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Adrenergic cholinergic interaction in rabbit isolated left atrium : effect of pertussis toxin treatment

University of British Columbia

In the atrial myocardium the biochemical basis of the functional interaction between muscarinic and beta adrenoceptor agonists is not clear. Although muscarinic agonists can antagonise beta adrenoceptor agonist-induced increases in cAMP levels, this effect is not considered proportional to the effect of muscarinic agonists on beta adrenergic agonist-induced increases in force of contraction. It is believed that the ability of muscarinic agonists to exert a cAMP-independent, direct negative inotropic response may also contribute in the process of their interaction with beta adrenoceptor agonists. The purpose of the present study was to further explore the role played by cAMP in the process of the interaction between the muscarinic agonist, carbachol, and the beta adrenoceptor agonist, isoproterenol. The interaction was studied in electrically driven rabbit left atrial strips after removing the inhibitory effect of muscarinic agonists on beta agonist-induced cAMP generation using pertussis toxin. Pertussis toxin is known to uncouple various inhibitory receptors (muscarinic, alpha-2 adrenergic, opiate etc) from the adenylate cyclase system. The effect of pertussis toxin treatment on the force of contraction and cAMP levels of rabbit left atria in response to carbachol and isoproterenol, alone and in combination, was studied. The role played by cGMP in the interaction between isopro- terenol and carbachol was also studied, since cGMP has been suggested to be the second messenger mediating the effects of muscarinic receptor activation. The interactions of carbachol with forskolin or phenylephrine (in the presence of 1 uM timolol to block the beta receptor-mediated component of its effect) were also studied in the presence and absence of pertussis toxin, since it has been reported that carbachol can overcome the positive inotropic responses to forskolin and phenylephrine in a cAMP-independent manner. It was observed that pertussis toxin treatment attenuated the inhibitory effects of carbachol on isoprote-renol-induced increases in force of contraction and cAMP levels. The direct, cAMP-independent, negative inotropic response to carbachol was also attenuated by pertussis toxin treatment. However, pertussis toxin had a relatively greater effect on the response to carbachol in the presence of isoproterenol than on the direct negative inotropic response to carbachol. Carbachol lost almost completely its ability to overcome isoproterenol's effect on tension and cAMP levels while still retaining its ability to exert a direct negative inotropic response. This suggests that muscarinic antagonism of beta adrenoceptor agonist-induced cAMP generation may play a role in the process of the interaction between muscarinic and beta adrenoceptor agonists. Carbachol-induced cGMP levels, however, remained elevated even when pertussis toxin almost completely attenuated the inhibitory effect of carbachol on isoproterenol-induced tension. This suggests that carbachol-induced cGMP elevation may not be related to its effect on isoproterenol-induced increases in force of contraction. Pertussis toxin had a very similar effect on the direct negative inotropic response to carbachol and the ability of carbachol to overcome forskolin-induced tension. This, along with the fact that both responses to carbachol occured without any change in cAMP levels, either forskolin-induced or basal, suggests that both of these effects of carbachol may be mediated by a common mechanism. On the other hand, although no change in cAMP levels was seen in the process of the phenylephrine-carbachol interaction, the inhibitory effect of carbachol on the positive inotropic response to phenylephrine was almost totally lost after pertussis toxin treatment. At this stage it is not known exactly how carbachol antagonises the functional responses to alpha adrenergic receptor stimulation. Overall, from the results of the present study it appears that the ability of carbachol to antagonise isoproterenol-induced cAMP generation may play a role in the process of its functional interaction with isoproterenol.

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2010-09-09

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http://hdl.handle.net/2429/28322

Pharmaceutical Sciences

University of British Columbia

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