The action of agonists and antagonists at the histamine H1 receptor and receptor protection studies in guinea pig ileum

We have examined the ability of histamine and the competitive reversible antihistamines to protect the histamine H1 receptor against alkylation with the 2-haloalkylamines, phenoxybenzamine and SY-14. In isolated guinea pig ileum these irreversible antagonists produce a parallel shift in the dose-response curve to histamine with retention of the maximum response if they are used at concentrations less than about 10(-6)M. Treatment with these 2-haloalkylamines in the presence of a high concentration of histamine did not alter the blocking activity. Thus histamine appears to be unable to protect its own receptor against irreversible blockade. The competitive reversible antagonists, on the other hand, did provide effective protection against irreversible blockade. It is likely that the competitive reversible H1 receptor antagonists have at least some part of their attachment site in common with irreversible antagonists of the 2-haloalkylamine type, while the inability of histamine to provide self-protection suggests that its primary attachment site is different from that of the antagonists.     

The effects of methylxanthines, ethymizol, ephedrine and papaverine on guinea pig and dog trachea

The study was aimed to compare the effects of pentoxyphylline, aminophylline, choline theophyllinate and ethymizol on guinea pig and dog trachea with those of theophylline, papaverine and ephedrine. The effects of these drugs on the basal tension, on dose-response curves for muscle contraction produced by histamine and on cAMP level were investigated in guinea pig trachea, together with their influence on the resting and histamine-evoked mechanical and membrane activities of dog trachea. Like papaverine, pentoxyphylline did not alter the resting membrane potential, although it relaxed both tracheal preparations, and it antagonised the effects histamine and raised the cAMP level of the smooth muscle. The effects of ethymizol were similar to those of theophylline and its water soluble derivatives (aminophylline and choline theophyllinate). Whereas, ephedrine although it decreased the basal tension and inhibited histamine-evoked responses, also elicited substantial hyperpolarization of the smooth muscle membrane with no effect on the cAMP level. These findings are consistent with the hypothesis that cAMP has an important role in the action of some bronchodilator drugs; however, it is concluded that the possibility of contributing of their action on membrane potential to their action needs to be considered. The similarity of the potencies of ethymizol and pentoxyphylline to that of classical bronchodilators in inhibiting contraction of guinea pig and dog tracheal smooth muscle suggests that they may have a therapeutic value.     

Molecular dynamics simulations to explore the active/inactive conformers of guinea pig β2 adrenoceptor for the selective design of agonists or antagonists

It is well known that guinea pig β₂ adrenoceptors (Gβ₂ARs) and human β₂ adrenoceptors (Hβ₂ARs) have structural similarity. However, only one conformational state of Gβ₂ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ₂ARs by using putative active/inactive Hβ₂AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β₂AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ₂AR sites similar to those reported for Hβ₂ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0.66 in all cases. The highest values were for agonists on the active models and antagonists on the inactive model. The four Gβ₂AR models proved useful for analysing agonist/antagonist activity. The results suggest that the selection of an adequate model is dependent on the intrinsic activity of a given ligand.     

The deceleration of the heart frequency in the waterfleaDaphnia magna by adrenoceptor agonists and antagonists

Beat-to-beat measurements with 30 micro-seconds accuracy were carried out in order to determine the chronotropic effects on the heart ofDaphnia magna induced by adrenoceptor agonists and antagonists dissolved in water. Agonists and antagonists were either ineffective or lowered the heart frequency (P < 0.05). In addition, the negative chronotropic effect of the agonist epinephrine could not be blocked by the antagonist propranolol. It may, therefore, well be that the drug actions were not mediated through adrenoceptors.     

The activity of synthetic leukotriene C-1 on guinea pig trachea and ileum

The effects of synthetic leukotriene C-1 (LTC-1) on isolated guinea pig trachea and ileum have been determined and compared to histamine. LTC-1 produced a slow contraction of the trachea and the ileum with pD2 values of 8.7 +/- 0.1 (n = 14) and 8.5 +/- 0.1 (n = 13), respectively. In comparison, the pD2 values for histamine were 5.6 +/- 0.1 (n = 6) and 6.2 +/- 0.1 (n = 6), indicating LTC-1 was 2-3 orders of magnitude more potent. LTC-1 was antagonised by FPL 55712 with pA2 values of 6.9 +/- 0.1 (n = 5) and 6.4 +/- 0.1 (n = 7) on the trachea and ileum, respectively. Incubation with lipoxidase produced a time and enzyme dependent loss of biological activity and a concurrent shift in U.V. absorption spectrum.     

Release of leukotriene C4 from guinea pig trachea

Immunological (ovalbumin) and non-immunological (calcium ionophore A23187) stimulation of guinea pig trachea induces a prolonged contraction that is enhanced by indomethacin (8.5 microM) and inhibited by nordihydroguaiaretic acid (50 microM) pretreatment of the tissue. The mediator released by the above stimuli was identified as leukotriene C4 by reverse-phase high performance liquid chromatography, and quantitated by bioassay. Indomethacin, and/or arachidonic acid (32.8 microM) did not enhance the release, whereas nordihydroguaiaretic acid reduced the contraction and release of LTC4. The results demonstrate the hitherto unproved capability of the large airways to synthesize leukotrienes and emphasize the importance of examining their role in asthma.     

Contraction of guinea pig trachea by epidermal growth factor--urogastrone

To evaluate further the action of epidermal growth factor - urogastrone (EGF-URO) in smooth muscle systems, we examined the effect of the peptide on guinea pig tracheal strips. The cumulative addition of EGF-URO to the organ bath resulted in a concentration-dependent tonic contraction without tachyphylaxis. The half-maximal contraction was obtained at 13 +/- 3 ng/mL EGF-URO (2 nM). The maximum contraction at 100 ng/mL approached 60% of that induced by 1 microM histamine. No significant difference in the EGF-URO-induced contraction was observed in the presence or absence of a functional epithelium. Preincubation with 1 microM indomethacin for 20 min abolished the action of EGF-URO. The contractile effect of EGF-URO was not affected by yohimbine, propranolol, atropine, tetrodotoxin, and esculetin. However, mepacrine caused inhibition by 37 +/- 7% (mean +/- SEM for n = 3). Verapamil (10 microM) inhibited the EGF-induced response by 62 +/- 5% (mean +/- SEM for n = 4); the response was also absent in Ca-free (1 mM EGTA) buffer. However, the response was restored after the readdition of calcium. Our results suggest that EGF-URO can modulate tracheal smooth muscle contractility via a cyclooxygenase product and raise the possibility that EGF-URO might play a role in controlling pulmonary smooth muscle tone in vivo.     

Osmotic stimuli induce epithelial-dependent relaxation in the guinea pig trachea

Epithelium in airways, like endothelium in blood vessels, may regulate responses of adjacent smooth muscle. To study the intact trachea from guinea pigs we developed an in vitro preparation that permits independent stimulation from either the inner epithelial surface or the outer serosal surface. The whole guinea pig trachea was excised, cannulated, and perfused at a constant flow with Krebs-Henseleit (KH) solution that was in direct contact with the inner epithelial-lined surface. The outer serosal surface of the trachea was immersed in a separate system (bath) containing KH solution. Tracheal responses were assessed by measuring the pressure drop between the tracheal inlet and the outlet under conditions of constant flow. When the trachea was precontracted with carbachol or KCl, hyperosmolar stimuli (KCl, mannitol, urea, or NaCl) produced concentration-dependent relaxation when applied to the inner epithelial surface. Relaxation was not produced when the hyperosmolar stimulus was applied to the serosal surface and was markedly reduced or abolished when the epithelial surface had been physically damaged or removed. These results indicate that hyperosmotic stimuli induce epithelial-dependent relaxation of trachea. A defect in this mechanism may be partially responsible for the bronchoconstriction seen in asthmatic subjects after exercise.     

Tyr-MIF-1 and hemorphin can act as opiate agonists as well as antagonists in the guinea pig ileum.

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC50 was about 9 microM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with beta-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or beta-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.     

The effect of calcium antagonists on contractions of the sensitized and normal guinea pig ileum

The purpose of this study was to learn wether a number of Ca²⁺ antagonists were effective in reducing contractile response of the isolated ileum of the sensitized and normal guinea pig. Contractions of the normal ileum in response to LTD₄, acetylcholine, histamine, and potassium chloride were obtained before and after verapamil, diltiazen and papaverine. Ovalbumin-induced contractions of the ovalbumin-sensitized ileum were obtained in the presence of the three Ca²⁺ antagonists. In the normal ileum, all the Ca²⁺ antagonists were highly effective in diminishing the contractile responses to LTD₄, acetylcholine, histamine and potassium chloride. In the sensitized ileum, ovalbumin-evoked contractions, with subsequent release of a potent contractile mediator (presumably SRS-A), were Ca²⁺-dependent since verapamil, diltiazem and papaverine caused a concentration-related reduction of contractions. Thus, the influx of extracellular Ca²⁺ plays a key role in the contractile responses of the normal and sensitized guinea pig ileum when stimulated by various potent agonists acting on specific receptors or on the cell membrane.     

Nitric oxide regulatory role in sensitized guinea pig trachea

Nitric oxide (NO) has been cited to play an important regulatory role in airway function. Moreover, the NO synthase expression in models of inflammation is documented. The aim of this study was to investigate, in vitro, the NO modulation of cholinergic responses in sham-sensitized and ovalbumin-sensitized guinea pig trachea by using L-arginine (L-ARG), a precursor of NO synthesis, and L-Ng-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase.Our results showed that NO's ability to modulate cholinergic responses in oval-buminsensitized guinea pig trachea is lost. Indeed L-ARG and L-NAME modify acetylcholine sensitivity in sham-sensitized guinea pig but not in ovalbumin-sensitized guinea pig.     

Histochemical localization of carbonic anhydrase in the trachea of the guinea pig

 Tissue specimens from guinea pigs were examined using an enzyme-histochemical reaction to explore the presence of carbonic anhydrase (CA) activity in the trachea. CA activity was detected in a group of morphologically distinct epithelial cells, in goblet cells, and in glands of the tracheal mucosa. The epithelial cells showing CA activity were distributed singly and sparsely throughout the entire trachea. These cells showed a wide morphological variability and were clearly different from those forming the pseudostratified ciliated epithelium. Their number was higher in sections closer to the tracheal bifurcation than in those near the larynx. Although the nature of these cells is unknown, based on their morphological and histochemical characteristics and their distribution, they may represent a specialized chemoreceptor. To our knowledge, this is the first report of CA localized in tracheal epithelial cells. Accepted: 6 March 1996