Localization of bradykinin B2 receptors in sheep uterus.

The localization and characterization of bradykinin B2 receptors in sheep uterus was carried out using a radiolabelled B2 receptor ligand, 3,4-hydroxyphenypropionyl-D-Arg0-[Hyp3,Thi5,D-++ +Tic7,D-Oic8] bradykinin (HPP-HOE140). Competition of the radioligand, [125I]HPP-HOE140, from membrane preparations of anoestrus sheep uterus by bradykinin agonists and antagonists revealed the presence of high- and low-affinity binding sites with ligand specificity typical of the bradykinin B2 receptor. Using in vitro autoradiography on tissue sections, intense binding was visible over the superficial epithelial layer of the endometrium and inner third of the myometrium of anoestrus sheep uterus. Bradykinin B2 receptors in the myometrium were down regulated in pregnant sheep uterus. We demonstrate that binding studies using [125I]HPP-HOE140 offer high sensitivity and specificity for characterization, quantitation and localization of the bradykinin B2 receptors in tissues and offers new information on uterine bradykinin B2 receptors.

Characterization and localization of bradykinin B2 receptors in the guinea-pig using a radioiodinated HOE140 analogue.

The potent bradykinin B2 receptor antagonist analogue, [125I]HPP-HOE140, ([125I]-3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3, Thi5,D-Tic7, Oic8]bradykinin), was used to localize and characterize guinea-pig tissue bradykinin B2 receptors. Analysis of competition for the radioligand binding, using membrane preparations of lung, ileum and uterus, revealed the presence of a high- and low-affinity binding site: at the high-affinity site, the apparent Ki for the various bradykinin B2 receptor ligands ranged from 0.26 to 2.13 nM for HPP-HOE140, from 0.25 to 1.45 nM for HOE140, from 129 to 625 nM for D-Arg0[pHyp3,Phe7]bradykinin and from 0.05 to 1.11 nM for bradykinin. At the low-affinity site, the apparent Ki values ranged from 4.90 to 10.5 nM, from 1.23 to 1.90 nM, 4760 nM and from 2.01 to 62.1 nM, respectively. By contrast, the bradykinin Bi receptor antagonist, des-Arg9[Leu8]bradykinin did not compete for [125I]HPP-HOE140 binding from membrane preparations at concentrations up to 1 microM. Using in vitro autoradiography on tissue sections, intense binding was observed in the lamina propria of the villi of ileum and the arteriolar smooth muscle cells in lung. In the uterus, dense binding was found in the inner third of the myometrium and over epithelial cells of the glandular endometrium, while diffuse binding was observed throughout the endometrial stroma. In the brain, intense binding was observed in the nucleus of the solitary tract, spinal trigeminal tract and area postrema of the hindbrain, the middle cerebral arteries, and the choroid plexus of the third and lateral ventricles. Moderate binding was observed in the CA3 region of the hippocampus and posterior and ventroposterior thalamic nuclei. In the spinal cord, high-density binding occurred in the laminae I and II of the dorsal horn. Unlike previous autoradiographic localization studies of the bradykinin B2 receptor using radiolabeled bradykinin, the radiolabeled antagonist HPP-HOE140 did not bind to bradykinin-degrading peptidases, such as angiotensin-converting enzyme, and displayed subtype specificity. Therefore, binding studies with [125I]HPP-HOE140 offers high sensitivity and specificity for characterization, quantitation and localization of subtypes of bradykinin B2 receptors in tissues, and offers new information on uterine and brain bradykinin B2 receptors.

Localization of angiotensin converting enzyme by in vitro autoradiography in the rabbit brain.

The distribution of angiotensin converting enzyme was examined in the rabbit brain by in vitro autoradiography with the specific radiolabelled inhibitor 125I-351A. In the rabbit, the highest concentrations of radioligand binding were found in the choroid plexus, blood vessels, subfornical organ, vascular organ of the lamina terminalis, area postrema and inferior olive. High levels of binding were found throughout the basal ganglia, consistent with the results in all other species studied. In the midbrain the central gray and the superior colliculus displayed high levels of binding. In the medulla oblongata high levels of binding were associated with the nucleus of the solitary tract and dorsal motor nucleus of vagus, consistent with the pattern in other species. There was moderate labelling throughout both the cerebral and cerebellar cortices, which contrasts to the rat but is consistent with the situation in primates. Angiotensin converting enzyme (ACE) is more widely distributed in rabbit brain that in rat, human and Macaca fascicularis, and the results suggest ACE has a very general role in the metabolism of neuropeptides. Inhibitors of converting enzyme are very widely used in the treatment of hypertension and heart disease, and the rabbit should provide a useful model for examining the effects of these drugs in the brain.

Distribution of angiotensin II receptor binding in the spinal cord of the sheep.

The distribution of angiotensin II binding sites has been mapped at segmental levels throughout the spinal cord of the sheep using in vitro autoradiographic methods. Binding of 125I-[Sar1.Ile8] Ang II is most prominent in the lateral horns of the thoracolumbar and sacral regions containing the sympathetic and parasympathetic preganglionic neurons respectively. Binding is also present in the dorsal horns of the grey matter, in the central canal region, dorsal root ganglia and associated with non-neuronal elements such as the ependyma surrounding the central canal, and blood vessels. Displacement with receptor antagonists specific for AT1 and AT2 subtypes, indicates that angiotensin II receptors in the spinal cord are of the AT1 type. These data help to interpret the physiological actions of angiotensin II in the spinal cord, particularly with respect to its autonomic components.

Presence of angiotensin converting enzyme in the adventitia of large blood vessels.

BACKGROUND: Angiotensin converting enzyme (ACE) is present in the endothelial cells of all vascular beds. There are, however, many reports of converting enzyme activity in blood vessels not associated with the endothelium. METHODS: ACE was localized in large blood vessels of a number of mammals by in vitro autoradiography using the radioligand 125I-351A. To characterize this binding further, immunohistochemistry was performed on rabbit aorta using polyclonal antisera raised to two different preparations of rabbit lung ACE. RESULTS: In all of the blood vessels studied, which included the rabbit pulmonary artery, rabbit, dog and sheep aorta, human internal mammary artery and human saphenous vein, high levels of radioligand binding were found in endothelial cells, as expected. In addition, a very high density of punctate binding was observed interspersed between diffuse moderate labelling in the adventitia. Immunoreactivity was confined to the endothelium of both the intima and the vasa vasorum of the adventitia. The immunostaining correlated well with the autoradiography. The ACE inhibitors lisinopril and perindoprilat displayed similar high affinities in competing for the binding of 125I-351A to the endothelium and adventitia of the sheep aorta, suggesting that at these two sites the radioligand was binding to ACE. CONCLUSIONS: We find that ACE in the adventitia of large blood vessels is confined to the vaso vasorum. The results of this study help to explain the findings of many studies that ACE activity persists in endothelium-denuded blood vessels and also reveals a source of ACE distant from the luminal endothelial surface.