Neural pathways from the lamina terminalis influencing cardiovascular and body fluid homeostasis.

1. The lamina terminalis, a region of the brain with a high concentration of angiotensin AT1 receptors, consists of three distinct nuclei, the median preoptic nucleus, the subfornical organ and organum vasculosum of the lamina terminalis (OVLT). These latter two regions lack a blood-brain and detect changes in plasma angiotensin (Ang) II concentration and osmolality. 2. Efferent neural pathways from the lamina terminalis to the hypothalamic paraventricular and supraoptic nuclei mediate vasopressin secretion in response to plasma hypertonicity and increased circulating levels of AngII. 3. Studies using the neurotropic virus pseudorabies, which undergoes retrograde transynaptic neuronal transport following injection into peripheral sites, show that neurons in the lamina terminalis have efferent polysynaptic neural connections to the peripheral sympathetic nervous system. Some of these neurons have been shown to have polysynaptic connections to the kidney and to express AT1 receptor mRNA. We propose that circulating AngII acts at AT1 receptors in the subfornical organ and OVLT to influence the sympathetic nervous system. It is likely that the neural pathway subserving this influence involves a synapse in the hypothalamic paraventricular nucleus. 4. The lamina terminalis may exert an inhibitory osmoregulatory influence on renin secretion by the kidney. This osmoregulatory influence may be mediated by inhibition of renal sympathetic nerve activity and appears to involve a central angiotensinergic synapse. 5. The lamina terminalis exerts an osmoregulatory influence on renal sodium excretion that is independent of the renal nerves and is probably hormonally mediated.

Distribution of angiotensin II receptor subtypes in the rabbit brain.

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.

Localization and characterization of angiotensin II receptor binding sites in the human basal ganglia, thalamus, midbrain pons, and cerebellum.

Angiotensin II (Ang II) binding sites were localized in the thalamus, basal ganglia, midbrain, and pons of the human central nervous system by in vitro autoradiography, employing 125I-[Sar1, Ile8]angiotensin II as the radioligand. High-density binding occurs in the substantia nigra pars compacta, the interpeduncular nucleus and two of the raphe nuclei, the raphe magnus, and median raphe nucleus. Moderate densities occur in the caudate nucleus, putamen, bed nucleus of the stria terminalis, rostral linear nucleus, caudal linear nucleus, dorsal and paramedian raphe nuclei, locus coeruleus, and region of the subcoeruleus, oral dorsal paramedian nucleus, and A5/periolivary region. Low levels occur in the region between the subthalamic nucleus and the zona incerta, the mediodorsal thalamic nucleus, the central gray, the lateral and medial parabrachial nuclei, and the molecular layer of the cerebellum. The high density of Ang II receptor binding in the substantia nigra occurs over pigmented, presumably dopaminergic, neurons. The binding in this site, and in the striatum, is not observed in any of the other species we have studied. It displays similar pharmacological characteristics to the Ang II receptor binding site in other regions of the human brain. Overall we demonstrate a discrete pattern of Ang II receptor binding sites in the human brain, which shows a high correlation with the distribution observed in other mammalian species.

Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment.

Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. This complicates the use of ACE enzymatic activity as an index of inhibition in plasma or tissues after chronic administration of ACE inhibitors. We have, therefore, developed a method for ACE measurement by in vitro autoradiography using an 125I-labelled inhibitor to quantitate total ACE and the concentration of free (not inhibited) ACE in tissues after prolonged administration of ACE inhibitors to rats. Measurements made on unprocessed tissue sections reflect residual free ACE activity in the presence of the unlabelled inhibitor. In a parallel series of adjacent sections, the ACE inhibitor is dissociated from the enzyme by reversibly denaturing the enzyme by zinc chelation. This is followed by reconstitution of the active enzyme by zinc ion replacement and measuring total enzyme concentration. This technique permits measurement of the extent of ACE inhibition and induction. This method was evaluated in tissues of rats following chronic oral administration of lisinopril (10 mg/kg per day) for 2 weeks. The pattern of ACE inhibition was similar to that seen in our previous acute studies. However, induction of ACE was found to be organ specific; plasma total ACE increased 1.75-fold and total ACE in the lung increased by 30% compared with untreated animals, but there was no demonstrable change in total ACE concentration in the kidney, adrenal or aorta. Despite this, during chronic treatment with lisinopril, ACE activity in all of these organs was inhibited with low levels of free ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II receptor binding in the rat hypothalamus and circumventricular organs during dietary sodium deprivation.

The effect of dietary sodium intake on angiotensin II (Ang II) receptor binding in the rat brain was studied using quantitative in vitro autoradiography. After 2 weeks of sodium deprivation, the peripheral angiotensin system was activated as shown by increased plasma renin activity (4-fold) and plasma aldosterone concentration (approximately 40-fold). At the same time, Ang II receptor binding in the adrenal glomerulosa zone increased by 40%. Frozen brain sections prepared from 12 male Sprague-Dawley rats (6 control, 6 sodium-deprived) were incubated with 125I[Sar1, Ile8] Ang II, exposed to X-ray film, and Ang II receptor binding in individual brain nuclei was quantitated by computerized densitometry. Ang II binding in the area postrema was significantly suppressed in the sodium-deprived rats (60% of control; p less than 0.05). No change was observed in the other circumventricular organs studied, the subfornical organ or organum vasculosum of the lamina terminalis. Ang II binding in the solitary tract nucleus was not affected by the dietary salt treatment. In the hypothalamic paraventricular nucleus, there was a small (9%) but significant (p less than 0.001) increase in Ang II receptor binding in the sodium-deprived group. However, no change was observed in the hypothalamic median preoptic or suprachiasmatic nuclei, areas with similarly high Ang II receptor binding. These results suggest that only a limited subset of brain Ang II receptors respond to sodium deprivation and do so in a region-specific manner. These results support evidence that the central angiotensin system may contribute to the regulation of fluid and electrolyte homeostasis.

Differential angiotensin-converting enzyme inhibition in brain after oral administration of perindopril demonstrated by quantitative in vitro autoradiography.

To help elucidate potential sites for the central actions of a new angiotensin-converting enzyme (ACE) inhibitor, perindopril, ACE levels were measured in the brain of Sprague-Dawley rats by quantitative in vitro autoradiography after administration of the drug. Following acute oral administration of 1 mg/kg perindopril, ACE in the two circumventricular organs, the subfornical organ and organum vasculosum of the lamina terminalis, was markedly inhibited and had only partially recovered after 24 h. The ACE inhibition in the circumventricular organs did not correlate with the inhibition of ACE in plasma but with that of pressor response to intravenous angiotensin I. No or little change in ACE was observed in other brain structures which are rich in the enzyme, including the choroid plexus and basal ganglia. However, large doses of perindopril (up to 16 mg/kg) did progressively inhibit ACE in all brain structures measured, including the basal ganglia. These findings fit with the deficient blood-brain barrier known to occur in the circumventricular organs. These regions are rich in ACE and angiotensin II receptors and exhibit physiological responses to angiotensin II with effects on fluid, electrolyte, and blood pressure homeostasis. Combined with current observations, the circumventricular organs are potential targets for the centrally mediated actions of ACE inhibitors.

Localization of angiotensin II receptor binding in rabbit brain by in vitro autoradiography.

Binding of 125I-[Sar1,Ile8] angiotensin II (AII) to sections of brains from both wild and laboratory rabbits was determined by in vitro autoradiography. In the forebrain, specific high density binding was observed in the olfactory bulb, organum vasculosum of the lamina terminalis (OVLT), subfornical organ, median eminence, lateral septum, median preoptic nucleus and hypothalamic paraventricular, supraoptic and arcuate nuclei. In the midbrain, binding of the radioligand was observed in the interpeduncular and parabrachial nuclei, in the locus coeruleus, and ventrolateral pons. In the hind brain, there was dense binding of 125I-[Sar1,Ile8] AII to the nucleus of the solitary tract (NTS) and to both rostral and caudal parts of the reticular formation of the ventrolateral medulla oblongata. Weaker specific binding of the radioligand to the molecular layer of the cerebellum, to the nucleus of the spinal trigeminal tract, dorsal motor nucleus of the vagus, area postema, and to a band of tissue connecting the NTS to the ventrolateral medulla was also observed. Binding of the ligand to circumventricular organs such as the OVLT, subfornical organ, and median eminence suggests that these are sites in the brain of the rabbit at which blood-borne AII may exert influences on the central regulation of fluid balance and pituitary hormone secretion, although AII of neuronal origin could also act at these sites. Binding of the radioligand in several other brain regions suggests that angiotensin II of cerebral origin may be involved in a number of different aspects of brain function in the rabbit. The finding of dense binding in the NTS and ventrolateral medulla, which are involved in autonomic activity and are also sites of catecholamine-containing neurons, raises the possibility of angiotensin interaction with these neurons and involvement in autonomic function.

Comparative neuroanatomy of angiotensin II receptor localization in the mammalian hypothalamus.

1. The distribution of angiotensin II (AII) receptor binding sites in the hypothalamus of rat, rabbit, sheep and human was determined by in vitro autoradiography using 125I-[Sar1,Ile8]-AII as radioligand. 2. High receptor binding levels were observed in the continuum of tissue comprising the anterior wall of the third ventricle, including the subfornical organ, the median pre-optic nucleus and the organum vasculosum of the lamina terminalis. 3. High levels of binding sites were also found in the paraventricular and supra-optic nuclei, the median eminence and the arcuate nucleus. 4. These findings demonstrate sites in the hypothalamus of rat, rabbit, sheep and human where AII could exert its known actions on fluid and electrolyte balance, pituitary hormone release and cardiovascular function.

Angiotensin receptor binding in human hypothalamus: autoradiographic localization.

Binding of 125I-[Sar1,Ile8]angiotensin II in the human hypothalamus was mapped by in vitro autoradiography carried out on frozen sections of hypothalamus from two human brains. Regions showing the greatest specific binding of this radioligand were the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, median eminence, arcuate nucleus and paraventricular nucleus. These regions may be sites of angiotensin II receptors involved in the regulation of blood pressure, fluid balance and pituitary hormone secretion.

Angiotensin converting enzyme in rat brain visualized by quantitative in vitro autoradiography.

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.

Distribution of angiotensin converting enzyme in sheep hypothalamus and medulla oblongata visualized by in vitro autoradiography.

In vitro autoradiographic mapping of angiotensin converting enzyme (ACE) in sheep brain using the specific ACE inhibitor, 125I-351A, revealed very high densities of binding in large blood vessels and choroid plexus. In the a very high density of labelling occurred in the organum vasculosum of the lamina terminalis and median eminence and a high density in the subfornical organ and moderate density in supraoptic, suprachiasmatic, arcuate and paraventricular nuclei. All fiber tracts were unlabelled. In the medulla oblongata, a very high density of binding was detected in the area postrema and a high density in the nucleus of the solitary tract and dorsal motor nucleus of the vagus; a moderate density was found in the substantia gelatinosa of the spinal tract and the inferior olivary nucleus.

Autoradiographic localization of angiotensin receptors in the sheep brain.

Binding of [125I]-(Sar1,Ile8)angiotensin II (AII) to frozen sections of sheep brain was determined by in vitro autoradiography. Greatest AII-binding occurred in the organum vasculosum of the lamina terminalis, subfornical organ, median preoptic and periventricular nuclei situated in the anterior third ventricle wall. Other binding sites included the hypothalamic supraoptic and paraventricular nuclei and the medullary nucleus tractus solitarius. These regions may be central receptor sites for AII involvement in fluid and electrolyte balance and blood pressure regulation.