Abstract:
:The binding of the selective alpha 2-adrenoceptor antagonist radioligand [3H]rauwolscine ([3H]RAUW) to homogenates of cat frontal cerebral cortex and cat lumbar spinal cord was investigated. Experiments were performed at 20 degrees C in 50 mM Tris HCl/l mM Na2EDTA buffer (pH 6.9 at 20 degrees C). At this temperature, specific [3H]RAUW binding, defined as the difference between the amount of [3H]RAUW bound in the absence and presence of 1 microM rauwolscine or 1 microM rauwolscine or 1 microM yohimbine, reaches equilibrium values by approximately 60 min and is reversible with a mean t1/2 of dissociation of 15 min in cortex and 20 min in spinal cord. The kinetically determined Kd of [3H]RAUW (mean K-1/mean K1) was 0.59 nM and 1.68 nM in cortex and spinal cord, respectively. The results of equilibrium saturation experiments, routinely performed at [3H]RAUW concentrations between 0.1 nM and 6.0 nM, indicate that [3H]RAUW binds to saturable sites in both CNS regions of the cat. Scatchard plots of saturation isotherm data were consistently linear and the mean Kd value determined from 10 such experiments was 0.72 nM in frontal cortex and 0.82 nM in lumbar spinal cord. A mean Bmax value of 230 fmol/mg protein was determined for saturable [3H]RAUW binding sites in the cat frontal cortex. In teh cat lumbar spinal gray, a mean Bmax value for saturable [3H]RAUW binding sites of 75 fmol/mg protein was obtained. Saturable [3H]RAUW binding sites in the cat lumbar spinal gray are present at apparently equal density in dorsal and ventral horns. Inhibition experiments, performed at 0.2 nM or 0.4 nM [3H]RAUW, indicate that the binding sites labeled by [3H] RAUW possess a pharmacology characteristic of alpha-adrenoceptors. Thus, rauwolscine, yohimbine, and phentolamine compete for specific [3H]RAUW binding with high affinity and are much more potent inhibitors than corynanthine, prazosin, and propranolol. Mean Hill coefficients, calculated from logit-log plots of competition data, were close to one for all antagonists examined. L-Epinephrine and L-norepinephrine were 15-20 times more potent inhibitors of specific [3H]RAUW binding than were their corresponding D-isomers. The agonist inhibitor potency series: p-aminoclonidine = clonidine = L-epinephrine greater than L-norepinephrine much greater than isoproterenol, is that expected of alpha 2-adrenoceptor sites. Mean Hill coefficients efficients for all agonists were considerably less than one.(ABSTRACT TRUNCATED AT 400 WORDS)
journal_name
Brain Resjournal_title
Brain researchauthors
Howe JR,Yaksh TLdoi
10.1016/0006-8993(86)91045-0subject
Has Abstractpub_date
1986-03-12 00:00:00pages
87-100issue
1eissn
0006-8993issn
1872-6240pii
0006-8993(86)91045-0journal_volume
368pub_type
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