Walter Häuser

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.

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.

Differential Expression of AT1 Receptors in the Pituitary and Adrenal Gland of SHR and WKY

Abstract—The renin-angiotensin (ANG) system has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR). Because SHR are more susceptible to stress than normotensive Wistar-Kyoto rats (WKY), we measured the mRNA expression of AT1A, AT1B, and AT2 receptors in the hypothalamo-pituitary-adrenal (stress) axis of male SHR in comparison to age-matched WKY at prehypertensive (3 to 4 weeks), developing (7 to 8 weeks), and established (12 to 13 weeks) stages of hypertension. AT1A receptor mRNA was mainly expressed in the hypothalamus and adrenal gland. AT1B receptor mRNA was detected in the pituitary and adrenal gland. AT2 receptor mRNA was prominent only in the adrenal gland. When compared with WKY, SHR showed increased AT1A receptor mRNA levels in the pituitary gland at all ages in contrast to reduced pituitary AT1B receptor mRNA levels. In the adrenal gland of SHR, AT1B receptor mRNA levels were decreased at the hypertensive stages when compared with WKY. The reduced expression of adrenal AT1B receptor mRNA was localized selectively in the zona glomerulosa by in situ hybridization. No differences were observed between WKY and SHR in the expression of hypothalamic ANG receptors. ANG significantly increased plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in dexamethasone-treated SHR but not in WKY. The aldosterone response to ANG was similar in SHR and WKY. Our results suggest a differential gene expression of AT1A and AT1B receptors in the hypothalamo-pituitary-adrenal axis of SHR and normotensive WKY and imply the participation of AT1 receptors in an exaggerated endocrine stress response of SHR to ANG.

Increased angiotensin II AT1 receptor expression in paraventricular nucleus and hypothalamic-pituitary-adrenal axis stimulation in AT2 receptor gene disrupted mice

Angiotensin II AT2 receptor gene-disrupted mice have increased blood pressure and response to angiotensin II, behavioral alterations, greater response to stress, and increased adrenal AT1 receptors. We studied hypothalamic AT1 receptor binding and mRNA by receptor autoradiography and in situ hybridization, adrenal catecholamines by HPLC, adrenal tyrosine hydroxylase mRNA by in situ hybridization and pituitary and adrenal hormones by RIA in AT2 receptor-gene disrupted mice and wild-type controls.To confirm the role of adrenal AT1 receptors, we treated wild-type C57 BL/6J mice with the AT1 antagonist candesartan for 2 weeks, and measured adrenal hormones, catecholamines and tyrosine hydroxylase mRNA. In the absence of AT2 receptor transcription, we found increased AT1 receptor binding in brain areas involved in the regulation of the hypothalamic-pituitary-adrenal axis, the hypothalamic paraventricular nucleus and the median eminence, and increased adrenal catecholamine synthesis as shown by higher adrenomedullary tyrosine hydroxylase mRNA and higher adrenal dopamine, norepinephrine and epinephrine levels when compared to wild-type mice. In addition, in AT2 receptor gene-disrupted mice there were higher plasma adrenocorticotropin (ACTH) and corticosterone levels and lower adrenal aldosterone content when compared to wild-type controls. Conversely, AT1 receptor inhibition in CB57 BL/6J mice reduced adrenal tyrosine hydroxylase mRNA and catecholamine content and increased adrenal aldosterone content. These results can help to explain the enhanced response of AT2 receptor gene-disrupted mice to exogenous angiotensin II, support the hypothesis of cross-talk between AT1 and AT2 receptors, indicate that the activity of the hypothalamic-pituitary-adrenal axis parallels the AT1 receptor expression, and suggest that expression of AT1 receptors can be dependent on AT2 receptor expression. Our results provide an explanation for the increased sensitivity to stress in this model.

Effects of the AT1 Antagonist HR 720 in Comparison to Losartan on Stimulated Sympathetic Outflow, Blood Pressure, and Heart Rate in Pithed Spontaneously Hypertensive Rats

It has been demonstrated in isolated organs that angiotensin II mediates catecholamine release via presynaptically located AT1 receptor subtypes. In the present study, the relevance of AT1-mediated noradrenaline and adrenaline release in a whole-animal model, which reflects the peripherally sympathetic system (pithed rat), was investigated. Furthermore, the effects of a new AT1 antagonist, HR 720, are demonstrated with respect to its pre- and postsynaptic actions in comparison to the AT1 antagonist losartan. Dose-response curves to angiotensin II of blood pressure show a tenfold higher potency for HR 720 to compete for angiotensin II, thereby decreasing the maximum effects when compared with losartan. The electrically induced sympathetic outflow resulted in a dose-dependent increase after angiotensin II infusions. It could markedly be reduced with both AT1 antagonists, whereby HR 720 again was ten times more potent than losartan. Neither with HR 720 nor with losartan an agonistic activity could be demonstrated. The results indicate an AT1 receptor subtype mediated release of catecholamines in a whole-animal model. HR 720 is ten times more potent than the AT1 antagonist losartan and acts in a noncompetitive manner.

Increased AT1 receptors in adrenal gland of AT2 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.

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.

Centrally bradykinin B2-receptor-induced hypertensive and positive chronotropic effects are mediated via activation of the sympathetic nervous system.

OBJECTIVE The presence of bradykinin B2 receptors in the cardiovascular regulatory centres of the brain indicates that increase in mean arterial pressure (MAP) and heart rate after intracerebroventricular (i.c.v.) injections of bradykinin is mediated via stimulation of sympathetic nervous system. METHODS Adult Wistar- Kyoto (WKY) rats were instrumented chronically with an i.c.v. cannula, and the catheters were placed into the femoral artery and vein. Increasing doses of bradykinin (1 -300 pmol) were given i.c.v. and (i) MAP and heart rate, (ii) plasma dopamine, noradrenaline and adrenaline, and (iii) plasma arginine vasopressin (AVP) levels were determined. In addition, following blockade of peripheral alpha1 -adrenoceptors with prazosin (50 and 250 microg/kg i.v.) beta1-adrenoceptors with atenolol (10 mg/kg i.v.) or V1 -receptors with TMe-AVP (Manning compound) (10 microg/kg i.c.v. and 100 microg/kg i.v.) the effects of bradykinin (100 pmol i.c.v.) on MAP and heart rate were determined. RESULTS Bradykinin increased MAP and heart rate dose-dependently. The pressor effects of 100 pmol bradykinin i.c.v. were completely blocked by pretreatment with the specific B2 receptor antagonist Hoe 140 (3 pmol, i.c.v.). There was no change in plasma dopamine, noradrenaline, adrenaline or AVP levels after increasing doses of bradykinin. However, peripheral blockade of alpha1- and beta1-adrenoceptors reduced the bradykinin-induced increase in MAP and heart rate, whereas central and peripheral V1 receptor blockade did not alter the cardiovascular responses to i.c.v. bradykinin. CONCLUSION Our data suggest that the hypertensive and positive chronotropic effects induced by i.c.v. bradykinin are due to stimulation of sympathoneuronal rather than sympathoadrenal pathway in vivo.

Influence of Imidazolines on Catecholamine Release in Pithed Spontaneously Hypertensive Rats

The actions of the imidazoline derivatives clonidine, moxonidine, and rilmenidine and of the recently discovered clonidine-displacing substance agmatine on stimulation-induced norepinephrine overflow and epinephrine release were studied in pithed spontaneously hypertensive rats. All three imidazolines dose-dependently decreased norepinephrine overflow and led to an increase in epinephrine release when the highest dose of each compound was injected. The alpha 2-adrenoceptor antagonist rauwolscine shifted the dose-response curves of plasma norepinephrine concentrations to higher levels. Agmatine did not change norepinephrine overflow but increased epinephrine release into plasma after the highest dose administered. It is concluded that the investigated imidazolines decrease norepinephrine overflow via presynaptic alpha 2-adrenoceptors, whereas epinephrine release is mediated through putative imidazoline receptors on the adrenal medulla.

Norepinephrine Release Is Reduced by I1-Receptors in Addition to a2-Adrenoceptors

Abstract: In pithed spontaneous hypertensive rats, noradrenaline overflow was diminished by moxonidine even when a2‐adrenoceptors were blocked quantitatively using phenoxybenzamine, suggesting an I1‐receptor‐mediated mechanism of noradrenaline release. This hypothesis was confirmed, since the noradrenaline overflow was (1) increased under a2‐adrenoceptors blockade by the mixed I1/a2‐antagonists efaroxan or idazoxan, (2) still reduced by moxonidine when both a2‐ and I1‐receptors were blocked, and (3) diminished by agmatine after pretreatment with phenoxybenzamine, but not with AGN192403. An indirect ganglionic I1‐receptor‐mediated mechanism of noradrenaline release is supposed.