Cumulative Concentration-effect Curves Research Articles

Analysis of agonist dissociation constants as assessed by functional antagonism in guinea pig left atria

In electrically driven guinea pig left atria, positive inotropic responses to (−)-isoprenaline and the selective β 1-adrenoceptor agonist RO363 were obtained in the absence and in the presence of the functional antagonists adenosine, carbachol, gallopamil, nifedipine, and Ro 03-7894. Each of the functional antagonists reduced the maximum response to both agonists and produced nonparallel right-ward shifts in the cumulative concentration effect curves. For both agonists, dissociation constants (k a) were calculated using the equation described by Furchgott (1966) for irreversible antagonism. For RO363, which is a partial agonist with high agonist activity, the equations outlined for functional interaction by Mackay (1981) were also employed to calculate k a values. The k a values obtained by each method were compared with the dissociation constants (K D) for the two agonists determined from their ability to displace the radioligand (−)-[ 125l]iodocyano-pindolol from β 1-adrenoceptors in guinea pig left atrial membrane preparations. The estimates of k A varied substantially from K D values. The K D values were taken as more accurate estimates of the true values for the dissociation constants because a high degree of correlation exists between pK D and pD 2 values for a number of other β-adrenoceptor agonists that behave as partial agonists and between pK D and pK B values for a number of β-adrenoceptor antagonists. Thus it appears that there are serious limitations in the current theory for using functional antagonism as a means of obtaining agonist dissociation constants.

Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists.

The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration-effect curves in guinea-pig isolated tracheal ring preparations with and without an intact epithelium. Estimates of Emax were not significantly different in epithelium-denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium-stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. The autoradiographic localisation of binding sites for the non-selective beta-adrenoceptor ligand [125I]-iodocyanopindolol (I-CYP) showed that the epithelium of the guinea-pig trachea had a 75 +/- 16% greater density of beta-adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I-CYP binding. It was concluded that the reduced functional response of guinea-pig trachea to isoprenaline was probably not due to smooth muscle beta-adrenoceptor dysfunction. Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea-pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.

Effect of nifedipine on constriction of human tracheal strips in vitro.

1 Autopsy specimens of human trachealis muscle were used to investigate the effect of the calcium channel blocker, nifedipine, on airway smooth muscle constriction. With these tracheal strips, two consecutive cumulative concentration-effect curves to histamine (0.1-100 microM) obtained at a 60 min interval were highly reproducible. 2 We examined the effects of adding nifedipine (2.9 microM) to the incubation medium before the second histamine response. The concentration-effect relationships determined after nifedipine incubation were significantly different from control: the response to 100 microM histamine was reduced by approximately one-half (to 2.53 +/- 0.6 g; P less than 0.025), and the concentration of histamine causing 40% of the maximal control contraction (EC40) increased nearly ten fold (to 36.9 +/- 10.5 microM; P less than 0.02). 3 In two additional tracheal strips submaximally constricted with 10 microM histamine, nifedipine 2.9 microM caused complete relaxation to resting tension or below. 4 These results indicate a direct inhibitory effect of nifedipine on airway smooth muscle constriction and partial dependence of human trachealis muscle on calcium ion fluxes for initiation and maintenance of contraction. In addition, the results suggest a potential mechanism for the inhibitory effects of calcium channel blocking drugs in exercise-induced asthma.

The effects of hydrocortisone on responses to and extraneuronal uptake of (-)-isoprenaline in cat and guinea-pig atria.

1. (-)-Isoprenaline-induced positive inotropic effects were assessed in driven left atria and positive chronotropic effects in spontaneously beating right atria from guinea-pigs and cats. 2. Hydrocortisone produced dose dependent shifts to the left in cumulative concentration-effect curves to (-)-isoprenaline in left and right atrial preparations from the cat. 3. Maximal potentiations were produced by 160 muM and 40 muM hydrocortisone in cat left and right atria respectively. 4. No such effects were seen in similar preparations from the guinea-pig even in the presence of concentrations of hydrocortisone as high as 400 muM. 5. Hydrocortisone inhibited the extraneuronal uptake of [7 - 3H](-)-isoprenaline in a concentration dependent manner in both left and right atria of the cat, but not the guinea-pig. 6. These results are consistent with the idea that hydrocortisone potentiates responses to (-)-isoprenaline in cat atria by inhibition of the extraneuronal uptake process.