Effects of verapamil on activation of arachidonic acid metabolism in guinea-pig lungs

The effects of (±)-verapamil and its optical isomers on the activation of arachidonic acid (AA) in guinea-pig isolated perfused lungs were investigated. The calcium ionophore A23187 (6–15 nmol), histamine (20–50 nmol) and leukotriene E₄ (1–2 nmol) induced the release of thromboxane A₂ (TxA₂), which was detected by bioassay and by radioimmunoassay of the stable metabolite TxB₂. Racemic (±)-verapamil (0.4–40 μM) caused concentration-dependent inhibition of A23187-induced release of TxA₂ without affecting the conversion of exogenous AA to TxA₂. Both (+)-verapamil and (−)-verapamil (1–10 μM) caused concentration-dependent inhibition of histamine-induced release of TxA₂. In contrast, racemic (±)-verapamil did not inhibit leukotriene E₄ (LTE₄)-induced release of TxB₂. Calcium depletion (with 2 mM ethylenediamine tetra acetate) significantly reduced both histamine-induced release of TxB₂ from 8.6 ± 2.6 to 1.8 ± 0.8 ng/min (P < 0.05) and LTE₄-induced release of TxB₂ from 4.9 ± 0.9 to 0.5 ± 0.2 ng/min (mean±S.E.M.)(P<0.05). Since histamine stimulates phospholipase A₂ in guinea-pig lungs, these results suggest that (±)-verapamil inhibits phospholipase A₂ and that A23187 activates AA metabolism by stimulating phospholipase A₂. Although all three agents activate AA metabolism by calcium-dependent processes, LTE₄ may stimulate calcium entry via separate mechanisms because it is not inhibited by (±)-verapamil.     

Adenosine modulation of non-adrenergic non-cholinergic neurotransmission in isolated guinea-pig atria

Summary Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1–10 M) concentration-dependently reduced the cardic response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 M 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 M) and by adenosine deaminase (1 g/ml), but was reduced by dipyridamole (1 M). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.Send offprint requests to A. Rubino at the above address     

Beta-adrenoceptors invloved in inhibition of histamine release from sensitized guinea-pig lung.

Selective β-adrenoceptor agonists and antagonists were used to study the type of β-adrenoceptors involved in inhibiting antigen-induced histamine release from actively sensitized guinea-pig lung. Results obtained with six non-catechol β-adrenergic agonists were compared with those found in guinea-pig atrial (β₁) and tracheal (β₂) preparations. In terms of rank order the relative activities of the compounds differed in the three preparations. Dissociation constants (K_B values) for the cardioselective antagonist H93/26 were assessed using (?)-isoprenaline as an agonist. The K_B value for inhibition of histamine release was significantly different from, and intermediate between, the K_B values obtained in atria and trachea. In the guinea-pig tissues H35/25 was not a selective β-adrenoceptor antagonist; K_B values were not significantly different in the three preparations. The results using the β-adrenoceptor agonists and antagonists suggest that the β-receptors involved in inhibition of antigen-induced histamine release in the guinea-pig lung differ from those found in guinea-pig atria and trachea.     

Adenosine receptors involved in the inhibitory control of non-adrenergic non-cholinergic neurotransmission in guinea-pig atria belong to the A1 subtype

Summary We have previously shown that endogenous adenosine inhibits non-adrenergic, non-cholinergic (NANC) neurotransmission in isolated guinea-pig atria. In the present study the effect of adenosine analogues, such as N⁶cyclopentyladenosine (CPA), 5 N-ethylcarboxamide adenosine (NECA), 2 chloroadenosine (2-CADO), R- and S-phenylisopropyladenosine (R- and S-PIA) on the cardiac response to transmural nerve stimulation has been tested in order to characterize the subtype of adenosine receptor involved in the inhibitory control of NANC neurotransmission. The effect of the adenosine antagonist 8-phenyltheophylline (8-PT) was then tested against CPA and NECA.The prototypical A-1 selective agonist CPA was the most active agonist, reducing the response to the stimulation of NANC nerves with an IC₅₀ value of 2.8 nM; RPIA, NECA and 2-CADO showed IC₅₀ values of 9.5, 13.7 and 35 nM respectively. S-PIA was the least active agonist, showing an IC₅₀ value (306 nM) about 30-fold greater than that of R-PIA (9.5 nM). None of the agonists tested was able to modify cardiac response to exogenous CGRR Furthermore, 8-PT competitively antagonized the effect of CPA and NECA with very close pA₂ values (6.77±0.01 and 6.63±0.08 respectively). From these findings we concluded that prejunctional inhibitory adenosine receptors on capsaicin sensitive sensory nerves of cardiac tissue belong to the A-1 subtype. Send offprint requests to A. Rubino at the above address     

Effects of adenosine analogues on contractile response and CAMP content in guinea-pig isolated ventricular myocytes

Summary In the present study the effects of adenosine analogues were investigated on cAMP content and contractile response in guinea-pig ventricular myocytes. The adenosine analogues (–)-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamideadenosine (NECA) and (+)-N⁶-phenyl-isopropyladenosine (S-PIA) in the presence of 0.01 mol/l isoprenaline reduced contractile response concentration-dependently. R-PIA and NECA were about equipotent (IC₂₅: 0.01 mol/l and 0.039 mol/l respectively), while S-PIA was less potent (IC₂₅: 0.6 mol/l). Isoprenaline stimulated cAMP content was reduced by R-PIA (IC₂₅: 0.004 mol/l) and with lower potency by S-PIA (IC₂₅: 0.15 mol/l) but the extent of reduction of cAMP by R-PIA and S-PIA (to 55% and 64% respectively) was less than the reduction of contractile response (to 26% and 55% respectively). This suggests that the effects of R- and S-PIA on contractile response are only in part due to a reduction in cAMP content. In addition, NECA did not decrease cAMP content but decreased contractile response to the same extent as R-PIA. Similar results were obtained in the presence of the phosphodiesterase inhibitor Ro 20-1724. Time course studies revealed that the effects of R-PIA (1 mol/l) on cAMP content and contractile response coincided reaching steady state after 5 min and remained stable thereafter. The effects of NECA (1 µmol/l) on contractility also reached steady state within 5 min, whereas it did not change cAMP content. It is concluded that the reduction of contractility by adenosine analogues in the presence of isoprenaline can only in part be explained by a reduction of cAMP content. It is suggested that a cAMP-independent effect, possibly an activation of phosphatases, might be involved additionally.Abbreviations R-PIA (–)-N⁶-phenylisopropyladenosine - NECA 5-N-ethylcarboxamideadenosine - S-PIA (+)-N⁶-phenylisopropyl-adenosine - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone Send offprint requests to J. Neumann at the above address     

Pharmacological characterization of mediators and vagal influence in the acute allergic bronchoconstriction in guinea pigs

Summary The allergic bronchoconstriction in guinea pigs has been attributed mainly to the release of mast cell mediators. Histamine has been involved in the first minutes of the anaphylactic reaction and new-formed compounds in the subsequent response. In this asthma model the vagal influence has been sparsely investigated. In the present work we evaluated the pharmacological modification of the acute allergic bronchoconstrictor response in guinea pigs sensitized to ovalbumin through aerosol exposure. Pyrilamine (20 g/kg), diethylcarbamazine (a lipoxygenase inhibitor, 10 mg/kg) and dexamethasone (4 mg/kg) each reduced the antigen-induced bronchoconstriction throughout the 30 min studied. Indomethacin (3.1 mg/kg) did not modify the response to the antigen. Atropine (2 mg/kg) plus bilateral vagotomy also diminished this response from 5 min onward. On the other hand, from 5 min ahead pyrilamine-resistant bronchoconstriction was partially inhibited by dexamethasone, and it was almost completely blocked during all of the response when atropine plus bilateral vagotomy were added to dexamethasone. Dipyridamole (an inhibitor of the adenosine uptake, 0.4 mg/kg) enhanced the bronchoconstriction, though this was significant only in the 2–5 min time-interval of the response. These results suggest that histamine and vagal influence play an important role in the whole response to antigen, that other mediators, probably leukotrienes, participate in this response from 5 min onward, and that adenosine could exert a potentiation effect on this response. Send offprint requests to L. M. Montaño at the Instituto Nacional de Enfermedades Respiratorias     

Separate receptors mediating the positive inotropic and chronotropic effect of histamine in guinea-pig atria

The direct positive inotropic effect of histamine was studied on paced left atrial preparations from guinea pigs. Histamine (10^?8 to 10^?4 M) increased the maximum tension developed in left atria incubated at 35°C and driven at 2 Hz. The maximum increase in tension was 60% of that observed with norepinephrine. Metiamide (3 × 10^?5 M; a specific H₂-receptor antagonist) did not alter the inotropic response of left atria to histamine. However, tripelennamine (a typical H₁-receptor antagonist) competitively shifted the histamine inotropic dose—response curve to the right at concentrations from 10^?8 to 10^?7 M. Higher concentrations (3 × 10^?7 and 10^?6 M) caused little further additional shift to the right. The positive chronotropic effect of histamine on spontaneously beating atria was competitively antagonized by metiamide (10^?6 and 3 × 10^?6 M). These results demonstrate that in guinea-pig atria histamine increases myocardial contractility by an interaction with receptors closely related to classical H₁-receptors while its chronotropic effects is mediated by interaction with H₂-receptors.     

Different effector mechanisms for ATP and adenosine hyperpolarization in the guinea-pig taenia coli

The hyperpolarizations in response to ATP and adenosine in the guinea-pig taenia coli, measured by the sucrose-gap technique at room temperature, were compared in solutions of modified ionic composition. ATP hyperpolarization was increased in low chloride and in low potassium, but was reduced to 12% of control in calcium-free solution on second application of the agonist. The response to adenosine, however, was decreased in low chloride, unchanged in low potassium and was 45% of control in calcium-free solution. The different mechanisms for ATP and adenosine hyperpolarization provide evidence for the presence of separate receptors.     

Studies on the action and mechanism of action of oxyfedrine on isolated tracheal chains

Oxyfedrine, a β-sympathomimetic drug, did not affect isolated rat and rabbit trachea in concentration from 2.86 × 10^?8 to 2.86 × 10^?4 M, but on the guinea-pig trachea, it caused a dose dependent relaxation of natural tone in lower concentrations (1.79 × 10^?7 to 2.86 × 10^?6 M. In higher concentrations (1.14 × 10^?5 to 2.86 × 10^?4 M), however, a contraction was observed, which was also dose dependent. This contraction was not affected by atropine, lysergic acid diethylamide or by pretreatment with reserpine but was blocked by antihistaminics (isothipendyl and clemastine). Adrenaline, noradrenaline, phenylephrine and isoprenaline did not contract the guinea-pig trachea, whereas contractions were observed after high concentrations of norephedrine, amphetamine, ephedrine and tyramine. These contractions were also unaffected by reserpine pretreatment.It is concluded that the contraction of the guinea-pig trachea by oxyfedrine is related to its structural relationship to the phenylethylamines and might be due to histamine release, an action on histamine receptors or a histamine-like action.     

Cooling-induced contraction of trachea isolated from normal and sensitized guinea-pigs

Summary Fast (–7°C/min) cooling of guinea-pig isolated trachea produced a rapidly developing, transient contraction followed by relaxation. Cooling-induced contraction was dependent on temperature (30, 20 or 10°C) and responses in trachea obtained from actively sensitized guinea pigs were significantly greater (20 and 10°-C) than those observed in normal trachea. Cooling to 20°C was selected for subsequent experiments. Pre-treatment with sufficient concentrations of atropine, clemastine, cromoglycate, indomethacin, or nordihydroguaiaretic acid did not depress contraction to cooling in either normal or sensitized trachea. This indicates a direct effect of cooling. The contraction. produced by cooling was resistant to verapamil (1 mol/l) or dantrolene (0.3 mmol/l). Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium; all of them at 10–100 mol/l) inhibited contraction in sensitized and normal trachea. Activators of protein kinase C (phorbol 12,13-diacetate, 1 mol/l) enhanced while inhibitors (H-7, 20 mol/l; staurosporine, 10 mol/l) depressed cooling-induced contraction in both normal and sensitized tissues. Incubation (20 min) in a Ca²⁺ -free solution inhibited cooling-induced contraction in normal but not in sensitized trachea. Exposure to a low Na⁺ (25 mmol/l) or a K⁺-free medium abolished contraction to cooling in normal and sensitized trachea. Ouabain (0.1–10 mol/l) and vanadate (0.01–5 mmol/l) inhibited cooling-induced-contraction to a greater extent in normal than in sensitized trachea. Polymyxin B (0.5 mmol/l) selectively depressed responses to cooling in sensitized trachea. In a separate series of experiments, it was shown that sensitized trachea was hyperresponsive to ouabain and vanadate. Previous cooling to 20°C abolished responses to ouabain but only attenuated those to vanadate. These results are compatible with an enhancement of Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities in sensitized trachea and further support the notion that intracellularly stored Ca²⁺ plays a decisive role in the activation of sensitized tracheal muscle. Send offprint requests to J. L. Ortiz at the above address     

Adenosine-induced bronchoconstriction of isolated lung and trachea from sensitized guinea-pigs.

1. The bronchoconstriction of airway-perfused lungs and contraction of superfused tracheal spirals from guinea-pigs in response to adenosine were examined. 2. In lungs from untreated animals, adenosine had little effect unless the perfusion pressure was raised with carbachol (1.1 microM), when it caused a fall in perfusion pressure. However, if removed from guinea-pigs sensitized with ovalbumin (5 mg and 10 mg i.p. 14 and 12 days before use), adenosine was bronchoconstrictor, exerting bronchodilator effects only at high (1 mg) doses. The constrictor response to adenosine (300 micrograms) was significantly greater than that in lungs from untreated or sham-injected animals. 3. In superfused trachea from untreated animals, adenosine exerted only relaxant responses. In tissues from ovalbumin-sensitized guinea-pigs adenosine produced contractile responses, with relaxation appearing only at high (1 mg) doses. 4. Thus sensitization by antigen challenge revealed a bronchoconstrictor response of isolated airway preparations to adenosine. This is related to the clinical situation where only asthmatic subjects respond to adenosine by bronchoconstriction and suggests that the sensitization may destabilize inflammatory cells for mediator release by adenosine. 5. The response to a second exposure to adenosine was consistently reduced (lungs) or converted to a relaxation (trachea) indicating tachyphylaxis and consistent with a mediator release mechanism. 6. The P1-purinoceptor antagonist, 8-phenyltheophylline (3.9 microM), antagonized the relaxant responses to higher doses of adenosine. However, it did not affect the contractile responses to lower doses of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK₁, NK₂ and NK₃ receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle.
2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK₁ and NK₃ receptor-selective agonists.
3. Among the natural tachykinins neurokinin B (EC₅₀=3.2 nM; 95% c.l.=2.0–5.1; n=4) was the most potent, being about 40 and 25 fold more potent than substance P (EC₅₀=121.6 nM; 95% c.l.=94–157; P<0.01; n=4) and neurokinin A (EC₅₀=83.4 nM; 95% c.l.=62–112; P<0.01; n=4), respectively. Among the synthetic analogues the NK₃ receptor-selective agonist senktide (EC₅₀=1.1 nM; 95% c.l.=0.7–1.8; n=8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar⁹]substance P sulfone (NK₁ receptor-selective) (EC₅₀=130.4 nM; 95% c.l.=99–172; P<0.01; n=8), [βAla⁸]NKA (4–10) (NK₂ receptor-selective) (EC₅₀=120.1 nM; 95% c.l.=95–151; P<0.01; n=8) and septide (NK₁ receptor-selective) (EC₅₀=22.6 nM; 95% c.l.=18–28; P<0.01; n=8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar E_max, averaging about 50% of that produced by KCl (80 mM).
4. Atropine (1 μM) did not affect the responses to either NK₁ or NK₂ receptor-selective agonists, whereas it reduced the E_max of senktide by about 50%, without affecting its potency (EC₅₀). Tetrodotoxin (1 μM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK₁ and NK₂ receptor-selective antagonists GR 82334 and MEN 11420 (1 μM each), respectively.
5. GR 82334 (1 μM) blocked with apparent competitive kinetics septide- (apparent pK_B=7.46±0.10; n=5) and [Sar⁹]substance P sulfone- (apparent pK_B=6.80±0.04; n=4) induced contractions. MEN 11420 (30–300 nM), a novel potent NK₂ receptor antagonist, potently antagonized [βAla⁸]NKA (4–10), with competitive kinetics (pK_B=8.25±0.08; n=12: Schild plot slope=−0.90; 95% c.l.=−1.4; −0.35). The NK₃ receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (E_max inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration–response curve to methacholine (0.1–300 μM).
6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK₁, NK₂ and NK₃ receptors. The responses obtained by activating NK₁ and NK₂ receptors are atropine-resistant. The contraction obtained by stimulating NK₃ receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK₁/NK₂ receptors. The role of the NK₃ receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

     

A study of the effects of amodiaquine on the guinea-pig isolated ileum

Summary Amodiaquine (3.5·10^–7 mol/l), a 4-aminoquinoline antimalarial, increases the responses of the guinea-pig isolated ileum to direct (acetylcholine, histamine, barium chloride), partially (5-hydroxytryptamine, PGE₁ and F₂) and totally (electrical stimulations, nicotine) indirect agonists. Moreover it reverses inhibitions of the electrical induced contractions by acetylcholine antagonists (atropine), acetylcholine release blocking agents (morphine, tetrodotoxin, procaine, noradrenaline) and prostaglandin synthesis inhibitors (indomethacin, ibuprofen, flufenamine acid, hydrocortisone). From 2.7·10^–6 mol/l, it induces dose-related tonic contractions which are totally and reversibly abolished by indomethacin as well as by the prostaglandin antagonist, polyphloretin phosphate, but not by atropine, morphine and tetrodotoxin. This indicates that amodiaquine exerts both a direct muscular non selective ileal sensitization to various agonists and, but at higher concentrations, a contractile effect apparently dependent on prostaglandin synthesis and release.At even higher concentrations (5.4·10^–6 mol/l) an effect of amodiaquine on acetylcholine release was demonstrated by the observation of phasic contractions inhibited by atropine, morphine and tetrodotoxin.These effects are different from those found by us with several other antimalarial compounds in the same preparation.     

Discrimination by dipyridamole of two types of responses to adenosine and ATP of the carbachol-induced contraction of guinea-pig trachea

Two types of effect of purines on the carbachol-induced contraction of the guinea-pig trachea in the presence or absence of glucose were evaluated using dipyridamole, an adenosine uptake inhibitor. ATP exerted a greater relaxant effect on the carbachol (10^?6 M)-induced contraction than did adenosine in concentrations ranging from 10^?4 to 10^?3 M. These effects of purines were enhanced by pretreatment with dipyridamole (10^?5 M) but were virtually unaffected by the purine receptor antagonists theophylline (10^?5 M) and quinidine (10^?5 M). The contraction evoked by carbachol was gradually reduced and was almost abolished 4–5 h after exposure to the glucose-free solution. This reduction was almost eliminated by readmitting glucose, and was restored to a certain degree by introducing ATP or adenosine. ATP was less effective than adenosine. This effect of purines but not that of glucose, was markedly diminished by pretreatment with dipyridamole (3 × 10^?6 M to 10^?5 M). The restoration of the carbachol-induced contraction following introduction of purines or glucose was completely antagonized by atropine (10^?6 M). These results suggest that the extracellular effects of adenosine and ATP on the carbachol-induced contraction in normal solution are potentiated by dipyridamole, whereas the possible intracellular effects of these purines in the glucose-free solution are inhibited by this adenosine uptake inhibitor.     

Mechanism of the inhibitory actions of some spasmogens on the bradykinin relaxation of the guinea-pig ileum

The bradykinin relaxation of the acetylcholine-contracted guinea-pig ileum is decreased by increasing the concentration of acetylcholine. This finding prompted an investigation to determine whether other spasmogens can reduce the bradykinin relaxation, and the mechanisms involved.Linear inverse relationships were found between both the percentages of maximum contraction and concentrations of histamine and eledoisin, and the bradykinin relaxation. Eledoisin and histamine showed smaller regression equation slopes than acetylcholine. No relationship existed for angiotensin, though low non-spasmogenic doses in the presence of contraction by acetylcholine reduced the bradykinin relaxation. Morphine and atropine reduced the angiotensin contraction, and potentiated the bradykinin relaxation in its presence. Only atropine reduced the acetylcholine contraction and potentiated the bradykinin relaxation in its presence. Neither morphine nor atropine had any effect on the bradykinin reduction of its own responses.The magnitude of the bradykinin relaxation on the guinea-pig ileum is dependent on the manner of stimulation, rather than on the state of contraction; and, also is sensitive to regulation by agents that have their actions on the parasympathetic system. Angiotensin reduces the bradykinin relaxation by a mechanism involving its indirect parasympathomimetic effects. In contrast, the bradykinin reduction of its own relaxation is not via a parasympathetic mechanism, but probably is a direct action.     

Prostaglandin-versus alpha-adrenoceptor-mediated control of sympathetic neurotransmitter secretion in guinea-pig isolated vas deferens

Noradrenaline (NA) secretion of isolated superfused guinea-pig vas deferenswas studied by determination of total field stimulation-induced efflux of tritium, after preincubation with ³H-l-NA. The medium contained optimal concentrations of desmethylimipramine and normetanephrine to block local rebinding of free NA. Further addition of the two chemically different inhibitors of prostaglandin synthetase, 5,811,14 eicosatetraynoic acid or indomethacin, consistently enhanced the nerve stimulation-induced output of tritium at a frequency of 5/sec, but not at 10/sec. The α-adrenoceptor blocking agent, phenoxybenzamine further strongly elevated nerve stimulation-induced output of tritium. This rise was abolished by low concentrations of exogenous prostaglandin E₂. The results show that sympathetic neurotransmitter secretion in the guinea-pig vas deferens, during low frequency stimulation, is restricted by local formation of prostaglandin E. However and in addition, neurotransmitter secretion appears to be restricted by an α-adrenoceptor-mediated mechanism, which does not appear to depend on endogenous prostaglandin E as a chemical mediator.     

Effects of low temperature and pharmacological interventions on the responses of the isolated guinea-pig trachea

Summary Cooling the guinea-pig isolated trachea from 37°C to 20°C virtually abolished the response to CaCl₂ (in K⁺-depolarized tissues) and depressed that to histamine (about 75% reduction), KCl and 5-hydroxytryptamine (around 50% inhibition) while the response to acetylcholine remained unaffected. A further cooling to 10°C was necessary to inhibit acetylcholine-induced contractions. Hyporesponsiveness to spasmogens by cooling was not associated with subsensitivity (increased EC₅₀) except for 5-hydroxytryptamine. Contractile responses to KCl (50 mmol/l), histamine (1 mmol/l) and 5-hydroxytryptamine (0.1 mmol/l) in a Ca²⁺-free EGTA (0.1 mmol/l)-containing solution were inhibited by cooling to 20°C but responses to acetylcholine (1 mmol/l) in the same experimental conditions were not affected. Cooling to 20°C after treatment with an antagonist (ouabain 10 µmol/l, amiloride 0.1 mmol/l or vanadate 0.1 mmol/l) or after incubation in K⁺-free medium or low Na⁺ (25 mmol/l) solution produced the same or greater inhibitions of tracheal responses to spasmogens than cooling alone. The guinea-pig trachea treated with phorbol 12,13-diacetate (PDA; 1 µmol/l) and cooled to 20°C responded to spasmogens similarly to a trachea untreated with PDA at 37°C. In contrast, PDA (1 µmol/l) did not counteract the depressed responsiveness to histamine of ouabain (10 µmol/l)- or amiloride (0.1 mmol/l)- treated tracheal strips at 37°C. PDA (1 mol/l) enhanced tracheal contractions caused by KCl (50 mmol/l) in Ca²⁺-free medium at 20°C but failed to augment those to histamine in the same conditions. PDA (0.1 or 1 µmol/l) did not change the concentration-response curve for Ca²⁺ in skinned trachea. In conclusion, low temperature inhibits the responses to spasmogens in the guinea-pig trachea. Probably, reduced temperature interferes with extracellular and intracellular sources of Ca²⁺ which are differently affected by various spasmogens. Alteration by cooling of the electrogenic Na⁺ pump, Na⁺/Ca²⁺ exchange system, and Ca²⁺-ATPase may participate in the decrease of tracheal responsiveness to agonists. Reversion by PDA of the inhibitory effect of low temperature suggests a role for protein kinase C in the cooling-induced changes of tracheal responses. Correspondence to J. Cortijo at the above address     

Effects of endothelin-1 on the isolated guinea-pig ileum: role of Na+ ions

Summary Endothelin-1 induced an unusual transient biphasic effect (relaxation followed by contraction) in the isolated guinea-pig ileum. The contractile component of the response was concentration-dependent in the dose range studied, but the relaxant component was not. Neuronal mechanisms and cyclic GMP do not seem to be involved in the relaxing effect since this component was not affected by either tetrodotoxin, atropine or methylene blue. Endothelin-1 induced desensitization which was well characterized for the contractile component of the response, and was only partially reversed after a 3-h resting period. The effects of endothelin-1 were particularly sensitive to decreases in the Na⁺ gradient induced either by lowering the Na⁺ concentration in the medium or by treating the tissue with ouabain or by a previous treatment with acetylcholine. Amiloride partially inhibited the response induced by endothelin-1 indicating that Na⁺ channels and/or Na⁺/H⁺ exchange are probably involved in its action. Our results suggest that the contractile response induced by endothelin-1 in the isolated guinea-pig ileum depends greatly on Na⁺ ions, in contrast to the responses observed in vascular smooth muscle. Send offprint requests to N. Miasiro at the above address     

Differential effects of SKF 10,047 (N-allyl-normetazocine) on peristalsis and longitudinal muscle contractions of the isolated guinea-pig ileum

Summary The purpose of this study was to investigate the differential involvement of distinct types of opioid receptors in the modulation of intestinal peristalsis compared to electrically induced longitudinal muscle contractions.Like naloxone, the proposed -agonist and -antagonist SKF 10,047 (N-allyl-normetazocine) dose-dependently enhanced peristaltic circular muscle contractions in the isolated guinea-pig ileum. Pre-application of SKF 10,047 at a concentration which itself enhanced peristalsis by 20% on average strongly attenuated the inhibition of peristalsis produced by opioids previously proposed to act via -opioid-receptors in the guinea-pig ileum, i.e. normorphine, -endorphin, d-Ala²-d-Leu⁵-enkephalin and d-Ser²-l-Leu⁵-enkephalyl-Thr, but less strongly attenuated the inhibition produced by compounds suggested to act via -opioid-receptors in this tissue, i.e. ethylketazocine and dynorphin (1–13). In contrast to its effect on peristalsis, SKF 10,047 inhibited the electrically induced contractions of the myenteric plexus-longitudinal muscle preparation in a naloxonereversible fashion.It may be concluded that - and -opioid receptors are of a greater functional significance than -receptors in the control of peristalsis. -Receptors might participate predominantly in modulating the release of acetylcholine which underlies the electrically induced longitudinal muscle contraction.     

The effects of metoclopramide on acetylcholine release and on smooth muscle response in the isolated guinea-pig ileum

Summary The effects of metoclopramide on smooth muscle contraction and on release of acetylcholine were studied in the guinea-pig myenteric plexus longitudinal muscle preparation. Acetylcholine was determined either as endogenous acetylcholine, or as labelled transmitter from strips preloaded with ³H-choline.Metoclopramide caused an increase in resting tension of longitudinal muscle as well as an increase in resting output of either endogenous or labelled acetylcholine. Tetrodotoxin abolished the metoclopramide-evoked increase in transmitter release. The increase in smooth muscle tension was clearly related to the increase in resting output. The effects of metoclopramide on both longitudinal muscle contraction and resting release of labelled acetylcholine were prevented in the presence of a concentration of 5-hydroxytryptamine (5-HT) that desensitized 5-HT receptors. This suggests that metoclopramide stimulates neuronal 5-HT receptors and, thereby, facilitates acetylcholine release.Metoclopramide augmented the twitch-like contractions induced by field stimulation at 0.1 Hz. Contractions elicited at 1 Hz were only slightly enhanced. Similarly, metoclopramide facilitated only the release of labelled acetylcholine evoked by electrical stimulation at 0.1 Hz, but not that at 1 Hz. The facilitatory effetts of metoclopramide on twitch height and evoked release could not be attributed to a blockade of presynaptic inhibitory -adrenoceptors, dopamine or muscarine receptors.