Noradrenaline (NA) 1 microM and caffeine (CAF) 10 mM induce a contractile response in isolated rat aorta maintained at 37 degrees C either in the presence or absence of extracellular calcium. In Ca-free media the contractile response was reduced and contractile activity of CAF only occurred at 25 degrees C. NA induced a biphasic response in Ca-free medium, with a fast phasic contraction followed by a smaller more sustained contraction. The response induced by CAF consisted of a fast transient contraction which returned to a level below the resting tone. After washing, further addition of NA or CAF evoked no increase in smooth muscle tension. The influence of Mg-depletion in the extracellular medium on the contractile responses induced by NA and CAF in Ca-free medium was determined: similar response to NA or CAF in media with or without Mg after 2 min loading were observed, but after 15 min loading in Mg, Ca-free solution, the responses to NA or CAF were significantly higher than after incubation in Ca-free medium containing Mg. Differences were observed when contractile response to NA was elicited after the refilling process by loading the aortic strip in Ca-containing Mg-free solution. In this case, instead of recovering the magnitude of contraction there was a significant decrease. The existence of two independent intracellular Ca-pools releasable by NA, one of them also sensitive to CAF, is postulated. The refill of the Ca-store specific to NA is dependent on the presence of Mg in the extracellular medium. In contrast, the refill of the common Ca-pool releasable by NA and CAF is independent of the extracellular Mg, but its spontaneous emptying is temperature-, and Mg-dependent. This suggests the intermediacy of an enzymatic process to extrude the Ca-content of this store to the extracellular space.

Different and common intracellular calcium-stores mobilized by noradrenaline and caffeine in vascular smooth muscle.

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 μmol/l) or dantrolene (0.3 mmol/l). Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium; all of them at 10–100 μmol/l) inhibited contraction in sensitized and normal trachea. Activators of protein kinase C (phorbol 12,13-diacetate, 1 μmol/l) enhanced while inhibitors (H-7, 20 μmol/l; staurosporine, 10 μmol/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 μmol/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.

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

Responses to KCl and histamine and 45Ca movements were studied in trachea from normal and actively sensitized guinea-pigs. Sensitized tracheas were hyperresponsive and hypersensitive to KCl and histamine. 45Ca uptake experiments show that sensitized tracheal muscle behaves as normal except that the uptake of 45Ca in low concentration (0.03 mM) Ca2+ solution was higher and the number of binding sites for the high affinity component of 45Ca uptake (as estimated by Scatchard-coordinate plot) was augmented. Additionally, in sensitized tracheal muscle, incubation in low (0.03 mM) Ca2+ solution followed by La3+ wash-out resulted in a greater amount of residual 45Ca than in normal tissues. KCl, but not histamine, increased the La3+ resistant 45Ca content. This increase was greater in sensitized than in normal trachea. This demonstrates the existence of hyperreactivity and altered 45Ca movements in sensitized trachealis muscle.

https://erj.ersjournals.com/content/erj/4/4/450.full.pdf

Hyperreactivity and 45Ca movements in sensitized guinea-pig tracheal muscle

Effects of dantrolene on the responses to methylxanthines in the isolated guinea-pig trachea.

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contraction produced by caffeine or theophylline in PDA (5 microM)-treated tissues. 3. The ability of PDA (5 microM) to convert caffeine-induced relaxation into caffeine-induced contraction was retained in tissues pretreated with a combination of atropine (1 microM) and mepyramine (1 microM) and in tissues denuded of the airway epithelium. 4. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each relaxed trachea contracted with histamine (0.1 mM). The relaxation induced by caffeine, theophylline and isoprenaline was markedly reduced in the presence of PDA (5 microM) and the responses to high concentrations of caffeine and theophylline, but not those to isoprenaline, were reversed to contractions. Verapamil (10 microM) prevented the effects of PDA against caffeine- or theophylline-induced relaxation. 5. PDA (1 microM) enhanced the tracheal spasm produced by caffeine (10 mM) and theophylline (10 mM) in indomethacin (2.8 microM)-treated trachea maintained at 20 degrees C. This enhancement was reduced in the presence of verapamil (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Julio Cortijo

Papers

The spasmogenic effect of caffeine in trachealis isolated from control and actively sensitized guinea-pigs