Rat Cerebral Membranes Research Articles

Inhibition of 3H-imipramine binding by plasma from depressed and normal subjects

The inhibition by human plasma of 3H-imipramine ( 3H-IMI) specific binding to rat cerebral membranes was investigated using platelet-free plasma (PFP) from normal and depressed subjects. The specific binding of 3H-IMI at a ligand concentration of 4 n M decreased with increasing PFP volumes, reaching 50% inhibition following the addition of 10–13 μl PFP to a final incubation volume of 250 μl. The extent of this inhibition was the same using PFP from controls or depressed patients. The inhibitory activity was associated with plasma proteins. Scatchard analysis of 3H-IMI binding in the presence and absence of PFP indicated that the inhibitory effect was associated with an increased K d without an appreciable change in B max. It is suggested that an endogenous acceptor in the PFP, such as α 1-acid-glycoprotein may be responsible for the observed inhibition of 3H-IMI binding to the cerebral membranes.

Identification of aminopeptidase M as an enkephalin-inactivating enzyme in rat cerebral membranes

Two membrane-bound enkephalin-hydrolyzing aminopeptidase activities were partially purified from rat brain membranes. The first, which represents 90% of the total activity, was highly sensitive to both puromycin (Ki = 1 microM) and bestatin (Ki = 0.5 microM). The second was inhibited much more by bestatin (Ki = 4 microM) than by puromycin (Ki = 100 microM). The latter puromycin-insensitive aminopeptidase was found to resemble aminopeptidase M purified from rat kidney brush border membranes. Both displayed the same purification pattern and the same kinetic constants of substrates and inhibitors, and both were similarly inactivated by metal chelating agents. Moreover, antibodies raised in rabbits against rat kidney aminopeptidase M inhibited the aminopeptidase activities of both kidney and brain puromycin-insensitive enzymes at similar dilutions, while the brain puromycin-sensitive aminopeptidase activity was not affected. Thus, aminopeptidase M (EC 3.4.11.2) was found to occur in brain, and the role of this enzyme in inactivating endogenous enkephalins released from their neuronal stores is suggested.

Study of two alkylating derivatives: The p-isothiocyanato- and the p-methylisothiocyanato-clonidine

Pharmacological experiments with isolated rat aorta and radioligand binding studies in rat cerebral membranes were performed with the p-isothiocyanato (p-NCS) and p-methylisothiocyanato (p-CH 2-NCS) derivatives of clonidine in order to assess their selectivity for α 1- and α 2-adrenoceptors, and to characterie their ability to alkylate α-adrenoceptors. Preincubation of rat aortic strips with both derivatives produced non-parallel rightward shifts in the dose-response curves of noradrenaline and significantly depressed the maximum response in a manner characteristic of irreversible receptor antagonists. The p-CH 2-NCS derivative was slightly more potent than the p-NCS derivative. Further analysis of the data indicated that treatment of rat aorta with a 30 μM concentration of the p-CH 2-NCS derivative alkylated all but 2.4 percent of the α-adrenoceptors, whereas a 100 μM concentration of the p-NCS derivative was required to produce a similar degree of α-adrenoceptor alkylation. Radioligand binding studies indicate an apparent 2 fold α 2-adrenoceptor selectivity for the p-NCS derivative. In contrast, the p-CH 2-NCS derivative displayed 7 fold selectivity for α 1-adrenoceptors. Interestingly, both alkylating derivatives of clonidine produced dose-dependent contractile responses in rat aorta with pD 2 values of 6.30 and 5.56 for the p-NCS and p-CH 2-NCS derivatives, respectively, relative to a pD 2 of 7.67 for clonidine. The order of potency of the two alkylating derivatives of clonidine for producing contraction of rat aorta is the opposite of that for antagonizing the contractile effects of noradrenaline. The results suggest that the p-NCS and p-CH 2-NCS derivatives of clonidine non-competitively antagonize noradrenaline by irreversibly alkylating α-adrenoceptors.

Hemodynamic characterization of pinacidil in rats. Comparison with hydralazine.

Pinacidil (N"-cyano-N-4-pyridyl-N'-1,2,2-trimethylpropylguanidine monohydrate; P1134) is a new vasodilator drug with a direct relaxant effect on vascular smooth muscle. Its hemodynamic properties, in comparison with those of hydralazine, were studied in conscious normotensive and spontaneously hypertensive rats; anesthetized normotensive rats; pithed normotensive rats; pithed normotensive rats subjected to electrical stimulation of the spinal cord. Radioligand binding studies on rat cerebral membranes were carried out to study a possible affinity for pinacidil towards alpha 1- and alpha 2-adrenoceptors, respectively. The observations made in conscious and anesthetized rats suggest that both pinacidil and hydralazine are predominantly arterial vasodilators. In conscious animals reflex tachycardia was elicited by both drugs. Neither pinacidil nor hydralazine possessed substantial affinity for alpha 1- or alpha 2-adrenoceptors, as concluded from radioligand binding studies. Pinacidil interferes with the pressor response to postsynaptic alpha 2-adrenoceptor stimulation in pithed rats, possibly reflecting weak calcium antagonistic activity of the drug. Pinacidil did not interfere with the electrically induced release of noradrenaline from presynaptic sites. All results suggest that pinacidil is a direct-acting arteriolar dilator, which on a molar base is somewhat more potent than hydralazine.

Binding characteristics of [ 3H] guanfacine to rat brain α-adrenoceptors : Comparison with [ 3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 Ci mmole ) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [ 3H]clonidine (sp. act. 26.7 Ci mmole ). The nonspecific binding of [ 3H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [ 3H]clonidine. The specific binding of [ 3H] guanfacine (0.1–20 nM) and [ 3H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (−)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K D values of 3.9 ([ 3H]guanfacine) and 3.7 nM ([ 3H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 fmole mg protein for [ 3H]guanfacine and [ 3H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [ 3H] guanfacine, K d value and maximal number of binding sites were about twice as high as determined in the presence of excess (−)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [ 3H] guanfacine in rat brain possess the general characteristics of α 2-adrenoceptors. Selective antagonists of α 2-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α 1-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [ 3H]guanfacine was identical to their effectiveness in competing for [ 3H]clonidine specific binding sites. It is concluded that [ 3H]guanfacine labels the same α 2-adrenoceptor population in rat brain as [ 3H]clonidine. However, [ 3H]guanfacine seems not as suitable as [ 3H]clonidine for routine use in the direct identification of α 2-adrenoceptors in view of its relatively high nonspecific binding.

Quantitative assessment of heterogeneous 3H-spiperone binding to rat neostriatum and frontal cortex

Anomalies of the binding of 3Hspiperone to rat cerebral membranes have been examined. By employing a very low ligand concentration ( ∼ 25 pM 3Hspiperone ) we have demonstrated that even within the corpus striatum, 3Hspiperone appears to bind to multiple sites and that dopaminergic and serotonergic agents can selectively inhibit from these sites. In the corpus striatum, 75–80% of the 3Hspiperone specific binding can be inhibited with high affinity by dopaminergic drugs while some 20–30% is inhibited with high affinity by serotonergic compounds. The two 3Hspiperone sites, which we have shown to have affinities of 31 and 325 pM, may therefore represent dopaminergic and serotonergic sites. At higher concentrations of 3Hspiperone, however, the picture may be complicated by a further low affinity site. The great selectivity shown by dopaminergic agonists for the two 3Hspiperone sites explains the ‘flattened’ displacement curves reported for 3Hspiperone/agonist interactions. As dopaminergic agents show the greater affinity for the high affinity 3Hspiperone site, it is tempting to speculate that this site has the greatest association with the dopamine receptor.

A comparative study of [ 3H] haloperidol and [ 3H] spiroperidol binding to receptors on rat cerebral membranes

FEBS LettersVolume 87, Issue 1 p. 152-156 Full-length articleFree Access A comparative study of [3H] haloperidol and [3H] spiroperidol binding to receptors on rat cerebral membranes First published: March 01, 1978 https://doi.org/10.1016/0014-5793(78)80155-0Citations: 41AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References [1] Hornykiewicz O., Brit. Med. Bull., 29, (1973), 172– 178. [2] Hornykiewicz O., Ann. Rev. Pharmacol. Toxicol., 17, (1977), 545– 559. [3] Seeman P., Biochem. Pharmacol., 26, (1977), 1741– 1748. [4] Burt D.R., Creese I., Snyder S.H., Mol. Pharmacol., 12, (1976), 800– 812. [5] Enna S.J., Bennett J.P. jr, Burt D.R., Creese I., Snyder S.H., Nature, 263, (1976), 338– 341. [6] Janssen P.A.J., Van Bever W.F.M., Curr. Develop. Psychopharmacol., 2, (1975), 165– 184. [7] Fields J.Z., Reisine T.D., Crawford D., Enna S.J., Yamamura H.I., Trans. Am. Soc. Neurochem., 8, (1977), 193– [8] Lindvall O., Bjorklund A., Divac I., E. Costa G.L. Gessa Non-striatal Dopaminergic Neurons (1977), Raven Press New York 39– 46. Citing Literature Volume87, Issue1March 01, 1978Pages 152-156 ReferencesRelatedInformation