JoséL. Ortiz

Rolipram inhibits PAF‐induced airway microvascular leakage in guinea‐pig: a comparison with milrinone and theophylline

Summary— The effects of 3 phosphodiesterase (PDE) inhibitors, rolipram (PDE IV), milrinone (PDE III) and theophylline (nonselective) on PAF (50 ng kg−1; iv)‐induced airway vascular leakage have been evaluated in guinea‐pigs. Rolipram (3–300 μg kg−1; iv) reduced the increase in permeability induced by PAF at all airway levels whereas milrinone (10–1000 μg kg−1; iv) and theophylline (30 mg kg−1; iv) were without effects. The anti‐leakage activity of rolipram may be of therapeutic value in asthma.

Non-specific hyperreactivity to pharmacological stimuli in tracheal and lung parenchymal strips of actively sensitized guinea-pigs

The responsiveness of tracheal and lung parenchymal strips isolated from actively sensitized guinea‐pigs to CaCl2 (in K+‐depolarized tissue), KCl, acetylcholine and histamine was compared with that of strips from unsensitized animals. The concentration‐response curves to the mentioned agonists exhibited, in the sensitized group, a left upward displacement (greater maximal effect, lesser effective concentration 50% and a steeper slope) compared with those obtained in the unsensitized group. These results indicate the existence of a non‐specific increase in responsiveness in the airway smooth muscle from sensitized animals.

Histamine up-regulates phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in human neutrophils.

Abstract:Objective: To investigate whether histamine produces up-regulation of phosphodiesterase (PDE) activity with functional consequences in human peripheral blood neutrophils.¶Methods: PDE activity was studied by a radioisotopic method following anion-exchange chromatography. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of mRNA transcripts of PDE4 subtypes. Cyclic AMP (cAMP) levels were measured by enzyme-immunoassay, and superoxide generation by cytochrome c reduction.¶Treatment: Neutrophils were incubated for 4 h with histamine (1 μM).¶Results: PDE4 was the only isoenzyme activity increased in treated neutrophils. Kinetic analysis showed a ∼1.5-fold increase in Vmax without alteration of Km values. cAMP content in treated cells was higher than resting values (0.52 ± 0.07 vs. 2.75 ± 0.31 pmol/106 cells). RT-PCR showed increased expression of mRNA transcripts for PDE4B in histamine-treated cells. Functionally, up-regulation of PDE4 reduced the inhibition by prostaglandin E2 of zymosan-induced superoxide generation.¶Conclusion: Histamine up-regulates PDE4 activity and produces heterologous desensitisation of human neutrophils.

EFFECTS OF DANTROLENE ON THE RESPONSES TO METHYLXANTHINES IN THE ISOLATED GUINEA-PIG TRACHEA

The effect of dantrolene on the responses to methylxanthines (caffeine and theophylline) and a beta-adrenoceptor agonist (salbutamol) was studied in isolated guinea-pig trachea. Caffeine and theophylline (1 microM-10 mM) and salbutamol (1 nM-10 microM) produced concentration-dependent relaxation of spontaneous and stimulated (acetylcholine 1 mM) tone. Responses to high concentrations (10 mM) of caffeine and theophylline added to tracheal strips either unstimulated (spontaneous tone) or indomethacin (2.8 microM)-treated were reversed to contractions in the presence of dantrolene (3 microM-0.3 mM). This effect was not observed for salbutamol or when relaxant responses to the agonists were generated in pre-contracted (acetylcholine 1 mM) strips. Dantrolene inhibited concentration dependently the contraction evoked by caffeine (1 mM) in indomethacin (2.8 microM)-treated strips at 20 degrees C. This effect of dantrolene was attenuated in a low Ca2+ (0.8 mM) medium or when the bath temperature was lowered to 10 degrees C. Although an intracellular site of action cannot be excluded these results suggest that dantrolene mainly interferes with transmembrane Ca2+ movements in the guinea-pig trachea.

Modification by indomethacin of airway contractile responses in normal and sensitized guinea-pigs

Active sensitization of guinea-pigs resulted in an increase in responsiveness and sensitivity of tracheal and lung parenchymal strips to CaCl2 (in K+-depolarised tissue), KCl, acetylcholine and histamine. Indomethacin (5 microM) preferentially enhanced the response of tracheal strips from normal animals to histamine and to a lesser extent acetylcholine but not to CaCl2 or KCl. A similar trend was observed in sensitized tissues. Indomethacin pretreatment did not cause changes in responsiveness or sensitivity of lung parenchymal strips from normal or sensitized guinea-pigs to the agonists tested. It is concluded that immunological sensitization produced a non-specific hyperresponsiveness in trachea and lung parenchymal strips. Conversely, cyclooxygenase inhibition by indomethacin elicited a selective increase in the responsiveness to certain agonists in central but not in the peripheral airways.

Sources of Calcium for the Contraction Induced by Various Agonists in Trachealis Muscle from Normal and Sensitized Guinea Pigs

Active sensitization of guinea pigs resulted in an increase in the responsiveness and sensitivity of tracheal strips to CaCl2 in K+-depolarized tissue (Emax 0.81 +/- 0.22 g/mm2 and pD2 2.35 +/- 0.10 in normal vs. 0.98 +/- 0.01 g/mm2 and 3.10 +/- 0.08 in sensitized tissue; p less than 0.05), KCl (Emax 0.62 +/- 0.08 g/mm2 and pD2 1.71 +/- 0.03 in normal vs. 0.91 +/- 0.04 g/mm2 and 2.00 +/- 0.01 in sensitized tissue; p less than 0.05) and histamine (Emax 0.70 +/- 0.06 g/mm2 and pD2 5.08 +/- 0.06 in normal vs. 0.94 +/- 0.09 g/mm2 and 5.80 +/- 0.16 in sensitized tissue; p less than 0.05) but not to caffeine 10 mM (20 degrees C, indomethacin 2.8 microM). Generation of responses to these agonists in nonsensitized tissues bathed in a Ca2+-free medium resulted in the abolition of KCl-induced contraction and partial inhibition of the responses to histamine (60% inhibition) and caffeine (40% inhibition). The contraction of sensitized tracheal strips in response to histamine in 0 calcium was greater than that obtained in nonsensitized tissues (0.48 +/- 0.02 g/mm2 vs. 0.28 +/- 0.04 g/mm2, respectively; p less than 0.05). The caffeine-induced contraction of sensitized tracheae was independent of extracellular calcium. These results suggest that a greater calcium entry and/or intracellular calcium release may be an alteration underlying hyperreactivity of sensitized tissues to spasmogens.

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

SummaryCooling 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 μmol/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.

Effect of dantrolene sodium in isolated guinea-pig trachea

The effect of dantrolene sodium (3 microM-0.3 mM) on the spontaneous tone and responses to various contractile agonists was studied in isolated guinea-pig trachea. Dantrolene produced a concentration-related inhibition of the spontaneous tracheal tone, reaching a value of 94.8 +/- 4.8% of the relaxation induced by caffeine 10 mM. Removal of the epithelium did not affect the dantrolene-induced relaxation. Dantrolene did not alter the concentration-response curve for KCl and produced only small displacements of the concentration-response curves for CaCl2, acetylcholine and histamine, without affecting their maximal effects. Dantrolene dose relatedly inhibited the contraction induced by caffeine (1 mM) in Krebs solution containing indomethacin (2.8 microM) at 20 degrees C. The spasm induced by caffeine in Ca2(+)-free Krebs solution (20 degrees C, indomethacin 2.8 microM) was slightly depressed by dantrolene. Dantrolene did not depress the Ca2+ (1 microM)-induced contraction in skinned trachea. These results suggest that besides a possible intracellular site of action, the mechanism of the inhibitory effect of dantrolene in guinea-pig trachea may be related to interference with Ca2+ entry through pathways not susceptible to calcium channel blockers.

Effects of calcium antagonists on rat normal and skinned fundus.

Abstract— Calcium chloride (CaCl2) (0·1−25 Mm, in K+‐depolarized tissue), KCl (10−112 Mm) and acetylcholine (1 × 10−9 m−1 Mm) produced concentration‐dependent contractions of rat isolated fundus. Verapamil (0·01−100 μm), cinnarizine (1−100 μm), trifluoperazine (10−500 μm) and dantrolene (50−250 μm) each produced a concentration‐related rightward and downward shift of the log concentration‐effect curve for CaCl2. The rank order of potencies of these antagonists, measured as the IC50 against Ca2+ (25 Mm)‐induced contraction of depolarized fundus, was verapamil (2.5 μm) > cinnarizine (8·7 μm) > trifluoperazine (85·1 μm) > dantrolene (> 250 μm). Cinnarizine (0·5 Mm) and trifluoperazine (0·5 Mm), but neither verapamil nor dantrolene depressed Ca2+ (20 μm)‐evoked contraction of rat skinned fundus preparations. In intact preparations of rat fundus, verapamil had greater inhibitory effects on contractions produced by KCl than against those elicited by acetylcholine while trifluoperazine depressed to the same extent the responses to these two spasmogens. Dantrolene was without effect on contractions elicited by KCl or acetylcholine. Cinnarizine inhibited acetylcholine‐induced responses but enhanced contractions to KCl. Augmentation of KCl‐induced responses by cinnarizine is resistant to verapamil (1 μm). This enhancing effect of cinnarizine was not observed for KCl‐induced contraction of guinea‐pig fundus or rat gastro‐oesophageal sphincter. In the rat fundus, cinnarizine (1−100 μm) produced an additional and concentration‐related contraction when added on the plateau contraction to KCl (100 Mm). The enhancing effect and the direct contraction produced by cinnarizine are at least partly dependent on extracellular Ca2+. It is concluded that distinct differences exist between the calcium antagonists examined. The action of verapamil is restricted to the plasmalemma whereas cinnarizine and trifluoperazine also act on the intracellular contractile machinery. Dantrolene is scarcely effective as a calcium antagonist in rat fundus.

Responses to histamine and selective H2-receptor agonists in lung parenchymal strips from normal and sensitized guinea-pigs

Histamine produces concentration-dependent contractions of lung parenchyma strips obtained from normal and sensitized guinea-pigs. The responsiveness of the sensitized lung strips to histamine was significantly increased compared to normal tissues. Clemizole (0.1 μM) was equally effective as an H1-antagonist in normal (dose-ratio 9.12) and sensitized (dose-ratio 9.77) tissues.The concentration-response curves to histamine were displaced to the left by cimetidine (0.1 μM to 0.1 mM) with similar dose-ratios in normal and sensitized tissues. Cimetidine enhanced maximal responses to histamine only in normal lung strips. The effects of submaximal equieffective concentrations of histamine were augmented to the same extent by cimetidine (0.1 mM) in normal and sensitized tissues. The responses to histamine were not modified by indomethacin (5 μM).The responsiveness and sensitivity of sensitized lung strips to isoprenaline, impromidine, 4-methylhistamine and dimaprit were not different from those of normal tissues. Cimetidine yielded, as antagonist of dimaprit, similar pA2 values in normal and sensitized tissues.In conclusion, there is no experimental evidence in favour of the existence of an impairment of H2-receptor activity in sensitized airways. Hyperreactivity to histamine is probably due to differences between normal and sensitized tissues with respect to Ca2+ entry and/or intracellular Ca2+ release in response to H1-receptor activation.