Rat pineal alpha 1-adrenoceptor subtypes: studies using radioligand binding and reverse transcription-polymerase chain reaction analysis.
1. The pharmacological characteristics of alpha 1-adrenoceptor binding sites in rat pineal gland membranes, detected by use of a selective alpha 1-adrenoceptor antagonist ([125I]-iodo-2-[beta-(4-hydroxyphenyl) ethylaminomethyl]tetralone, [125I]-HEAT), were investigated with the alkylating agent, chloroethylclonidine (CEC), and in competition experiments with a number of adrenoceptor agonists and antagonists. 2. Chloroethylclonidine (CEC) treatment (10 microM, 10 min) of rat pineal membranes inactivated approximately 70% of specific [125I]-HEAT binding sites. Higher concentrations of CEC (up to 100 microM) or longer treatment periods (up to 40 min) were no more effective. 3. Adrenoceptor agonists and antagonists competitively inhibited [125I]-HEAT binding with Hill coefficients close to unity indicating a single alpha 1-adrenoceptor subtype is present. The affinity (Ki) of subtype selective agonists (oxymetazoline, SDZ NVI-085) and antagonists (5-methylurapidil, WB4101, benoxathian, phentolamine) was consistent with binding to an alpha 1B-adrenoceptor subtype. 4. The (-)- and (+)-enantiomers of niguldipine had an equal and low affinity for alpha 1-adrenoceptor binding sites both in untreated (log Ki-6.66 and -6.90 respectively) and CEC-treated membranes in which approximately 70% of sites had been inactivated (log Ki-6.41 and -6.86 respectively). This indicates that the small proportion of alpha 1-adrenoceptors insensitive to CEC are not alpha 1A-adrenoceptors. 5. mRNA was isolated from rat pinealocytes, cDNA was synthesized and then amplified by the polymerase chain reaction with alpha 1-adrenoceptor subtype specific primers. These experiments identified both alpha 1A- and alpha 1B-adrenoceptor mRNA, but not alpha 1D-mRNA in rat pinealocytes, although all three adrenoceptor subtypes were readily identified in rat brain cortex. 6. These data indicate that although both alpha 1A- and alpha 1B-adrenoceptor mRNAs are present in the pineal the major subtype of alpha 1-adrenoceptor expressed is the alpha 1B.
Sugden
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Biomedical Sciences Division, King's College London.
Anwar
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N
Klein
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DC
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D003000:Clonidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004146:Dioxanes / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D010627:Phenethylamines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010870:Pineal Gland / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000145:classification* / Q000378:metabolism / Q000502:physiology; D044024:Tetralones; D014158:Transcription, Genetic
D000317:Adrenergic alpha-Antagonists; D004146:Dioxanes; D010627:Phenethylamines; D018340:Receptors, Adrenergic, alpha-1; D044024:Tetralones; C051731:chlorethylclonidine; C048008:2-(beta-(3-iodo-4-hydroxyphenyl)ethylaminomethyl)tetralone; C010654:(2-(2',6'-dimethoxy)phenoxyethylamino)methylbenzo-1,4-dioxane; D003000:Clonidine