Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD₄ > LTC₄ > K⁺ > histamine > acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca²⁺-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca²⁺-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca²⁺-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca²⁺) the Ca²⁺ entry blockers nifedipine (N) ? D-600 > verapamil (V) > diltiazem (D) almost completely abolished the contractions to K⁺ but blocked only a component of the maximum response to the other agonists. After exposure to Ca²⁺-free Krebs' solution for 45 minutes, any residual contractions to LTC₄ & LTD₄, were reversed by low concentrations of N (0.3 μM) or D-600 (2.1 μM). Leukotrienes appear to mobilize a superficial and a bound store of Ca²⁺ which gains entry through at least two types of Ca²⁺ channels (or mechanisms), one of which is blocked by N and D600. K⁺-induced contractions appear to be dependent on superficial and tightly bound Ca²⁺ but entry is solely through channels which are blocked by the Ca²⁺ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca²⁺ free Krebs' solution but contractile activity was virtually abolished in Ca²⁺ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca²⁺-free Krebs' solution were blocked by low dose N (0.3μM) or D600 (2.1 μM). These findings suggest a major role for extracellular Ca²⁺ during spasmogen-induced contraction in this tissue.     

Characterization of alpha1-adrenoceptor subtypes mediating noradrenaline-induced contraction of rat epididymal vas deferens in calcium-free medium.

The alpha1-adrenoceptor subtype mediating noradrenaline (NA)-induced contractions of rat epididymal vas deferens in Ca2+-free/EGTA (1 mM) medium was studied using competitive antagonists. The effects of chloroethylclonidine (CEC) was investigated in Ca2+-free and normal Krebs' medium and RT-PCR was used to identify alpha1-adrenoceptor specific mRNA in epididymal vas deferens. In Ca2+-free medium, NA evoked sustained contractions but was less potent (pD2, 5.9) than in normal Krebs' medium (pD2, 7.3). The contractions in Ca2+-free medium were inhibited by prazosin (pA2, 9.3), 5-methylurapidil (pA2, 8.4), spiperone (pA2, 7.6) and BMY 7378 (pK(B), 6.8) consistent with activation of alpha1A-subtype. Repeated pretreatment with CEC (100 microM) reduced the potency of NA and maximum contractions in normal and Ca2+-free media. CEC-sensitivity in normal Krebs' medium was enhanced by prior treatment with phenoxybenzamine. mRNA for alpha1a- and alpha1d- but not alpha1b-adrenoceptors were detected in epididymal vas deferens. These results suggest that NA contracts the tissue in Ca2+-free medium by the stimulation of alpha1A-adrenoceptors. Two factors affecting CEC-sensitivity of NA-induced contractions in this tissue are discussed.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Histaminergic effect of crude papaya latex on isolated guinea pig ileal strips.

In the present study, we investigated the effect of the crude latex of Carica papaya L. (CPX) on isolated guinea pig ileal strips. CPX (0.5-512 microg/ml) caused concentration-dependent contraction of ileal strips suspended in Tyrode solution. The concentration of atropine (0.69 microM) that significantly blocked the contractile effect of acetylcholine on the isolated guinea pig ileum showed no significant effect on CPX- and histamine-induced contractions of the ileal strips. Mepyramine (87.6 nM) significantly blocked the contractile effect of histamine and CPX on the ileum. The same concentration of mepyramine, however, had no significant effect on acetylcholine-induced contraction of the isolated ileal strips. Removal of Ca2+ from the bathing medium abolished ileal contractions induced by acetylcholine, histamine and CPX. All the test substances were able to provoke ileal contractions after replacement of the Ca(2+)-free solution with Tyrode solution. Furthermore, 10(-5) M of nifedipine, a Ca(2+)-entry antagonist, reversibly inhibited the contractile effect of all the test substances on the ileal strips. Results of this study together appear to show that CPX-induced contraction of the isolated guinea pig ileum is mediated via H1-receptors and dependent on extracellular Ca2+ influx.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

Calcium pools mobilized during excitation-contraction coupling in flounder intestinal smooth muscle

The effects of Ca2+ free solutions (0 mM Ca2+/5mM EGTA) and low Ca2+ media (no added Ca2+) on the 100 mM K+ and 10(-5) M ACh contractions of the flounder intestine were examined. Ca2+-free solutions abolished the K+-contractions and reduced the normal ACh response to a smaller transient event. Low Ca2+ media blocked the prolonged tonic phase of the K+-contractions more readily than the initial phasic component. It was concluded that both ACh and K+ stimulate Ca2+ entry into the cell and that the phasic component of the K+-contraction relies on a more tightly bound extracellular Ca2+-fraction than that utilized during the tonic phase. ACh can also mobilize an intracellular Ca2+-store. Ultrastructural studies suggested that this ACh-releasable intracellular Ca2+-store may reside on the inner surface of the plasma membrane or within the peripheral S.R.     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Subcellular mechanism of desensitization of the ATP-induced Ca2+ mobilization in human umbilical vein endothelial cells: role of intracellular Ca2+ stores

Confluent monolayers of human umbilical vein endothelial cells subcultured on glass coverslips were loaded with the fluorescent Ca2+ indicator, fura-2. Changes in fura-2 fluorescence were detected by means of a fluorescence spectrophotometer. Both ATP and ADP (0.3-100 microM) caused a concentration-dependent transient peak response of the intracellular free calcium concentration ([Ca2+]i), followed by a lower sustained response. AMP and adenosine did not induce detectable changes in [Ca2+]i. The sustained response to ATP was abolished by superfusion with the Ca2(+)-free solution (with 1 mM EGTA), while the transient peak response was uninfluenced. The transient peak response to ATP (30 microM) was inhibited by pre-exposure to ATP in a graded manner depending on the concentration of ATP. The response to ATP recovered after washout for 20 min with the solution containing Ca2+, but not with the Ca2(+)-free solution. The transient peak response to ATP was markedly reduced by preceding exposure to histamine, while the response to histamine was not influenced by pre-exposure to ATP. These findings indicate that depletion and refilling of the ATP-sensitive intracellular Ca2+ store may be responsible for the desensitization and recovery of the ATP-induced [Ca2+]i response. The pharmacological characteristics of the ATP-sensitive intracellular Ca2+ store seem different from those of the histamine-sensitive store.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonism of leukotriene (LT) C4-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cyclooxygenase inhibitor indomethacin. NDGA (30 microM) as well as indomethacin (5 microM) inhibited LTC4-induced contractions. But, in the presence of indomethacin NDGA was ineffective to inhibit the LTC4 response, whereas two other lipoxygenase inhibitors, phenidone (3-30 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM), markedly inhibited it. The antagonist action of an LTD4 receptor antagonist FPL55712 against LTC4-induced contractions was significantly reduced by NDGA (10-30 microM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect on the LTC4 antagonism in the presence of indomethacin, but 0.3 microM phenidone and 1 microM ETYA which did not inhibit the LTC4 response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC4, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LTC4 response was not inhibited by indomethacin, 0.3 microM phenidone and 1 microM ETYA. In the presence of a gamma-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC4 antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 microM histamine- and 30 microM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H1-blocker pyrilamine. These results suggest that some putative non-prostanoids are involved in LTC4-induced contractions of guinea-pig trachea and which regulate the effects of LTD4 antagonism and beta-adrenoceptor activation.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement

The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Antagonistic effect of KC-404, a new anti-asthmatic agent, on leukotriene D4-induced contractile responses in isolated guinea pig smooth muscles

The inhibitory effects of KC-404, a novel clinically available anti-asthmatic drug, on leukotriene(LT) D4-, LTC4-, histamine- and acetylcholine(ACh)-induced contractile responses in isolated guinea pig lung parenchymal, tracheal and ileal longitudinal strips were compared using an organ bath system. In lung parenchyma, KC-404 antagonized LTD4 in a competitive fashion, whereas it antagonized histamine noncompetitively. The pA2 value against LTD4 was 7.39. KC-404 hardly antagonized LTC4 and ACh. A ranked order of potency estimated from its minimum effective concentrations (MEC) was LTD4 greater than histamine greater than LTC4 greater than ACh. In trachea, KC-404 antagonized LTC4 and LTD4 in a competitive fashion, while it antagonized histamine noncompetitively. The pA2 values against LTC4 and LTD4 were 5.99 and 6.51, respectively. KC-404 hardly antagonized ACh. A ranked order of the potency estimated from MEC was LTD4 greater than LTC4 greater than histamine greater than ACh. The pA2 values of KC-404 against LTD4 in lung parenchyma and trachea were little or not altered, while its inhibitory effect on histamine-induced contraction in trachea was markedly diminished by the pretreatment of tissues with indomethacin. In ileum, KC-404 noncompetitively antagonized all of the agonists used. A ranked order of the potency estimated from pD2 values was LTD4 divided by LTC4 greater than histamine greater than ACh. These results suggest that KC-404 is a selective antagonist of LTD4 and that it might interact with LTD4 receptor in airway smooth muscles but not in ileum. Another possibility that the drug might interact with LTD4 specific excitation-contraction coupling mechanism was also discussed.     

Cholinergic innervation to the upper esophageal sphincter muscle in the eel,with special reference to drinking behavior

To elucidate innervation in the upper esophageal sphincter (UES) muscle of the eel, a key muscle in swallowing, repetitive electrical field stimulation (EFS; 30 mA, 40 V, 300 micros, 10 Hz, 10 trains) was employed. Anatomically, the eel UES muscle consists of striated fibers. The EFS-induced contraction of the UES was completely blocked by tetrodotoxin and curare, and abolished in Ca2+ -free Ringer solution. These results suggest that the EFS stimulates nerve fibers specifically and releases acetylcholine as a neurotransmitter. In fact, acetylcholine and carbachol constricted the UES in a concentration-dependent manner. Even after blocking neuronal firing with tetrodotoxin, acetylcholine constricted the UES muscle, suggesting the existence of acetylcholine receptors on the UES muscle cells. Both EFS- and carbachol-evoked contractions of the UES were blocked by curare at a lower concentration than by atropine or hexamethonium, suggesting that the acetylcholine receptor is nicotinic. Even in Ca2+ -free Ringer solution, a direct current stimulus (2 s duration) constricted the UES muscle to an extent similar to that in the presence of Ca2+, indicating that the muscle contraction itself does not need extracellular Ca2+, i.e., the muscle can be constricted by a release of Ca2+ from the sarcoplasmic reticulum.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.     

LTD4 induces hyperresponsiveness to histamine in bovine airway smooth muscle: role of SR-ATPase Ca2+ pump and tyrosine kinase

Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D(4) (LTD(4)) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD(4), but not leukotriene C(4) (LTC(4)), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC(4) nor LTD(4) modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca(2+) ([Ca(2+)](i)) and contraction, histamine or carbachol produced a transient Ca(2+) peak followed by a plateau, along with a contraction. LTD(4) increased the histamine-induced transient Ca(2+) peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD(4) were not modified by staurosporine or chelerythrine but were abolished by genistein. Western blot showed that carbachol, but not histamine, caused intense phosphorylation of extracellular signal-regulated kinase 1/2 and that LTD(4) significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca(2+) channel participation in the hyperresponsiveness to histamine was discarded because LTD(4) did not modify the [Ca(2+)](i) changes induced by KCl. In tracheal myocytes, LTD(4) enhanced the transient Ca(2+) peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca(2+) refilling. Genistein abolished this last LTD(4) effect. Partial blockade of the SR-ATPase Ca(2+) pump with cyclopiazonic acid reduced the Ca(2+) transient peak induced by histamine but not by carbachol. These results suggested that LTD(4) induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca(2+) pump activity. L-type Ca(2+) channels seemed not to be involved in this phenomenon.