Estimation of agonist dissociation constants at central presynaptic alpha-2 autoreceptors in the absence of autoinhibition.
The agonist dissociation constants (KA) and relative efficacies of UK-14304, norepinephrine (NE) and clonidine at presynaptic release-modulating alpha-2 adrenoceptors were determined in rat cerebral cortex slices using the irreversible alpha-2 adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (0.8 mg/kg i.p.) to reduce the receptor pool. Eighteen hours after treatment, the slices were incubated with [3H]NE, superfused in the presence of reuptake inhibitor and stimulated electrically with short bursts of 4 pulses delivered at 100 Hz (pseudo-one-pulse; POP) or trains of 36 or 72 pulses delivered at 3 Hz. EEDQ treatment did not affect the overflow of radioactivity evoked by POP, but greatly facilitated overflow at 36 or 72 pulses/3 Hz indicating that the autoinhibition seen under the latter conditions is totally lacking with POP stimulation. KA values determined using 4 pulses/100 Hz were 136, 50 and 625 nM for UK-14304, clonidine and NE, respectively. At 36 or 72 pulses/3 Hz the values were higher by a factor of up to 3. The percentage of receptors active after EEDQ treatment was found to be 5.5 to 8.2% and was not influenced by conditions of stimulation. Receptor reserves were estimated to be about 65% for UK-14304 and NE and 40% for clonidine. The efficacies of UK-14304 and clonidine relative to NE were 1 and 0.5, respectively. The data indicate that KA values for agonists at presynaptic alpha-2 autoreceptors are inevitably underestimated if the released transmitter causes inhibition of release in addition to the drug under investigation.
Agneter
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Institute of Pharmacology, University of Vienna, Austria.
Drobny
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Singer
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EA
D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D003000:Clonidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D066298:In Vitro Techniques; D008297:Male; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D011804:Quinolines; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D000068438:Brimonidine Tartrate; C000470:EEDQ; D003000:Clonidine; D009638:Norepinephrine