Interactions of orexins/hypocretins with adrenocortical functions.

The neuropeptides orexin A and B (hypocretin-1 and -2) are involved in numerous central regulation processes such as energy homeostasis, sleeping behaviour and addiction. The expression of orexins and orexin receptors in a variety of tissues outside the brain and the presence of orexin A in the circulation indicate the existence of an additional peripheral orexin system. Furthermore, it is well established that orexins exert an influence on the regulation of the hypothalamus-pituitary-adrenal axis, acting both on its central and peripheral branch. In rat and human adrenal cortices the expression of both orexin receptors has been verified with a predominance of OX(2)R. The local expression of orexin receptors was observed to be gender specific and to be modified by plasma glucose and insulin concentrations, nutritional status as well as gonadal steroids. Various studies consistently demonstrated orexin A to enhance glucocorticoid secretion of rat and human adrenal cortices, while orexin B was found to be either less potent or ineffective. On the contrary, the influence of orexins on adrenocortical aldosterone production and cell proliferation is still more controversial. Recent findings indicate that orexins stimulate adrenocortical steroidogenesis by augmenting transcription of selective steroidogenic enzymes and proteins such as steroidogenic acute regulatory protein. Both, G(q) and G(s), signalling pathways with a downstream activation of MAP kinases appear to be involved in this regulation.

Changes in the brain serotonin satiety system in transgenic rats lacking brain angiotensinogen.

In transgenic rats, TGR(ASrAOGEN)680, with reduced glial expression of angiotensinogen, changes in brain angiotensinogen are associated with reductions in serotonin (5-HT) content and/or 5-HT metabolism as determined in various brain regions, including the hypothalamus. These rats showed an anxious phenotype upon a first behavioural screen. The present study aimed to extend the search for functional consequences of changes in brain 5-HT with respect to feeding behaviour in these transgenic rats. In feeding experiments, rats were treated with the anorectic drug fenfluramine to probe for functional changes in the serotonergic satiety system. Fenfluramine (0.3 mg/kg, i.p.) reduced food intake in TGR(ASrAOGEN)680 rats whereas the minimal effective dose in wild-type rats was 3 mg/kg, i.p. Although, in the cortex, no differences were apparent in the expression of serotonin 5-HT(1A), 5-HT(1B), 5-HT(2C) receptor and 5-HT transporter mRNAs between TGR(ASrAOGEN)680 and wild-type rats, the expression of mRNAs for the 5-HT(2C) receptor and 5-HT transporter mRNA were significantly higher in the hypothalamus of TGR(ASrAOGEN)680 rats compared to wild-type rats. No differences were found in the mRNA levels for hypothalamic 5-HT(1A) and 5-HT(1B) receptors between TGR(ASrAOGEN)680 and wild-type rats. Taken together, these findings suggest that the transgenic effect on the brain 5-HT system is paralleled by functional changes of the serotonergic feeding system.

Orexins (hypocretins) and adrenal function.

The recently discovered neuropeptides orexin A and B regulate feeding behavior, neuroendocrine and autonomic functions, and sleep-wakefulness by central mechanisms. The expression of orexins and orexin receptors in various peripheral organs and the presence of orexin A in blood indicate the existence of a peripheral orexin system. In rat and human adrenal glands, both OX (1) and OX (2) receptor subtypes have been described with a predominant expression of OX (2) receptors in the adrenal cortex. In male rats, adrenocortical OX (2) receptors are much higher expressed than in female rats. Various experimental data demonstrate a stimulatory effect of orexins on the secretion of adrenocortical steroids, mainly on glucocorticoids. Some results also suggest the regulation of catecholamine synthesis and release by orexins. Whether the gender-dependent expression of adrenocortical OX (2) receptors has functional correlates awaits future clarification. As plasma orexin appears to rise during hunger and hypoglycemia, orexins may link adrenal functions with energy homeostasis.

Kinin B1 and B2 receptor mRNA expression in the hypothalamus of spontaneously hypertensive rats.

The central hypertensive effects induced by bradykinin are known to be mediated via B2 receptors, which are present constitutively in the brain. B, receptors are rapidly upregulated during inflammation, hyperalgesia, and experimental diabetes. The hypothalamus plays an important role in the regulation of cardiovascular homeostasis, and all components of kallikrein-kinin system have been identified in this area. Therefore, we analyzed the mRNA expression of B1 and B2 receptors in the hypothalamus of spontaneously hypertensive rats (SHR) by RT-PCR. Male SHR were studied at three different ages corresponding to the three phases in the development of hypertension: (i) 3-4 (prehypertensive), (ii) 7-8 (onset of hypertension), and (iii) 12-13 weeks (established hypertension) after birth, and compared with age-matched Wistar-Kyoto (WKY) rats. At all ages tested, B2 receptor mRNA levels in the hypothalamus of SHR were higher than age-matched WKY rats (p < 0.001). However, the B1 receptor mRNA levels were higher at the established phase of hypertension only. We conclude that B1 and B2 receptor mRNA are differentially expressed in the hypothalamus of SHR and may play different roles in the pathogenesis of hypertension: upregulation of B2 receptor mRNA from early age may participate in the pathogenesis of hypertension, whereas an upregulation of B1 receptor mRNA in the established phase of hypertension may reflect an epiphenomenon in essential hypertension.

Increased AT(1) receptors in adrenal gland of AT(2) receptor gene-disrupted mice.

Angiotensin II (Ang II) AT(2) receptor-gene disrupted mice have increased systemic blood pressure and response to exogenous Angiotensin II. To clarify the mechanism of these changes, we studied adrenal AT(1) receptor expression and mRNA by receptor autoradiography and in situ hybridization in female AT(2) receptor-gene disrupted mice (agtr 2-/-) and wild-type controls (agtr 2+/+). We found high expression of AT(1) receptor binding and mRNA in adrenal zona glomerulosa of female wild-type mice. AT(2) receptors and mRNA were highly expressed in adrenal medulla of wild-type mice, but were not detected in zona glomerulosa. There was no AT(2) receptor binding or mRNA in adrenal glands of AT(2) receptor-gene disrupted mice. In these animals, AT(1) receptor binding and mRNA were increased in adrenal zona glomerulosa and AT(1) receptor mRNA was increased in the adrenal medulla when compared with wild-type animals.The present data support the hypothesis of an interaction or cross talk between AT(2) and AT(1) receptors in adrenal gland. The significant increase in AT(1) receptor expression in the absence of AT(2) receptor transcription may be partially responsible for the increased blood pressure and for the enhanced response to exogenously administered Angiotensin II in this model.

Prepro-orexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats.

Orexins are produced specifically by neurons located in the lateral hypothalamus. Recent results suggested peripheral actions of orexins. Therefore, we analyzed the mRNA expression of prepro-orexin and the orexin receptor subtypes OX(1) and OX(2) in peripheral rat tissues. Using real-time quantitative RT-PCR we detected significant amounts of prepro-orexin mRNA in testis, but not in ovaries. OX(1) receptor mRNA was highly expressed in the brain and at lower levels in the pituitary gland. Only small amounts of OX(1) receptor mRNA were found in other tissues such as kidney, adrenal, thyroid, testis, ovaries, and jejunum. Very high levels of OX(2) receptor mRNA, 4-fold higher than in brain, were found in adrenal glands of male rats. Low amounts of OX(2) receptor mRNA were present in lung and pituitary. In adrenal glands, OX(2) receptor mRNA was localized in the zona glomerulosa and reticularis by in situ hybridization, indicating a role in adrenal steroid synthesis and/or release. OX(1) receptor mRNA in the pituitary and OX(2) receptor mRNA in the adrenal gland were much higher in male than in female rats. In the hypothalamus, OX(1) receptor mRNA was slightly elevated in female rats. The differential mRNA expression of orexin receptor subtypes in peripheral organs indicates discrete peripheral effects of orexins and the existence of a peripheral orexin system. This is supported by the detection of orexin A in rat plasma. Moreover, the sexually dimorphic expression of OX(1) and OX(2) receptors in the hypothalamus, pituitary, and adrenal glands suggests gender-specific roles of orexins in the control of endocrine functions.

Orexin (hypocretin) gene expression in rat ependymal cells.

The expression of prepro-orexin (PPO) mRNA in the rat brain was investigated by in situ hybridization histochemistry. In the lateral and posterior hypothalamic areas, which are considered to produce exclusively PPO mRNA, we found high levels of PPO mRNA expressions. We also localized PPO mRNA hybridization signals at lower levels around the lateral ventricles, the third and fourth ventricle. Cellular analysis by emulsion autoradiography revealed the expression of PPO mRNA in the ependymal cell layer. Our results demonstrate that beside the lateral and posterior hypothalamus PPO mRNA is expressed in ependymal cells.

Increased AT(1) receptor expression and mRNA in kidney glomeruli of AT(2) receptor gene-disrupted mice.

The proposed feedback between angiotensin II AT(2) and AT(1) receptors prompted us to study AT(1) receptor expression in kidneys of male AT(2) receptor-gene disrupted mice (agtr2 -/y). In wild-type (agtr2 +/y) mice, AT(1) receptor binding and mRNA is abundant in glomeruli, and AT(1) receptor binding is also high in the inner stripe of the outer medulla. AT(2) receptors are scarce, primarily associated to cortical vascular structures. In agtr2 -/y mice, AT(1) receptor binding and mRNA were increased in the kidney glomeruli, and AT(1) receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT(2) receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT(1) receptor expression. AT(1) upregulation alone may explain the AT(2) gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT(1)/AT(2) receptor feedback could have clinical significance because AT(1) antagonists are widely used in medical practice.

Gerbil angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development.

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT1 Angiotensin II receptors. The recently cloned gerbil AT1 receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT1 receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region. We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life. The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT1 receptors in the development and function of the corticohippocampal system.

Local renin-angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells.

NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.

Ischemia-induced neuronal cell loss is associated with loss of atypical angiotensin type-1 receptor expression in the gerbil hippocampal formation.

The hippocampal formation of Mongolian gerbils expresses high amounts of atypical angiotensin II type-1 receptors. We studied the expression of these receptors by in situ hybridization using specific [35S]-labeled riboprobes and by receptor autoradiography using [125I]Sarcosine1-angiotensin II. Angiotensin II receptor mRNA was found in the pyramidal cell layer of the CA1, CA2 and CA3 subfields, with the highest expression in the CA2 subfield, and in the granular cell layer of the dentate gyrus. Angiotensin II binding was detected in the stratum oriens and stratum radiatum of the CA1 and CA2 subfields, in the stratum oriens of the CA3 subfield, and in the molecular layer of the dentate gyrus. We then studied the effect of ischemia on hippocampal angiotensin II receptor expression, 1, 4 and 15 days after bilateral occlusion of the common carotid arteries for 5 min. No changes in angiotensin II receptor mRNA or binding were detected 1 day after ischemia. Delayed, progressive loss of angiotensin II mRNA and binding occurred 4 and 15 days after ischemia, in the CA1, CA2 and CA3 subfields. The decline was faster in the CA1 subfield, and paralleled the loss of neurons after ischemia. In the dentate gyrus, angiotensin II receptor mRNA and angiotensin II binding were not changed when compared to sham operated controls. The decrease of angiotensin II receptor expression may reflect the loss of angiotensin II receptor-producing neurons rather than a down-regulation of receptor expression.

The angiotensin AT1 receptor antagonist CV-11974 regulates cerebral blood flow and brain angiotensin AT1 receptor expression.

We studied cerebral blood flow autoregulation by laser Doppler flowmetry, and expression of brain angiotensin II AT1 receptors by quantitative autoradiography, after administration of an angiotensin AT1 receptor antagonist, CV-11974 (Candesartan, 0.5 or 1.0 mg/kg.day) for two weeks via subcutaneously implanted osmotic pumps in adult normotensive Wistar Kyoto and spontaneously hypertensive male rats (SHR). In SHR, the autoregulation curve was shifted towards higher blood pressures, when compared with that of normotensive Wistar Kyoto rats. Administration of CV-11974 shifted the autoregulation curve toward lower blood pressures in both Wistar Kyoto and SHR, partially normalizing the autoregulation curve in SHR. CV-11974 treatment markedly decreased the expression of AT1 receptors in Wistar Kyoto rats, both in areas outside the blood brain barrier (subfornical organ, 95% decrease) and inside the blood brain barrier (nucleus of the tractus solitarius, 87% decrease, and paraventricular nucleus, 96% decrease). Our results demonstrate that blockade of AT1 receptors tends to normalize the shift to higher pressures in the autoregulation curve of genetically hypertensive rats, and has a profound modulatory role in brain angiotensin II AT1 receptors.

Molecular cloning and pharmacological characterization of an atypical gerbil angiotensin II type-1 receptor and its mRNA expression in brain and peripheral tissues.

In the gerbil brain, most of the [125I]Sarcosine1-Angiotensin II binding sites are atypical, not sensitive to displacement with selective Angiotensin II AT1 and AT2 receptor ligands. A similar atypical binding profile exists in the gerbil kidney, where binding is highly expressed. We isolated a 2197 base pair clone from a gerbil kidney cDNA library which encodes a 359 amino acid protein with higher than 90% homology to other mammalian angiotensin II AT1 receptors. When expressed in COS-7 cells, stimulation by Angiotensin II of both the cloned gerbil receptor or the human AT1 receptor enhanced IP3 production to a similar degree. In COS-7 cells, the gerbil receptor also had a ligand affinity profile similar to that of the human AT1 receptor, but showed greatly reduced affinity for losartan (IC50=3480+/-174 nM). In the gerbil brain, in situ hybridization revealed receptor mRNA in circumventricular organs, selective hypothalamic, midbrain and brain stem areas, and in the hippocampus, where high mRNA expression was detected in the stratum pyramidale of the CA1 and CA2 subfields, and in the stratum granulosum of the dentate gyrus. The expression pattern of receptor mRNA corresponded well with that of atypical [125I]Sar1-Ang II binding. In situ hybridization and Southern blot experiments using riboprobes against the open reading frame and the 3'-untranslated region of the cloned gerbil Ang II receptor cDNA suggest that gerbils have, like other rodents, two AT1 receptor subtypes. The receptor mRNA distribution of the cloned gerbil Ang II receptor corresponds to the distribution of AT1A receptors described in other rodent species.

Characterization and distribution of angiotensin II receptor subtypes in the mouse brain.

We localized and characterized angiotensin II receptor subtypes (AT1 and AT2) in the mouse brain, with the use of autoradiography after incubation with [l25I][Sar1]-angiotensin II or [125I]CGP 42112 and displacement with selective angiotensin AT1 (losartan and candesartan) or angiotensin AT2 (CGP 42112(1) and PD 123319(2)) receptor ligands. In the mouse, the receptor subtype affinity for the different ligands was similar to that of the rat. The receptor subtype distribution was also similar to that in the rat, with some notable exceptions, such as the presence of angiotensin AT1 but not AT2 receptors in the locus coeruleus, and the expression of angiotensin AT1 receptors in the caudate putamen. These results confirm that careful consideration of the specific distribution of receptor subtypes in different species, even those closely related such as the mouse and the rat, should be conducted before meaningful comparisons could be proposed. Our data also form the basis for future studies of mouse models such as those with angiotensin receptor gene deficiencies.

Chemical lesion of the inferior olive reduces [125I]sarcosine1-angiotensin II binding to AT2 receptors in the cerebellar cortex of young rats.

In young rats, AT2 receptors and AT2 receptor mRNA are discretely localized in neurons of the inferior olive, with highest expression in the medial nucleus. We previously detected AT2 receptor binding, but not AT2 receptor mRNA, in the molecular layer of the cerebellar cortex. To determine whether AT2 receptors are expressed in climbing fiber terminals which arise to the molecular layer from the inferior olive and innervate Purkinje cells, we chemically destroyed olivary neurons of 2-week-old rats by intraperitoneal (i.p.) injection of the neurotoxin 3-acetylpyridine. Lesions of the inferior olive reduced [125I]Sar1-Ang II binding to AT2 receptors and AT2 receptor mRNA levels in this area by 50%, and produced a similar decrease in AT2 receptor binding in the molecular layer of the cerebellar cortex. The extent of binding reduction was similar 3 days and 7 days after the lesion. 3-Acetylpyridine lesions did not change [125I]Sar1-Ang II binding to AT1 receptors in the molecular layer of the cerebellar cortex or AT1 receptor mRNA levels in Purkinje cells. AT2 receptor binding and AT2 receptor mRNA levels in the deep cerebellar nuclei were also not affected by 3-acetylpyridine. Our results support the hypothesis that AT2 receptors are produced by inferior olivary neurons and transported through climbing fibers to the molecular layer of the cerebellar cortex. The high expression of AT2 receptors in the inferior olivary-cerebellar pathway during a crucial time in postnatal development of climbing fiber-Purkinje cell connectivity suggest a role of AT2 receptors in the development of this pathway.

Angiotensin II AT1A receptor mRNA expression is induced by estrogen-progesterone in dopaminergic neurons of the female rat arcuate nucleus.

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

Water deprivation upregulates ANG II AT1 binding and mRNA in rat subfornical organ and anterior pituitary.

We studied angiotensin II (ANG II) receptor subtype expression in selected brain nuclei and pituitary gland after water deprivation by in vitro receptor autoradiography using 125I-labeled [Sar1]ANG II and by in situ hybridization using 35S-labeled AT1A, AT1B, and AT2 receptor-specific riboprobes. In control rats we found binding to AT1 receptors in the subfornical organ, paraventricular nucleus, median eminence, and anterior pituitary; AT1A mRNA expression in the subfornical organ and paraventricular nucleus; and AT1B mRNA expression in the anterior pituitary. No receptor mRNA was found in the median eminence. AT1 receptors and AT1A receptor mRNA levels were increased in the subfornical organ, and, in the anterior pituitary, AT1 receptors and AT1B receptor mRNA were increased, only after 5 days of water deprivation. No significant changes occurred after 1 or 3 days of water deprivation, and no regulation of ANG II receptor expression was detected in other brain areas. Our results show that prolonged water deprivation selectively regulates AT1 receptor expression and AT1A and AT1B receptor mRNA levels in the subfornical organ and anterior pituitary, respectively, supporting a role for these receptors during sustained dehydration.

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus.

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [125I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [125I]Sar1-Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [125I]Sar1-Ang II binding was displaced by the AT1 receptor antagonist losartan, indicating the presence of AT1 receptors. In the paraventricular nucleus [125I]Sar1-Ang II binding was displaced by Ang II (Ki = 7.6 X 10(-9)) and losartan (Ki = 1.4 X 10(-7)) but also by the AT2 receptor ligand PD 123319 (Ki = 5.0 X 10(-7)). In addition, a low amount of AT2 receptor binding was detected in the paraventricular nucleus using [125I]CGP42112 as radioligand, and the binding was displaced by Ang II (Ki = 2.4 X 10(-9)), CGP42112 (Ki = 7.9 x 10(-10)), and PD123319 (Ki = 2.2 x 10(-7)). ACE, Ang II, and AT1 as well as AT2 receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.

Increased endothelin ET(A) receptor expression in rat carotid arteries after balloon injury.

Endothelins are vasoactive peptides and are believed to act as vascular smooth muscle mitogens. Vascular injury results in medial smooth muscle migration and proliferation with the formation of a neointima. Using quantitative autoradiography, we examined the expression of endothelin receptor subtypes ET(A) and ET(B) in the rat carotid artery 2, 8, and 16 days after balloon-catheter injury. At two and eight days after balloon catheterization, ET(A) receptor expression was significantly increased in the media of the injured vessel when compared to that in the media of the intact vessel. The enhanced expression of receptors returned to normal levels by 16 days after the injury. Neointimal cells also expressed ET(A) receptors at a lower level than that expressed by the injured media 8 days after injury, and continued to express ET(A) receptors 16 days after the injury. ET(B) receptors were not detectable in the media or the neointima at any time after the injury. Our results suggest the ET(A) receptors may have a significant role in injury induced vascular smooth muscle proliferation and neointima formation.

Cellular localization and expression of the serotonin transporter in mouse brain.

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.