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Central administration of angiotensin IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle1-Ang IV or LVV-haemorphin-7, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle1-Ang IV or LVV-haemorphin-7 to achieve learner criterion is significantly reduced compared with controls (P < 0.001 and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (1) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either 100 pmol or 1 nmol of Nle1-Ang IV or 100 pmol LVV-haemorphin-7 performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle1-Ang IV or LVV-haemorphin-7 made fewer errors (P < 0.01 and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle1-Ang IV (100 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (10 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammon's horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.

Angiotensin converting enzyme is localized in the monkey (Macaca fascicularis) brain by in vitro autoradiography using the radiolabelled inhibitor, [125I]351A. This radioligand binds with high affinity and specificity to monkey cortical sections. Specific inhibitors of converting enzyme, lisinopril and perindoprilat complete for the radioligand binding to monkey cortex sections with inhibitory constants of 10 nM. High concentrations of angiotensin converting enzyme occur in most components of the basal ganglia including the caudate nucleus, putamen, the internal and external globus pallidus, nucleus accumbens, ventral pallidum and the reticular part of the substantia nigra. The distribution of converting enzyme in the caudate nucleus and putamen is heterogeneous, with prominent patches of higher activity. The patches in the caudate nucleus correspond closely with the acetylcholinesterase-poor striosomes. In the hypothalamus, very high levels of angiotensin converting enzyme occur in the median eminence and the pituitary stalk and high concentrations occur in the supraoptic and suprachiasmatic nuclei. Moderate, diffuse binding is observed in the median preoptic nucleus, the medial preoptic area, and in the anterior, lateral, ventromedial, posterior and arcuate nuclei. In the circumventricular organs, the subcommissural and subfornical organs exhibit high levels of angiotensin converting enzyme. The organum vasculosum of the lamina terminalis and the pineal body display moderate enzyme activities whereas the area postrema is devoid of labelling. The interpeduncular nucleus and, in the hippocampal formation, the molecular layer of the dentate gyrus are also intensely labelled. High levels of angiotensin converting enzyme activity are also detected throughout the cerebral cortex with laminations of higher activity corresponding to cell dense layers of the cortex. Layered binding is also present in the cerebellar cortex, with the most intense labelling in the molecular layer. Moderate concentrations of converting enzyme also occur in the paraventricular, medial habenula, lateral habenula and central median nuclei of the thalamus, the amygdala, the central gray, the locus coeruleus, the parabrachial nucleus and dorsal tegmental nucleus. The dorsal vagal complex, inferior olivary nucleus and the caudal subnucleus of the spinal trigeminal nucleus all display high levels of binding. Moderate, diffuse labelling is found throughout the reticular region and is also present in the gracile and cuneate nuclei. Although the overall distribution of angiotensin converting enzyme in the monkey brain resembles that in the rat, there are some striking differences. These include the high levels of binding throughout the monkey cerebral cortex and in the interpeduncular and suprachiasmatic nuclei.

Angiotensin converting enzyme was localized in rat brain by quantitative in vitro autoradiography using an [125I]labelled converting enzyme inhibitor called "351A". This radioligand was found to bind with high affinity and specificity to angiotensin converting enzyme. Very high levels of converting enzyme were observed in the ventricular choroid plexus, ependyma of all ventricles and large and medium blood vessels, subfornical organ, and organum vasculosum of the lamina terminalis. High levels of converting enzyme were found in the basal ganglia including caudate putamen, nucleus accumbens, globus pallidus, entopenduncular nucleus and substantia nigra pars reticulata. The neurosecretory nuclei, paraventricular nucleus and supraoptic nucleus, as well as the median eminence and posterior pituitary displayed high levels of the enzyme. In the amygdala, basolateral, lateral, basomedial, medial and anterior cortical nuclei showed moderate converting enzyme activity. The medial habenula and molecular layer of the dentate gyrus showed high levels of activity. In the cerebellum, dense labelling was observed in the Purkinje cell layer. Moderate levels of converting enzyme occurred in the gelatinosus subnucleus of the caudal part of the nucleus of the spinal tract of the trigeminal. There was a close correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the neurosecretory nuclei (paraventricular and supraoptic nuclei) and median eminence and this suggests a role of angiotensin converting enzyme in the production of angiotensin II in this system. There was also a good correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the subfornical organ, median preoptic nucleus, and organum vasculosum of the lamina terminalis, structures abutting the anterior wall of the third ventricle which are implicated in fluid and electrolyte homeostasis. A striking discrepancy occurs in the basal ganglia which is reported to contain very little angiotensin II or angiotensin II receptors but is very rich in angiotensin converting enzyme. It is concluded that the enzyme may act to convert circulating angiotensin I to angiotensin II in circumventricular organs; generate intraneuronal angiotensin II in pathways such as the hypothalamic-hypophyseal tract; and process neuropeptides other than angiotensin II in regions such as basal ganglia.

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.

Vasopressin may act in the brain as a neurotransmitter or neuromodulator to influence blood pressure, memory, body temperature and brain development. In order to localize probable central nervous system sites for these actions, we have used 125I-labelled 1-d(CH2)5, 7-sarcosine-8-arginine vasopressin, a specific V1-receptor antagonist, and in vitro autoradiography to map brain vasopressin binding sites. High levels of binding were found in the choroid plexus, blood vessels, lateral septum, bed nucleus of stria terminalis, accumbens nucleus, central nucleus of amygdala, stigmoid hypothalamic nucleus, suprachiasmatic nucleus, arcuate nucleus, nucleus of the solitary tract, area postrema and parts of the hippocampus, thalamus, superior colliculus, and inferior olivary nuclei. Many of these regions are known to be vasopressin-sensitive and to contain vasopressin fibres. Significantly there was no binding to the paraventricular nor the supraoptic nuclei. Displacement of the radioligand from the lateral septum with unlabelled vasopressin analogues gave a rank order of potencies: d(CH2)5-D-Tyr2(Et)Val4-desGly9-arginine-vasopressin approximately equal to d(CH2)5-Tyr2-(Me)arginine-vasopressin approximately equal to arginine-vasopressin approximately equal to d(CH2)5-Sar7-arginine-vasopressin greater than [1-deamino, 8-D-arginine]-vasopressin approximately equal to oxytocin much greater than vasopressin4-9, consistent with binding to V1 receptor subtype. These studies confirm and extend previous findings of V1 receptors in the rat brain. In particular, several new regions of vasopressin receptor binding have been identified, possibly due to the advantages of a radioiodinated ligand with high receptor affinity without binding to neurophysins. Future study of these regions may prove fruitful in elucidating the central actions of vasopressin.

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [125I]-[Sar1,Ile8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.