David J. Casley

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography.

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammons horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.

Salt restriction reduces hyperfiltration, renal enlargement, and albuminuria in experimental diabetes.

The effects of dietary salt restriction on the renin angiotensin system, glomerular filtration rate (GFR), renal size, and albuminuria were assessed in streptozotocin diabetic rats. Two series of experiments were performed: one short-term with severe salt restriction and the second long-term with moderate salt restriction. The first studied the effect of a very-low-salt diet for 4 weeks on GFR, renal size, and plasma angiotensin II concentration in diabetic and control rats. Diabetic and control male Sprague-Dawley rats received either a very-low-salt (0.005% NaCl) or a normal-salt (0.4% NaCl) diet. Diabetes was associated with a 49% increase in GFR, a 34% increase in kidney weight, and an 85% reduction in plasma angiotensin II when compared with control rats (P < 0.001). Sodium restriction in diabetic rats reduced GFR, restored plasma angiotensin II to control values, and retarded kidney growth when compared with diabetic rats receiving a normal sodium diet. GFR correlated negatively with plasma angiotensin II (r = −0.65, P < 0.001) and positively with kidney weight (r = 0.66, P < 0.001). In the second experiment, serial measurements of albuminuria and GFR were performed in control, diabetic, and saltrestricted (0.05% NaCl) control and diabetic rats over 24 weeks. Albuminuria showed a continuous rise in the diabetic rats when compared with control rats. Salt restriction attenuated the increase in albuminuria over the whole study period as well as reducing blood pressure and kidney weight in the diabetic rats. In conclusion, sodium restriction was associated with a lower GFR and kidney weight after 4 weeks and reduced levels of albuminuria, kidney weight, and blood pressure after 24 weeks in diabetic rats. Salt restriction may have an important role in the prevention and treatment of diabetic nephropathy.

Role of Angiotensin Receptor Subtypes in Mesenteric Vascular Proliferation and Hypertrophy

The aim of this study was to explore the regulation of angiotensin receptors after chronic infusion with angiotensin II (Ang II) and to clarify the relative roles of the angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in the mediation of Ang II-induced mesenteric vascular hypertrophy. In male Sprague-Dawley rats, Ang II infusion at a dose of 58.3 ng/min by subcutaneous osmotic minipumps for 14 days led to increased mesenteric weight and wall:lumen ratio of the vessels and proliferation of smooth muscle cells. These vascular changes were attenuated by either valsartan, an AT(1) receptor antagonist, at a dose of 30 mg. kg(-1). d(-1) by gavage, or PD123319, an AT(2) receptor antagonist, at a dose of 830 ng/min by intraperitoneally implanted osmotic minipumps. Ang II infusion was associated with hypertension, which was prevented by valsartan, but not PD123319. (125)I-Sar(1), Ile(8) Ang II binding to mesenteric vasculature was increased after Ang II infusion. Valsartan treatment was associated with reduced Ang II binding to both receptor subtypes, whereas PD123319 was associated with reduced Ang II binding to only the AT(2) receptor subtype. These findings suggest that the trophic and proliferative effects of Ang II on the mesenteric vasculature are mediated by both AT(1) and AT(2) receptors.

Distribution of AT4 receptors in the Macaca fascicularis brain

Angiotensin IV (Val Tyr Ile His Pro Phe), administered centrally, increases memory retrieval and induces c-fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT1 and AT2 receptors and is known as the AT4 receptor. These observations suggest that the AT4 receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [125I]angiotensin IV to map AT4 receptors in the macaca fascicularis brain. The distribution of the AT4 receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT4 receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT4 receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT4 receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT4 receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT4 receptor in the primate brain.

Vascular expression of angiotensin type 2 receptor in the adult rat: influence of angiotensin II infusion.

Objective The aim of this study was to investigate the relative role of the angiotensin type 1 (AT1) and type 2 (AT2) receptors in mediating angiotensin II-induced regulation of AT2 receptor in mesenteric artery. Design Sprague–Dawley rats were infused with either angiotensin II or vehicle for 14 days at a dose of 58.3 ng/min. Ang II-infused rats were allocated to receive either an AT1 antagonist, valsartan at a dose of 30 mg/kg per day or the AT2 receptor antagonist PD123319 at a dose of 830 ng/min. Methods Gene and protein expression of the AT2 receptor in the mesenteric vasculature was assessed by quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry and by in vitro autoradiography with a specific radioligand, 125I-CGP 42112B. Results The AT2 receptor mRNA and protein were detected in the mesenteric artery from adult rats. Both nuclear emulsion and immunohistochemical staining showed expression of the AT2 receptor in the adventitial and medial layers. Compared to control rats, angiotensin II infusion was associated with a significant increase in the AT2 receptor expression. Valsartan treatment significantly reduced AT2 receptor gene expression, with no significant effect of PD123319 on this parameter. Conclusions This study confirms that the presence of the AT2 receptor in mesenteric arteries in adult rats, shows an up-regulation of the AT2 receptor following angiotensin II infusion and suggests a role for the AT1 receptor in this regulation. In view of the recently demonstrated effects of the AT2 receptor, these findings may be relevant to the role of the AT2 receptor in the pathophysiology of vascular remodeling.

Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat.

Ang-(1-7) (angiotensin-1-7), a peptide product of the recently described ACE (angiotensin-converting enzyme) homologue ACE2, opposes the harmful actions of AngII (angiotensin II) in cardiovascular tissues, but its role in liver disease is unknown. The aim of the present study was to assess plasma levels of Ang-(1-7) in human liver disease and determine its effects in experimental liver fibrosis. Angiotensin peptide levels were measured in cirrhotic and non-cirrhotic patients with hepatitis C. The effects of Ang-(1-7) on experimental fibrosis were determined using the rat BDL (bile-duct ligation) model. Liver histology, hydroxyproline quantification and expression of fibrosis-related genes were assessed. Expression of RAS (renin-angiotensin system) components and the effects of Ang-(1-7) were examined in rat HSCs (hepatic stellate cells). In human patients with cirrhosis, both plasma Ang-(1-7) and AngII concentrations were markedly elevated (P<0.001). Non-cirrhotic patients with hepatitis C had elevated Ang-(1-7) levels compared with controls (P<0.05), but AngII concentrations were not increased. In BDL rats, Ang-(1-7) improved fibrosis stage and collagen Picrosirius Red staining, and reduced hydroxyproline content, together with decreased gene expression of collagen 1A1, alpha-SMA (smooth muscle actin), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), ACE and mas [the Ang-(1-7) receptor]. Cultured HSCs expressed AT1Rs (AngII type 1 receptors) and mas receptors and, when treated with Ang-(1-7) or the mas receptor agonist AVE 0991, produced less alpha-SMA and hydroxyproline, an effect reversed by the mas receptor antagonist A779. In conclusion, Ang-(1-7) is up-regulated in human liver disease and has antifibrotic actions in a rat model of cirrhosis. The ACE2/Ang-(1-7)/mas receptor axis represents a potential target for antifibrotic therapy in humans.

Ischaemia causes externalization of endothelin-1 binding sites in rat cardiac membranes.

Specific, high affinity binding sites for iodinated endothelin-1 ([125I]-ET-1) were identified in crude plasma and light membrane fractions harvested from aerobically perfused and ischaemic rat hearts, to determine whether the ischaemia-induced increase in binding site density (Bmax) involves externalization of the sites. In crude plasma membranes Bmax increased after 60 min ischaemia, from 113.5 +/- 2.15 to 180.6 +/- 4.67 fmol/mg protein (p less than 0.01). In the light membranes, the Bmax fell, from 94.7 +/- 8.70 to 63.80 +/- 6.26 fmol/mg protein (p less than 0.05). Hill coefficients and selectivity of both membrane fractions were unchanged. These results are interpreted as meaning that ischaemia causes externalization of cardiac [125I]-ET-1 binding sites.

Evidence for activation of the renin–angiotensin system in the human prostate: increased angiotensin II and reduced AT1 receptor expression in benign prostatic hyperplasia

The expression and cellular localization of angiotensin II (Ang II) and AT1 receptor proteins were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by immunohistochemistry. In the normal prostate, Ang II immunoreactivity was localized to the basal layer of the epithelium and AT1 receptor immunostaining was found predominantly on stromal smooth muscle and also on vascular smooth muscle of prostatic blood vessels. Ang II immunoreactivity was markedly increased in hyperplastic acini in BPH compared with acini in the normal prostate (normal: 7.4±0.2%, n=5 vs. BPH: 22.7±1.9%, n=5, p<0.001). However, AT1 receptor immunoreactivity was significantly decreased in BPH compared with the normal prostate [normal: 16.4±2.2%, n=4 vs. BPH: 9.4±1.3%, n=5, p<0.05 (p=0.025)]. The present study demonstrates the presence of Ang II peptide in the basal layer of the epithelium and AT1 receptors on stromal smooth muscle, suggesting that Ang II may mediate paracrine functions on cellular growth and smooth muscle tone in the human prostate. Furthermore, AT1 receptor down‐regulation in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. These data extend previous findings in support of the novel concept that overactivity of the renin–angiotensin system (RAS) may be involved in the pathophysiology of BPH. Copyright

Interaction between atrial natriuretic peptide and the renin angiotensin aldosterone system: Endogenous antagonists

The biologic actions of the cardiac peptide hormone atrial natriuretic peptide (ANP) of vasorelaxation, diuresis and natriuresis, suppression of aldosterone, vasopressin release, and thirst are the opposite of those of the renin angiotensin system. This close relationship is further strengthened by the complementary localization of their receptors in the brain, adrenal gland, vasculature, and kidney. In many physiologic situations including postural changes, volume expansion, water immersion, high altitude, and lower body negative pressure, the plasma levels of ANP and angiotensin II change inversely. In congestive heart failure, renin and aldosterone levels may initially be suppressed by high levels of ANP. Similarly the low renin levels associated with increasing age and with elderly hypertensive patients, may be the result of the elevation of plasma ANP that occurs with aging. ANP may thus be the endogenous antagonist of the renin angiotensin aldosterone system. These two opposing systems allow fine-tuning of volume and pressure by the body.

Localization of angiotensin IV binding sites to motor and sensory neurons in the sheep spinal cord and hindbrain

In the sheep spinal cord, a high density of [125I]angiotensin IV binding sites was localized to the perikaryon and processes of all somatic motor neurons, the autonomic motor neurons in the lateral horns of thoracic and lumbar segments and all dorsal root ganglia, but was low in lamina II of all dorsal horns. At supraspinal levels, [125I]angiotensin IV binding was abundant in numerous motor associated regions, with weaker binding observed in the sensory regions. This wide distribution pattern suggests an important role for the binding site in the central nervous system.