 4. The effects of CTX in the rat bladder were also studied with a whole range of frequencies of field stimulation (0.1-50 Hz). Maximal inhibition was observed toward contractions elicited at frequencies of 2-5 Hz. At low frequencies the inhibitory effects of CTX and atropine were almost additive while at high frequencies of stimulation a large component of the atropine-sensitive response was CTX-resistant. 5. In the rat isolated proximal duodenum, field stimulation in the presence of atropine and guanethidine produced a primary relaxation followed by a rebound contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure to calcium-free medium protects sensory fibers by capsaicin desensitization

Capsaicin (1 μM) produces a tetrodotoxin-resistant contraction of the rat isolated urinary bladder ascribable to neuropeptide release from sensory nerves. A second application of capsaicin (1–10 μM) up to 5 h from the first one was ineffective, indicating complete desensitization. However, if the first exposure to capsaicin was made after a prolonged incubation in a Ca-free medium containing EDTA (0.1 mM), a second application of capsaicin (1 μM) was still able to induce a contraction, thus indicating protection from desensitization.

Pharmacological evidence for the possible coexistence of multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle

Contractile responses to neurokinin alpha and neurokinin beta were characterized and compared with those to substance P (a SP-P agonist) and eledoisin (a SP-E agonist) in isolated rabbit iris sphincter. Neurokinin alpha and neurokinin beta as well as substance P and eledoisin produced atropine- and tetrodotoxin-resistant contractions of the iris sphincter in nanomolar concentrations, and the rank order of sensitivity was eledoisin greater than substance P = neurokinin alpha = neurokinin beta. After prolonged cold-storage of the preparations, responses to capsaicin, a releaser of tachykinins from sensory nerve endings, were nearly absent, but responses of considerable magnitude to carbachol and the tachykinins persisted. On wash-out of the tachykinins, responses faded at characteristic rates (neurokinin alpha greater than eledoisin greater than neurokinin beta much greater than substance P). From the Schild analyses, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-substance P, a potent substance P antagonist, competitively antagonized the response to substance P, had no significant effect on the response to neurokinin beta, and antagonized the response to neurokinin alpha and eledoisin in a more complex manner. Taken together, these results suggest that there coexist multiple receptor sites for mammalian tachykinins in rabbit iris sphincter smooth muscle.