Elaine M. Richards

Immunohistochemical mapping of angiotensin AT1 receptors in the brain

A new approach to study angiotensin receptor distribution in the brain has been taken by developing antibodies to partial sequence of the angiotensin II (AII) type-1 receptor subtype (AT1) and demonstrating the presence of receptors with immunohistochemical staining. The antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor revealed distinctive punctate immunoreactive staining on cell bodies. The cell bodies were distributed in the forebrain in paraventricular and supraoptic nuclei, the organum vasculosum lamina terminalis, median preoptic area and subfornical organ. In the brainstem, the entire locus coeruleus was stained, together with the adjacent mesencephalic and motor nuclei of the trigeminal nerve. The auditory system including the cochlear nucleus and superior olivary nuclei were stained. In the medulla, all the structures involved in blood pressure control were stained including the nucleus of the solitary tract, the 12th nerve nuclei, the rostroventral lateral area and the nucleus ambiguous. Sites where AT2 receptors are located were not stained or staining was limited to specific area such as the medial accessory nucleus of the inferior olive. Immunocytochemical staining of AT1 receptors provides a new and more precise approach to the cellular localization of AII receptors.

Angiotensin II Type 2 Receptor-Mediated Apoptosis of Cultured Neurons from Newborn Rat Brain1

Angiotensin II (Ang II) type 2 (AT2) receptors are highly expressed in neonate brain and may have a role in developmental processes such as apoptosis. Concurrent activation of c-Jun N-terminal kinase (JNK) and inhibition of Erk mitogen-activated protein kinase activities is important for apoptosis in many cells, and we previously demonstrated that stimulation of AT2 receptors causes decreased mitogen-activated protein kinase activity in neurons cultured from newborn rat hypothalamus and brain stem. Using such cultures we have employed terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and internucleosomal DNA fragmentation to assess the role of AT2 receptors in neuronal apoptosis. Ang II (100 nM; 4-72 h) alone produced no significant neuronal apoptosis, and AT2 receptor activation did not stimulate JNK activity. However, exposure of cultures to UV radiation (6 J/m2/sec for 4 sec) to stimulate JNK elicited neuronal apoptosis that was significantly enhanced by Ang II, an effect that was abolished by the AT2 receptor antagonist PD 123,319 (1 microM) or the serine/threonine phosphatase inhibitor okadaic acid (3 nM). Additionally, Ang II enhanced the UV radiation-induced decrease in the levels of the DNA repair enzyme poly-(ADP-ribose) polymerase. These data indicate that Ang II, via AT2 receptors and activation of a serine/threonine phosphatase, contributes to neuronal apoptosis.

Angiotensin II Type 2 Receptor‐Mediated Stimulation of Protein Phosphatase 2A in Rat Hypothalamic/Brainstem Neuronal Cocultures

Abstract: Recent studies have suggested a role for an inhibitory guanine nucleotide binding (Gi) protein and protein (serine/threonine) phosphatase 2A (PP2A) in the angiotensin II type 2 (AT2) receptor‐mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of angiotensin II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Angiotensin II elicited time (30 min–24 h)‐ and concentration (10 nM‐1 µM)‐dependent increases in PP2A activity in these cells, an effect mimicked by the AT2 receptor ligand CGP‐42112A. These effects of angiotensin II and CGP‐42112A involve AT2 receptors, because they were inhibited by the AT2 receptor‐selective ligand PD 123,319 (1 µM) but not by the angiotensin II type 1 receptor antagonist losartan (1 µM). Furthermore, the stimulatory effects of angiotensin II and CGP‐42112A on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (200 ng/ml; 24 h), indicating the involvement of a Gi protein. These effects of angiotensin II and CGP‐42112A appear to be via activation of PP2A, and western blot analyses revealed no effects of either peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a cellular target modified following neuronal AT2 receptor activation.

Angiotensin II type 1 receptor-modulated signaling pathways in neurons

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin, secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in shortterm changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II, are discussed. It will be apparent from this discussion that the signaling pathways involved in these events, are often distinct.

Central pressor action of neurotensin in conscious rats.

The effects of neurotensin upon blood pressure in conscious rats were examined after intracerebroventricular (i.v.t.) or intravenous (i.v.) administration of this peptide. Whereas i.v. injected neurotensin (0.1–2.0 μ/) was depressor, i.v.t. injected neurotensin (1 μg and above) was pressor. Peripheral depressor responses could not be repeated in the same animal due to tachyphylaxis, but central pressor responses were repeatable without reduction in magnitude, showing that the two effects were separate entities. Thyrotropin-releasing hormone (TRH), which is reported to be a potent neurotensin antagonist, completely abolished the neurotensin depressor response, and attenuated the central pressor action. TRH did not alter the central pressor effect of another peptide, angiotensin II (All). The potent AH receptor antagonist saralasin, while abolishing the central pressor effect of AH, was completely without effect upon the neurotensin-induced pressor response. These results indicate that i.v.t. injected neurotensin and AH stimulate a rise in blood pressure via different receptors. The alpha-adrenergic antagonists phentolamine, prazosin, or yohimbine (injected i.v.t.) were able to abolish or attenuate the pressor response due to i.v.t.- neurotensin, suggesting involvement of the sympathetic nervous system in this response. These results are discussed in relation to the central pressor actions of other neuropeptides. (Hypertension 4: 888–893, 1982)

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-1 (AT1) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [3H]-DuP753 binding and AT1 receptor mRNA.

Angiotensin II stimulates protein phosphatase 2A activity in cultured neuronal cells via type 2 receptors in a pertussis toxin sensitive fashion.

Recent studies have suggested a role for an inhibitory G protein (Gi) and protein phosphatase 2A (PP2A) in the angiotensin II (Ang II) type 2 (AT2) receptor mediated stimulation of neuronal K+ currents. In the present study we have directly analyzed the effects of Ang II on PP2A activity in neurons cultured from newborn rat hypothalamus and brainstem. Ang II elicited time (30 min-24 h)- and concentration (10 nM -1 microM)-dependent increases in PP2A activity in these cells. This effect of Ang II involved AT2 receptors, since it was inhibited by the AT2 receptor selective ligand PD123319 (1 microM), but not by the Ang II type 1 receptor antagonist losartan (1 microM). Furthermore, the stimulatory effects of Ang II on PP2A activity were inhibited by pretreatment of cultures with pertussis toxin (PTX) (200 ng/ml; 24 h) indicating the involvement of an inhibitory G-protein; and by cycloheximide (CHX) (1 microgram/ml; 30 min) indicating a requirement for protein synthesis. These effects of Ang II appear to be via activation of PP2A, since Western Blot analyses revealed no effects of this peptide on the protein levels of the catalytic subunit of PP2A in cultured neurons. In summary, these data suggest that PP2A is a key component of the intracellular pathways coupled to neuronal AT2 receptors.

Prostaglandin Endoperoxide Synthase-2 Abundance Is Increased in Brain Tissues of Late-Gestation Fetal Sheep in Response to Cerebral Hypoperfusion:

Objective: To determine the mechanism by which cerebral hypoperfusion enhances prostanoid secretion by fetal brain tissues. Methods: Studies were performed on five intact and five carotid sinus—denervated sheep fetuses (124-136 days) exposed to 10 minutes of cerebral hypoperfusion. Plasma collected from lingual artery and sagittal sinus, and microdialysates collected from brain stem and hypothalamus were assayed for prostanoid production. Fetal hypothalamus, cerebral cortex, hippocampus, cerebellum, and brain stem were collected from intact animals and 30 minutes after cerebral hypoperfusion for the expression, activity, and distribution of prostaglandin endoperoxide synthase-1 (PGHS-1), PGHS-2, and thromboxane synthase. Results: Thromboxane B2 increased significantly in sagittal sinus compared with arterial blood, but PGE2 did not change. Thromboxane B2 decreased in brain stem and hypothalamus microdialysates, and prostaglandin E2 increased in these regions. PGHS-2 immunoreactive protein levels in brain tissues increased in the cerebral hypoperfusion fetuses compared with those of the intact animals. By contrast, PGHS-1 and thromboxane synthase protein levels did not change between these two groups. Prostaglandin endoperoxide synthase activity in brain tissues decreased with the increased levels of immunoreactive PGHS-2. Conclusions: 1) Prostanoids are produced in response to cerebral hypoperfusion, 2) the increase in the production of prostanoid responses to cerebral hypoperfusion is associated with the decrease in activity of, and therefore, the “suicide” inactivation of PGHS, and 3) PGHS-2 is the predominant form of PGHS, whose synthesis is induced by cerebral hypoperfusion in the fetal brain.

Cortisol stimulates proliferation and apoptosis in the late gestation fetal heart: differential effects of mineralocorticoid and glucocorticoid receptors

We have previously found that modest chronic increases in maternal cortisol result in an enlarged fetal heart. To explore the mechanisms of this effect, we used intrapericardial infusions of a mineralocorticoid receptor (MR) antagonist (canrenoate) or of a glucocorticoid receptor (GR) antagonist (mifepristone) in the fetus during maternal infusion of cortisol (1 mg·kg⁻¹·day⁻¹). We have shown that the MR antagonist blocked the increase in fetal heart weight and in wall thickness resulting from maternal cortisol infusion. In the current study we extended those studies and found that cortisol increased Ki67 staining in both ventricles, indicating cell proliferation, but also increased active caspase-3 staining in cells of the conduction pathway in the septum and subendocardial layers of the left ventricle, suggesting increased apoptosis in Purkinje fibers. The MR antagonist blocked the increase in cell proliferation, whereas the GR antagonist blocked the increased apoptosis in Purkinje fibers. We also found evidence of activation of caspase-3 in c-kit-positive cells, suggesting apoptosis in stem cell populations in the ventricle. These studies suggest a potentially important role of corticosteroids in the terminal remodeling of the late gestation fetal heart and suggest a mechanism for the cardiac enlargement with excess corticosteroid exposure.

Pharmacology and Physiology of Ovine Corticosteroid Receptors

The aim of these studies was to characterize the ovine corticosteroid receptors (MR, mineralocorticoid receptors and GR, glucocorticoid receptors) in ovine hippocampus and brainstem. Adrenal-intact and adrenalectomized ewes were studied; adrenalectomized ewes were killed 47 ± 9 h after steroid withdrawal, when symptoms of hypotension and/or hyperkalemia became evident. RT-PCR, immunoblotting and pharmacologic studies indicated the presence of both MR and GR in hippocampus and brainstem. Competitive binding studies using 3H-cortisol in brain tissue showed that the ovine MR binds cortisol, aldosterone and progesterone with equal affinity. Differences in receptor availability in intact and adrenalectomized ewes, along with determination of the binding affinity (Kd) of MR and GR, suggested that MR occupancy is about 90%, whereas GR occupancy is about 30%, in normal animals. There was a significant increase in protein level of MR in brainstem, and the appearance of a higher molecular weight band for MR in hippocampus following steroid withdrawal, however no significant change in mRNA was detected by semiquantitative RT-PCR for either MR or GR in hippocampus or brainstem following steroid withdrawal. These studies suggest that physiological ligands of MR in the sheep brain include progesterone and cortisol, and that, as in other species, affinity of MR for cortisol is greater than that of GR.