The A-1 antigen: A novel marker in experimental peripheral nerve injury

Expression of the Schwann cell phenotype is regulated by signals from the adjoining axon. After axotomy, the Schwann cell ceases the production and maintenance of the myelin sheath and assumes phagocytic properties necessary to digest its own myelin. The molecular mechanisms responsible for this behavior remain unclear. A monoclonal antibody termed BIKS was produced after the immunization of mice with guinea pig lymphoid tissue. This antibody recognizes a cytoplasmic vesicle-associated molecule (A-1 antigen) which is abundant in all tissue macrophages but is also expressed in small amounts in normal Schwann cells. Following axotomy, the A-1 antigen appears to be translocated from a perinuclear site to accumulate in large quantities around myelin ovoids in Schwann cells, as well at the nodes of Ranvier-sites where Wallerian degeneration is known to commence. The level of the antigen remains high when axons are prevented from regeneration. During repair of crush injury, however, the level of antigen drops concomitant with the ingrowth of regenerating axons, suggesting axonal control of A-1 antigen expression. © 1993 Wiley-Liss, Inc.     

The distribution of angiotensin II binding sites in rodent brain

The distribution of specific angiotensin II (AII) binding capacity of several brain regions, pituitary, and adrenals was determined in 6 rodent species namely rats, mice, hamsters, kangaroo rats, gerbils and degus. Rats and mice had similar distributions with the highest levels of binding observed in the area postrema, septum and superior colliculi. Low levels were seen in the cortex, cerebellum, striatum and hippocampus. Other areas had intermediate levels. The distribution of AII binding in gerbils and degus was strikingly different from rats and mice. In these species, little or no binding could be detected in the brain. Additionally, the level of binding in degu adrenals was extremely low when compared to the binding observed in the adrenals of the other species. The distribution of AII binding sites in hamsters and kangaroo rats, although similar in some ways to rats and mice, had several major differences. Both had much higher levels of specific binding in cerebellum, striatum, and the hippocampus areas which had low levels of AII binding in rats an mice. Hamsters were the only species to exhibit significant specific binding in the cortex. The kangaroo rats had an unusual distribution of receptors with an apparent lack of specific binding in midbrain and area postrema.     

Rat brain cells in primary culture: Characterization of angiotensin II binding sites

The binding kinetics of angiotensin II (ANG II) have been studied in primary cultures from fetal rat brain. Binding of [125I]ANG II to rat brain cells in culture is time-, pH- and cell concentration-dependent. The binding is saturable, reversible, and 90--95% specific. Binding follows first-order kinetics, with values for K1 and K-1 of 4.9 x 10(6)M-1 S-1 and 3.33 x 10(4)S-1 respectively. Scatchard analysis reveals the presence of a single class of binding sites with Ka of 1.0 x 10(9)M-1 and an average of approximately 6 x 10(3) sites per cell. [125I]ANG II recovered from incubation medium under the conditions of the binding assay or after dissociation from cells is not significantly degraded as judged by gel filtration on Sephadex G-25 and radioreceptor assay. ANG II analogs compete with [125I]ANG II for binding, with potencies in general paralleling previously established biological activities. Of 5 analogs tested, (Ile8)-ANG II was almost equipotent with ANG II while (Dval3)-ANG II was least potent in the competitive binding assay. These data fulfill criteria for the identification of specific angiotensin II receptors in cells from mammalian brain.     

Rat brain angiotensin II receptors: Effects of intracerebroventricular angiotensin II infusion

Angiotensin II (Ang II) was infused into a lateral cerebral ventricle of male Sprague-Dawley rats and its effects on blood pressure, water balance and specific [125I]Ang II binding to brain and adrenal tissues were studied. The infusion was maintained at a rate of 500 ng/microliter/h for 6 days using subcutaneously implanted osmotic minipumps. A control group was infused intracerebroventricularly (i.c.v.) with 0.9% saline at a rate of 1 microliter/h for 6 days. Angiotensin II treated rats showed a four-fold increase in water intake and urine volume and a moderate increase in blood pressure; these effects were not observed in rats given saline i.c.v. There was no significant difference in [125I]Ang II binding site density or binding affinity in either the hypothalamus-thalamus-septum-midbrain (HTSM) or the brainstem between Ang II-treated and saline-treated groups. In addition, [125I]Ang II binding sites in the adrenals were also unaffected by i.c.v. infusion of Ang II. The results suggest that brain Ang II receptors are unresponsive to increased Ang II levels in cerebrospinal fluid.     

Glia-derived nexin/protease nexin-1 is expressed by a subset of neurons in the rat brain

Glia-derived nexin/protease nexin-1 (GDN/Pn-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespreat expression during pre-and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor. © Wiley-Liss, Inc.     

Expression of a novel angiotensin II receptor subtype in gerbil brain

Angiotensin II receptors are highly localized in adult gerbil brain. Apparent receptor number is high in subfornical organ, vascular organ of the lamina terminalis, nucleus of the solitary tract, hippocampus, and in the anterior pituitary gland. In the hippocampus, binding is localized to the stratum oriens, radiatum, the lacunar molecular layers of the CA1 subfield, and the molecular layer of the gyrus dentatus, with a medial to lateral and anterior to posterior gradient in receptor expression. Binding is absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyrus dentatus, areas rich in angiotensin IV binding. Characterization in the hippocampus revealed the presence of a high affinity receptor, sensitive to incubation with the guanine nucleotide GTPγS, and displaced by angiotensin II = angiotensin III < Sar¹-Ile⁸-angiotensin II, but not by angiotensin IV or other angiotensin fragments, the AT₁ receptor antagonist losartan, or the AT₂ ligands CGP 42112 or PD 123177. In other brain areas, binding was equally insensitive to displacement by AT₁ or AT₂ ligands, with the exception of binding in the olfactory bulb, which was totally displaced by CGP 42112 and PD 123177, but not by losartan. In the gerbil, most of the brain and pituitary angiotensin II receptors are different from the AT₁ AT₄ and AT₄ subtypes, and should be considered ‘atypical’ until further characterization.     

Differential expression of neuropsin and protease M/neurosin in oligodendrocytes after injury to the spinal cord

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis.     

Role of brain angiotensin II in renal nerve inhibition elicited by volume expansion in the conscious rabbit

Losartan (10 microg/25 microl) or vehicle was injected into the fourth brain ventricle prior to volume expansion (VE) with Haemaccel (2 ml/min for 30 min). RSNA was reduced by a maximum of 45% in response to the VE following vehicle and by 33% following losartan. There was no significant difference between the treatments in RSNA, nor in the blood pressure and heart rate responses. We conclude that endogenous angiotensin II does not make a major contribution to the reflex reduction in RSNA initiated by VE.     

Different effects of chronic K depletion on forebrain and peripheral angiotensin II receptors in young rats

K+ depletion stimulates the circulating renin-angiotensin system and affects the regulation of peripheral angiotensin II receptors. The effects of K+ depletion on the regulation of central angiotensin II receptors are unknown. We studied the effects of selective K+ depletion (less than 0.05% in diet for 16 days) on angiotensin II receptor number in kidneys, adrenal glands, and selected brain areas of young rats. K+ depletion caused a significant increase in plasma renin activity and significantly decreased angiotensin II receptor number in the kidney glomeruli and medulla, and in the adrenal zona glomerulosa and adrenal medulla. In the brain, the angiotensin II receptor number was unchanged in the subfornical organ and the hypothalamic paraventricular nucleus after 16 days of K+ depletion. An additional NaCl supplementation (0.02% in the drinking water) to K(+)-depleted rats produced a decrease in plasma renin activity but failed to affect subfornical organ or paraventricular angiotensin II receptor number. Our results suggest that in young animals, K+ depletion has a significant impact on the peripheral renin-angiotensin system without affecting the density of forebrain angiotensin II receptors.     

Chronic peripheral administration of the angiotensin II AT1 receptor antagonist Candesartan blocks brain AT1 receptors

Brain Angiotensin II, through stimulation of brain AT₁ receptors, regulates pituitary hormones and autonomic activity. We have administered the insurmountable AT₁ antagonist Candesartan, s.c. via osmotic minipumps for 14 days, to determine whether peripheral chronic AT₁ blockade affects AT₁ receptor binding and mRNA in the brain. Peripherally administered Candesartan (0.1, 0.5 or 1.0 mg/kg per day) inhibits AT₁ binding in adrenal gland zona glomerulosa and kidney glomeruli. In addition, Candesartan dose-dependently decreases AT₁ binding in brain areas outside (subfornical organ and area postrema) and inside (paraventricular nucleus of the hypothalamus and nucleus of the solitary tract) the blood–brain barrier. Conversely, peripheral treatment with Candesartan does not affect AT_1A receptor mRNA, the predominant receptor subtype expressed in these areas, or Angiotensin II binding to AT₂ receptors in the locus coeruleus or inferior olive. Our results demonstrate that chronic peripheral treatment with selective, potent AT₁ antagonists not only inhibits peripheral but also brain AT₁ receptors. These central effects may play a role in the antihypertensive effects of the AT₁ antagonist Candesartan.     

An angiotensin II type 1 receptor blocker can preserve endothelial function and attenuate brain ischemic damage in spontaneously hypertensive rats

Hypertension reduces endothelial nitric oxide synthase (eNOS) expression and leads to endothelial dysfunction. However, few studies have demonstrated the influences of hypertension on eNOS function in the cerebral cortex. The present study investigates the influences of hypertension on endothelial function in the cerebral cortex and the protective effects of antihypertensive agents against brain ischemia through the preservation of endothelial function. Five‐ and ten‐week‐old male Wistar rats and spontaneously hypertensive rats (SHR) were used for experiments. Five‐week‐old SHR received olmesartan, hydralazine, or vehicle for 5 weeks in drinking water. eNOS activation in the cerebral cortex was evaluated by analyzing levels of total and Ser¹¹⁷⁷‐phosphorylated eNOS protein by Western blot. Blood pressure of 10‐week‐old SHR without treatment was clearly high, and the ratio of phospho‐eNOS/total eNOS protein was significantly low. Five‐week treatment with olmesartan or hydralazine suppressed the elevation of blood pressure and the reduction of phosphorylated eNOS‐Ser¹¹⁷⁷ in SHR, and olmesartan was more effective in maintaining phosphorylation of eNOS‐Ser¹¹⁷⁷ than hydralazine. To assess the contribution of eNOS to maintaining cerebral blood flow (CBF), we monitored CBF by laser‐Doppler flowmetry after L‐N⁵‐(1‐iminoethyl)ornithine (L‐NIO) infusion. CBF response to L‐NIO was preserved in olmesartan‐treated SHR but not in hydralazine‐treated SHR. Furthermore, infarct volume 48 hr after transient focal brain ischemia in olmesartan‐treated SHR was significantly reduced compared with vehicle‐treated SHR. These findings indicate that chronic prehypertensive treatment with olmesartan could attenuate brain ischemic injury through the maintenance of endothelial function in the cerebral cortex in SHR. © 2010 Wiley‐Liss, Inc.     

Localization of immunoreactive angiotensin II in the hippocampus and striatum of rat brain

Angiotensin II (ANG II) was estimated by radioimmunoassay in extracts of rat brain. In extracts of whole brain the mean content was 108 ± 16fmol/g and estimates of ANG II in kidney and plasma were similar to previous reports. A regional distribution of ANG II was determined. Hippocampus had the highest concentration and cortex the lowest. The concentrations relative to cortex were: hippocampus, 8; striatum, 5; cerebellum, 4; hypothalamus:thalamus:septum:midbrain (HTSM), 3; and medulla, 3.     

Differential modulation of angiotensin II and hypertonic saline-induced drinking by opioid receptor subtype antagonists in rats

Opioid modulation of ingestion includes general opioid antagonism of different forms of water intake, μ₂ receptor modulation of deprivation-induced water intake and δ₂ receptor modulation of saccharin intake. Water intake is stimulated by both central administration of angiotensin II (ANG II) and peripheral administration of a hypertonic saline solution; both responses are reduced by general opioid antagonists. The present study examined whether specific opioid receptor subtype antagonists would selectively alter each form of water intake in rats. Whereas systemic naltrexone (0.1–2.5 mg/kg, s.c.) reduced water intake induced by either peripheral ANGII (500 μg/kg, s.c.) or hypeptonic saline (3 ml/kg, 10%), intracerebroventricular (i.c.v.) naltrexone (1–50 μg) only inhibited central ANGII (20 ng)-induced hyperdipsia. Both forms of drinking were significantly and dose-dependently inhibited by the selective κ antagonist, nor-binaltorphamine (Nor-BNI, 1–20 μg). Whereas both forms of drinking were transiently reduced by the μ-selective antagonist, β-funaltrexamine (β-FNA, 1–20 μg), the μ₁ antagonist, naloxonazine (40 μg) stimulated drinking following hypertonic saline. The δ₁ antagonist, [d-Ala², Leu⁵, Cys⁶]-enkephalin (DALCE, 1–40 μg) significantly reduced drinking following ANGII, but not following hypertonic saline; the δ antagonist, naltrindole failed to exert significant effects. These data indicate that whereas κ opioid binding sites modulate hyperdipsia following hypertonic saline, μ₂, δ₁ and κ opioid binding sites modulate hyperdipsia following ANGII. The μ₁ opioid binding site may normally act to inhibit drinking following hypertonic saline.     

Cerebral microvascular inflammation in DOCA salt-induced hypertension: role of angiotensin II and mitochondrial superoxide

Angiotensin II-mediated hypertension (HTN) is accompanied by a pro-inflammatory and pro-thrombotic state in the cerebral microvasculature. Whether comparable phenotypic changes are elicited in other models of HTN remains unclear. Using wild-type mice with deoxycorticosterone acetate (DOCA) salt-induced HTN and intravital microscopy, we observed significant increases in the adhesion of both leukocytes and platelets in cerebral venules, compared with uninephrectomized control mice, without an accompanying increase in blood-brain barrier permeability. The cell-cell interactions in hypertensive mice were more pronounced after ischemic stroke, but no difference in infarct size was detected. The blood cell recruitment was largely prevented in the following groups of DOCA salt mice: losartan (angiotensin II AT1 receptor blocker) treated, AT1 receptor knockout mice, tempol (a membrane-permeable oxygen radical scavenger) treated, and mito-TEMPO (a mitochondria-targeted antioxidant) treated. A similar pattern of protection was noted in mice subjected to ischemic stroke. The blunted cell recruitment responses were not accompanied by reductions in blood pressure (BP). These findings implicate mitochondria-derived oxygen radicals and angiotensin II in the cerebral inflammation associated with DOCA salt HTN and suggests that BP per se is not a critical determinant of the phenotypic changes that accompany HTN, even after ischemic stroke.     

Nigral and striatal regulation of angiotensin receptor expression by dopamine and angiotensin in rodents: implications for progression of Parkinson's disease

The basal ganglia have a local renin–angiotensin system and it has been shown that the loss of dopaminergic neurons induced by neurotoxins is amplified by local angiotensin II (AII) via angiotensin type 1 receptors (AT1) and nicotinamide adenine dinucleotide phosphate (NADPH) complex activation. Recent studies have revealed a high degree of counter‐regulatory interactions between dopamine and AII receptors in non‐neural cells such as renal proximal tubule cells. However, it is not known if this occurs in the basal ganglia. In the striatum and nigra, depletion of dopamine with reserpine induced a significant increase in the expression of AT1, angiotensin type 2 receptors (AT2) and the NADPH subunit p47^phox, which decreased as dopamine function was restored. Similarly, 6‐hydroxydopamine‐induced chronic dopaminergic denervation induced a significant increase in expression of AT1, AT2 and p47^phox, which decreased with L‐dopa administration. A significant reduction in expression of AT1 mRNA was also observed after administration of dopamine to cultures of microglial cells. Transgenic rats with very low levels of brain AII showed increased AT1, decreased p47 ^phox and no changes in AT2 expression, whereas mice deficient in AT1 exhibited a decrease in the expression of p47 ^phox and AT2. The administration of relatively high doses of AII (100 nm ) decreased the expression of AT1, and the increased expression of AT2 and p47^phox in primary mesencephalic cultures. The results reveal an important interaction between the dopaminergic and local renin–angiotensin system in the basal ganglia, which may be a major factor in the progression of Parkinson’s disease.     

Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension

Angiotensin II plays a central role in the pathogenesis of hypertension and of related cardiovascular disorders by binding to and activating angiotensin II receptors (AT₁ receptors). Sensitization to the vasopressor response of angiotensin II is a key feature in many cardiovascular disorders. However, underlying mechanisms responsible for angiotensin II hypersensitivity are barely understood. Because angiotensin II responsiveness of AT₁ receptors can be specifically modified by AT₁/B₂ receptor dimerization, we determined the AT₁ receptor dimerization status in an experimental model of hypertension. AT₁/B₂ receptor heterodimers were abundant on renal mesangial cells isolated from spontaneously hypertensive rats compared with that on cells from normotensive controls. Heterodimerization of AT₁ with B₂ receptors was correlated with high levels of B₂ receptor protein on kidneys and on mesangial cells of hypertensive rats, as determined in immunoblot with receptor-specific antibodies. Specific inhibition of AT₁/B₂ receptor heterodimers revealed that these receptor heterodimers mediated an enhanced angiotensin II-stimulated Gα_q/11 activation and an increased endothelin-1 secretion of mesangial cells from hypertensive rats. Thus, AT₁/B₂ receptor heterodimerization contributes to angiotensin II hyperresponsiveness of mesangial cells in experimental hypertension.     

Evidence for an angiotensin II-like material and for a rapid metabolism of angiotension II in the rat brain

Radioimmunoassay and radioreceptor assay for angiotensin II (AII) have bee n developed to detect AII-like material in rat brain extracts using HCl extraction and boiling. The amount of AII-like material found was270 ± 39fmol/brain with radioimmunoassay and67 ± 7.8pmol/brain with radioreceptor assay. However, chromatographic separation by gel filtration on a Sephadex G25 column revealed that this material was not authentic AII, but of higher molecular weight. Column chromatography on Sephacryl S300 combined radioimmunoassay permitted us to show that the major part of the AII-like material had a molecular weight of about 10,000.To test the hypothesis that very rapid degradation of AII could explain the difficulty in detecting endogenous AII in the rat brain, we studied the metabolism of AII using HPLC analysis of the in vitro degradation of [³H]AI and [³H]AII (20 nM) by brain homogenates. HPLC analysis showed no detectable [³H]AII generation from [³H]AI. [³H]AI and [³H]AII yielded the same [³H]metabolites corresponding to two peaks α and β. Nevertheless, by adding an excess of unlabeled Ileu⁵-AII, which competitively inhibits AII-angiotensinase activity it was possible to detect the formation of [³H]AII from [³]AII from [³H]AI.We suggest that very low levels of AII could coexist with a higher molecular weight AII-coke compound in the rat brain and thet very rapid degradation of AII may account for the difficulty in detecting this peptide in the brain.