Changes in affinity states during down-regulation of muscarinic receptors in tracheal smooth muscle of organophosphate-treated swine.
1. Subacute (daily) treatment of male swine with the organophosphate acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) resulted in tolerance to the effects of DFP within 5-6 days. 2. Subacute administration of DFP resulted in a 98% inhibition of tissue cholinesterase after 7 days and in a decrease of [3H]quinuclidinyl benzilate [( 3H]QNB) binding sites in homogenates of tracheal smooth muscle by 77%. The maximal density of receptors (Bmax) decreased from 1.8 +/- 0.4 to 0.5 +/- 0.1 pmole mg-1 protein. There was no significant change in the dissociation constant (Kd) for [3H]QNB binding. 3. Pirenzepine displacement of [3H]QNB binding was best described by a single binding site model, with a Ki of 230 +/- 40 nM. This value was unchanged following seven days of DFP treatment (250 +/- 30 nM). The low affinity for this M1 antagonist suggests that there is predominantly a single population of [3H]QNB binding sites of the M2 subtype in tracheal smooth muscle. 4. Carbachol displacement of [3H]QNB binding yielded data best fit by a two-binding site model. The dissociation constants were KiL = 210 +/- 60 microM (61 +/- 1%) and KiH = 1.2 +/- 0.4 microM (39 +/- 1%) respectively (n = 7) for the low and high affinity states. Seven-day treatment with DFP reduced the percent of high affinity receptors to 25 +/- 4%. 5. Addition of Mg++ to the incubation medium prevented this shift in the proportion of low and high affinity receptors. Gpp(NH)p and Mg++ together decreased the proportion of the high affinity receptors when added to the incubation medium in control tissue (to 25%), but not tissue from 7-day DFP-treated swine. NEM increased the proportion of muscarinic receptors in the high affinity state both for controls and for the DFP-treated swine, in both cases yielding receptors with identical binding properties. 6. Thus, subacute administration of DFP causes not only a decrease in the number of receptors, but also a change in the affinity of the receptors for agonists which is related to the interaction of the guanine nucleotide binding protein and the muscarinic receptor.
Yang
C M
CM
Department of Pharmacology/Toxicology, University of Mississippi Medical Center, Jackson 39216-4505.
Mohan
P M
PM
Dwyer
T M
TM
Farley
J M
JM
D000818:Animals; D002217:Carbachol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000633:toxicity*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism; D013552:Swine; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism
D011976:Receptors, Muscarinic; D007531:Isoflurophate; D006165:Guanylyl Imidodiphosphate; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D008274:Magnesium; D005033:Ethylmaleimide