Metabolic dependency of ionophore A23187-induced contraction of ileal longitudinal smooth muscle

1. Tonic contraction in response to K+ is well known to be highly dependent on aerobic metabolism in ileal muscle. The ionophore A23187 (10(-5) M) induced an initial contraction and successive rhythmic contraction in ileal muscle, but did not induce tonic contraction. This study, therefore, was performed to investigate the metabolic dependency during contraction induced by A23187. 2. Under hypoxic conditions, A23187 (10(-5) M) induced an initial contraction accompanied by an increase in lactate release. However, it induced only small rhythmic contractions and decreases in ATP, phosphocreatinine (PCr) and glycogen contents. 3. In glucose-free medium, A23187 (10(-5) M) induced an initial contraction and concomitant significant decrease in the ATP and glycogen contents. However, it did not induce successive rhythmic contractions. In 'glycogen-depleted muscle' stimulated repeatedly with 60 mM K+ in glucose-free medium, 60 mM K+ induced a phasic contraction without tonic contraction. A23187 induced no contraction under these conditions. 4. These results suggested that the initial contraction induced by A23187 was dependent on endogenous glycogen, while successive rhythmic contractions were dependent on aerobic metabolism after supply of external glucose to the ileal muscle.     

Cooling-induced contraction in ileal longitudinal smooth muscle of guinea-pig

The ileal longitudinal smooth muscle developed a transient contraction on cooling from 37 degrees C to 1 degree C in normal Ca2+ (2.5 mM) medium. The transient contraction was not inhibited by pretreatment with the Ca2+ antagonist, D-600 (1 X 10(-6)M). The contractions were sustained by cooling to 1 degree C in the presence of added Ca2+ greater than 10 mM. After the pretreatment with D-600, when the muscle incubated in normal medium with added 20 mM Ca2+ had been cooled to 1 degree C, a phasic response was only seen. However, D-600 did not inhibit the sustained contraction at 1 degree C after incubation in the presence of added 20 mM Ca2+. It is suggested that the transient and sustained contraction at 1 degree C is maintained by Ca2+ release from a cellular site, probably the cell membrane and it requires more calcium for the sustained tension.     

Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle

1 In guinea-pig ileal longitudinal muscle, muscarinic partial agonists, 4-(N-[3-chlorophenyl]-carbomoyloxy)-2-butynyl-trimethylammonium (McN-A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca(2+) concentration ([Ca(2+)]c) with a higher EC(50) than that of the full agonist carbachol. The maximum response of [Ca(2+)]c or tension was not much different among the three agonists. The Ca(2+) channel blocker nicardipine markedly inhibited the effects of all three agonists 2 The contractile response to any agonist was antagonized in a competitive manner by M(2) receptor selective antagonists (N,N'-bis[6-[[(2-methoyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride and 11-[[2-[(diethlamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one), and the apparent order of M(2) antagonist sensitivity was McN-A343>pilocarpine>carbachol. M(3) receptor selective antagonists, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN-A343, while darifenacin had a similar action in the case of pilocarpine. Both M(3) antagonists behaved in a competitive manner in the case of the carbachol response. 3 McN-A343 failed to release Ca(2+) from the intracellular stores, and the Ca(2+)-releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca(2+) sensitivity of the contractile proteins. 4 McN-A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. 5 The results suggest that muscarinic agonist-evoked contractions result primarily from the integration of Ca(2+) entry associated with the increased spike discharge and myofilaments Ca(2+) sensitization, and that Ca(2+) store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M(2) and M(3) receptors plays a crucial role in mediating the contraction response.     

Characteristics of 5-hydroxytryptamine receptors involved in contraction of feline ileal longitudinal smooth muscle

A number of studies have demonstrated that 5-hydroxytryptamine (5-HT) can induce muscle contraction or relaxation response and enhance secretion in the gastrointestinal tract via a multiplicity of 5-HT receptor subtypes. In the present study, we investigated the pharmacological characterization of the 5-HT-induced contractile response in longitudinal smooth muscle isolated from the feline ileum. Addition of 5-HT into muscle chambers enhanced the basal tone and spontaneous activity in a concentration-dependent manner. The neurotoxin tetrodotoxin did not alter the 5-HT-induced contraction of the longitudinal muscles. Neither atropine nor guanethidine affected the contraction. The 5-HT agonists, 5-methylserotonin hydrochloride and mosapride, also evoked concentration-dependent contractions. The 5-HT-induced contraction was enhanced by the 5HT(2) receptor antagonist ketanserin and the 5-HT(3) receptor antagonist ondansetron but was inhibited by the 5-HT(1) receptor antagonist methysergide and 5-HT(4) receptor antagonist GR113808. These results indicate that 5-HT(1) and 5-HT(4) receptors may mediate the contraction of the 5-HT-induced response and 5-HT(2) and 5-HT(3) receptors may mediate 5-HT-induced relaxation in feline ileal longitudinal smooth muscles.     

Mechanism of cadmium-induced contraction in ileal longitudinal muscle of guinea-pig.

1 The mechanism of cadmium (Cd2+)-induced contraction was studied in isolated ileal longitudinal muscle of guinea-pig. 2 CdCl2 (1x10(-8) to 1x10-4M)) caused a transient contraction which subsided within approximately 6 min of application. The contraction was reproducible and dependent on the concentration. The dose-response curve was bell-shaped. A maximal response was observed at concentrations of 5x10(-6) to 1x10(-5) M. 3 The contractile effect was inhibited to some degree at 20 degree C or by tetrodotoxin (0.1 microgram/ml), hyoscine (o.1 microgram/ml), but completely inhibited by Ca2+ -removal from the medium. 4 Cd2+ increased the output of [14C]-acetylcholine biosynthesized from [14C] by the preparation depending on the concentration. The increase terminated within the first 6 min and was reduced by tetrodotoxin (0.1 microgram/ml) or by removal of Ca2+ from the medium. 5 Both the contractile and transmitter releasing effects of Cd2+ were dependent on the concentration of external Ca2+. Strontium ions were able to replace Ca2+ -induced transmitter release. 6 It is suggested that Cd2+ contracts ileal longitudinal muscle through a release of cholinergic transmitter from the parasympathetic nerve terminals, which is dependent on external Ca2+. It also has a smaller hyoscine-resistant contractile effect, presumably due to a direct action on smooth muscle cells.     

Differential inhibitory mechanism of Fe2+ and Fe3+ on contraction of ileal longitudinal smooth muscle.

The mechanism of the inhibition of K(+)-induced contraction caused by ferrous (Fe(2+)) and ferric (Fe(3+)) ions were analysed in guinea-pig ileal longitudinal muscle and taenia coli. Fe(2+)increased the threshold for Ca(2+)-induced contraction in Ca(2+)-free, K(+)-depolarized taenia coli. However, Fe(3+)reduced the size of the maximal response to Ca(2+)without shifting the dose-response curves in taenia coli. Both 10 mM Fe(2+)and 2 mM Fe(3+)caused significant decreases in Ca uptake, as determined by the La method, during K(+)-induced ileal contraction. After treatment with 10 mM Fe(2+)in a state of cell membrane depolarization with K(+)for 30 min, the ileal K(+)-induced tonic contraction was completely restored by washing with medium containing EDTA, a chelator of divalent cations, and Fe(2+)remaining in muscle was almost eliminated by washing. In contrast, after treatment with 2 mM Fe(3+)in K(+)medium, K(+)-induced contraction was reversed only to a slight degree by washing with medium containing deferoxamine, a chelator of trivalent cations, and Fe(3+)in muscle largely remained despite the washing. These results suggest that Fe(2+)binds to the ileal surface membrane and reduces the contraction in response to K(+)mainly by inhibiting Ca(2+)influx. Fe(3+)may exert an inhibitory action on intracellular sites, in addition to the interference of Ca(2+)influx at the cell membrane.     

Species differences in inhibitory effect of Li+ on high K+-induced contraction in the ileal longitudinal smooth muscle

The effects of Li+ substitution for external Na+ in the medium on a 60 mN KCl (60K+)-induced contraction were compared in the ileal longitudinal smooth muscles isolated from monkey, rabbit, guinea pig and rat. Li+ substitution inhibited the 60K+-induced contraction in the ileum of all the animal species. Greater inhibitory effect was observed with the increase in external Li+ concentration and the increase in incubation period with Li+. Intracellular Li+ contents of these muscles also increased with the increase in external Li+ concentration and the incubation period. Thus, a good correlation was obtained between Li+-induced inhibition of 60K+-induced contraction and the increase in Li+ content. However, close examination revealed that the contraction in monkey and rat ileum was inhibited by much lower Li+ accumulation than that in guinea pig and rabbit ileum, suggesting the existence of species difference in the sensitivity of the ileal smooth muscle to the inhibitory effect of Li+ substitution, and a mechanism for the species differences in the inhibitory action of Li+-substitution was discussed.     

Palytoxin-induced K+ efflux from ileal longitudinal smooth muscle of the guinea-pig

In guinea-pig ileal longitudinal smooth muscle, both palytoxin (PTX) and carbachol (CCh) increased K+ efflux with an EC50 of 1.8 X 10(-10) M and 4.1 X 10(-7) M, respectively. Atropine (10(-6) M) did not inhibit the K+ efflux due to PTX (3 X 10(-9) M), but completely inhibited the efflux due to CCh (10(-5) M). External Ca2+ removal and verapamil (10(-5) M) did not change the PTX-induced K+ efflux, although the CCh-induced K+ efflux was inhibited about 77% and 71%, respectively. PTX-induced K+ efflux was reduced to 31% by a depletion of intracellular Ca2+. Tetraethylammonium (15 mM) inhibited the K+ efflux due to PTX or CCh to 61% or 75%, respectively. The PTX-induced K+ efflux was also inhibited by cymarin (3 X 10(-8) M), ouabain (10(-5) M) and digitoxin (10(-5) M). These results suggest that the PTX-induced K+ efflux is less dependent on Ca2+ influx than that due to CCh. Furthermore, the binding sites for PTX in the ileal muscle of guinea-pig may be Na+, K+-ATPase, as has been suggested in other types of cells.     

Temperature high sensitivity of managanese uptake in ileal longitudinal smooth muscle of guinea-pig

1. Mn²⁺ (5 mM) inhibited completely the K⁺ (60 mM)-induced ileal tonic tension to the base line, however, the tension increased progressively to the above level of the original K⁺ tonic response after 3 hr of the Mn²⁺ application at 37°C.2. At 30, 32 or 34°C, the tensions which developed after 3 hr of the addition of 5 mM Mn²⁺ in the high-K⁺ medium was 0, 21, 48% of their original K⁺ tonic levels, respectively.3. The tension development and manganese uptake in the presence of Mn²⁺ in the high-K⁺ medium was highly dependent on the temperature in very narrow range of 32–37°C in the suspending medium.     

Changes in K(+)-induced contractions in ileal longitudinal smooth muscle of guinea-pig, induced by monoiodoacetic acid.

The ileal responses to 40 mM [K+]o were changed in the period after administration of monoiodoacetic acid (IAA). The phasic contraction to K+ appeared in the period before the development of a rigor, after the administration of 1 mM IAA. However, the response to K+ was absent during the development of a rigor to IAA during decreasing tissue ATP concentrations.     

Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release.

Neurotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig ileum, but was only 22% as effective as histamine. Neostigmine (0.1 micrometer) increased the effectiveness of neurotensin 4-fold. Atropine (0.1 micrometer) completely inhibited the contracting effect of neurotensin measured in the presence of neostigmine, whereas hexamethonium (0.1 micrometer) was without effect. It is concluded that in the longitudinal smooth muscle of the guinea-pig ileum, the contracting effect of neurotensin is mediated by the release of acetylcholine from nerve endings in response to a postganglionic stimulation of cholinergic nerve fibers.     

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.     

Transmembrane sodium and potassium gradients modulate histamine secretion induced by ionophore A23187

Histamine secretion was induced from rat peritoneal mast cells by calcium ionophore A23187 in the presence of various extracellular calcium concentrations. Transmembrane sodium and potassium gradients were altered by cold pretreatment of mast cells or through the inhibition of sodium-potassium ATPase by the use of ouabain or potassium-deprivation. Such pretreatments led to a parallel shift to the left of the extracellular calcium concentration-histamine secretion curve, i.e. to an apparent decrease of extracellular calcium requirement for the ionophore-induced histamine release. These effects were fully reversed by warming mast cells, by washing out ouabain or by adding potassium. Metabolic inhibition of mast cells prevented the ionophore-induced secretion in all the experimental conditions described. Secretion observed in the absence of added calcium was inhibited by short term treatment of cells with 5 X 10(-6) M EGTA or EDTA provided magnesium was absent from the assay medium. Data show that ionophore A23187 was able to induce secretion in the presence of micromolar concentrations of extracellular calcium, when the efficiency of the ionophore was not decreased by extracellular magnesium and when transmembrane sodium and potassium gradients were altered.     

Neurobehavioral effects of the calcium ionophore A23187

The divalent cationic ionophore A23187 (calimycin) facilitates the transport of calcium ions across biological membranes, resulting in an increase of cytosolic calcium. A23187 has been used extensively in vitro to activate calcium-dependent neurocellular processes. Because of its potential usefulness as a neurotoxicological probe, our laboratory conducted a series of studies to characterize the neurofunctional consequences of A23187 in the intact organism. In addition to approximating the LD50, the effects of acute parenteral administration of A23187 on conditioned avoidance, nociceptive shock threshold, open-field activity, consummatory behavior, body temperature and neuromotor function, including general activity, coordination, balance and grip strength, were assessed in the rodent. The LD50 of A23187, administered intraperitoneally to adult male rats, was 9.2 mg/kg. The predominant overt signs of toxicity included lethargy, limb weakness and apnea. Lower doses, from 0.5 to 0.03 mg/kg, produced a variety of more subtle neurobehavioral effects, including a selective depression of motor activity, a moderate elevation of shock threshold, altered conditioned avoidance behavior and hypothermia.     

Vanadate activates Rho A translocation in association with contracting effects in ileal longitudinal smooth muscle of guinea pig

We characterized the effects of vanadate, an inhibitor of tyrosine phosphatase, on the tension, the level of myosin light chain (MLC) phosphorylation, and Rho A activation in intact ileal longitudinal smooth muscle of the guinea pig to study the role of tyrosine phosphorylation in contraction signaling. Vanadate exerted a sustained contraction with a slow onset of tension development, in a concentration-dependent manner. The contractile effects of vanadate were accompanied by increases in the level of MLC phosphorylation. The tyrosine kinase inhibitor genistein; the MLC kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9); and the Rho kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632) inhibited the vanadate-induced contraction and MLC phosphorylation. Vanadate caused Rho A translocation from the cytosol to the membrane fraction, which was inhibited by genistein, but not by ML-9 and Y-27632. These data indicate that vanadate induces Rho A activation probably via protein tyrosine phosphorylation and the subsequent contraction through increases in the level of MLC phosphorylation.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1.

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

1. The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.
2. α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED₅₀ values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.
3. Although the contraction caused by α,β-mATP was strongly diminished by Ca²⁺-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.
4. UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.
5. The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.
6. In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.
7. These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release.

     

Experimental investigations on the contraction induced by lead in arterial smooth muscle

Previous observations suggested that the turnover of tissue calcium might be involved in the mechanism of smooth muscle contraction induced by lead.In the present investigations, the effect of lead on calcium exchangeability has been studied in the isolated rat tail artery. Experimental results suggest that the mechanism of lead action might be identified with a tissue calcium accumulation and with a lead-to-calcium competition. Evidence exists that the site of action is located in the cell membrane, where lead inhibits the processes of calcium extrusion, and in the intracellular calcium stores, whose calcium binding capacity is lowered; both processes induce an increase of the cellular exchangeable calcium available for contraction.