Rat pineal α1-adrenoceptor subtypes: studies using radioligand binding and reverse transcription-polymerase chain reaction analysis (original) (raw)

1996, British Journal of Pharmacology

The pharmacological characteristics of a,-adrenoceptor binding sites in rat pineal gland membranes, detected by use of a selective a,-adrenoceptor antagonist ([I25I]-iodo-2-[#-(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 gM, 10 min) of rat pineal membranes inactivated-70% of specific ['251]-HEAT binding sites. Higher concentrations of CEC (up to 100 gM) or longer treatment periods (upto 40 min) were no more effective. 3 Adrenoceptor agonists and antagonists competitively inhibited [1251]-HEAT binding with Hill coefficients close to unity indicating a single a1-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 xB-adrenoceptor subtype. 4 The (-)and (+)-enantiomers of niguldipine had an equal and low affinity for a,-adrenoceptor binding sites both in untreated (log K-6.66 and-6.90 respectively) and CEC-treated membranes in which-70% of sites had been inactivated (log Ki-6.41 and-6.86 respectively). This indicates that the small proportion of a,-adrenoceptors insensitive to CEC are not aIA-adrenoceptors. 5 mRNA was isolated from rat pinealocytes, cDNA was synthesized and then amplified by the polymerase chain reaction with ac-adrenoceptor subtype specific primers. These experiments identified both aIA-and C(IB-adrenoceptor mRNA, but not aCD-mRNA in rat pinealocytes, although all three adrenoceptor subtypes were readily identified in rat brain cortex.