Evidence for an intrarenal renin-angiotensin system in the rainbow trout, Oncorhynchus mykiss

Physiological and molecular approaches were used to investigate the existence of an intrarenal renin-angiotensin system (RAS) in rainbow trout. Inhibition of angiotensin-converting enzyme by captopril (5 x 10(-4 )M) rapidly decreased vascular resistance of the trunk of the trout, perfused at 19 mmHg, resulting in an increased perfusate flow rate and a decreased intrarenal dorsal aortic pressure. A profound diuresis occurred in the in situ perfused kidney and reflected both increased glomerular filtration rates and decreased water reabsorption (osmolyte reabsorption was unchanged). Renal and vascular parameters recovered once captopril treatment was stopped. Diuretic and vascular effects of captopril on the in situ trout kidney concur with an inhibition of known vasoconstrictor and antidiuretic actions of angiotensin II. However, at a higher perfusion pressure (28 mmHg), captopril had no effect on intrarenal aortic pressure or perfusate and urine flow rates, suggesting that the trout intrarenal RAS is activated by low perfusion pressures/flows. Existence of the renal RAS in trout was further supported by evidence for angiotensinogen gene expression in kidney as well as liver.     

The brain renin-angiotensin system: a critical analysis.

The concept of a brain renin-angiotensin system originated with the observation that the components necessary for the formation of angiotensin II are present in the central nervous system. This observation has been confirmed and extended, and it is now frequently assumed that there is a functional brain renin-angiotensin system. However, careful analysis of the available evidence has revealed a number of significant problems. It appears that most of the renin-like activity measured in extracts of brain is due to the acid protease cathepsin D; this is unlikely to function as an angiotensin-forming enzyme in vivo. Experiments involving central administration of renin substrate have not provided convincing evidence for a significant renin-renin substrate interaction in vivo. Attempts to demonstrate the presence of angiotensin in the brain have been plagued with problems of specificity and it is still not clear if the peptide is actually present in the central nervous system. These problems do not rule out the possibility that there is a brain renin-angiotensin system, but more definitive evidence is required before it can be concluded that such a tensin system exists.     

Evidence for a role for a uterine renin-angiotensin system in decidualization in rats.

Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.     

Cellular organization of the brain renin-angiotensin system

A model of intracellular Ang II formation (Figure 1) implies that angiotensinogen neurons exist and that CNS Ang II acts both as a neurotransmitter as well as a neurohormone. Such a mechanism is consistent with the immunocytochemical localization of a fraction of brain Ang II in neurosecretory vesicles. To date, several dozen peptide neurotransmitters and neurohormones have been studied. Those assigned to peptidergic systems follow the generalized pathway of biosynthesis shown in Figure 1. In peptidergic systems, a prohormone and all of its processing enzymes are synthesized in the rough endoplasmic reticulum of a cell and move into the Golgi apparatus (Figure 1: #1-3). In the Golgi the prohormone and processing enzymes are packaged into the same vesicle (#3). These secretory vesicles then migrate toward the plasma membrane, frequently via axonal or dendritic projections to terminals. Within these cytoplasmic vesicles and prior to release, the processing enzymes are activated (#4) and the prohormone enzymatically processed, yielding the active peptide (#5-6). Only then do the vesicles fuse with the plasma membrane (in a calcium-dependent process), releasing their contents (#7-8). Once released, the active peptide migrates across the extracellular space and interacts with specific cell surface receptors to initiate a response (#9). Finally, receptor-bound peptide degradation is initiated by receptor-mediated endocytosis (#10-11). For angiotensin peptides to be produced intracellularly, the cell must present only one secretory pathway for Golgi packaging of renin and angiotensinogen; otherwise current theories of protein sorting would predict that these two proteins would be segregated even if synthesized within the same cell. Small quantities of co-packaged renin and angiotensinogen occurring via "spill-over" between compartments seems an unsatisfactory process for a regulated hormone system. Figure 2, depicting an extracellular mechanism for producing Ang II in the brain, has also been proposed. The mechanism of extracellular angiotensin formation is consistent with the molecular information encoded within the component proteins, known mechanisms of protein secretio, well-defined systemic renin-angiotensin enzymatic cascades, and demonstration of all the components of the renin-angiotensin system in the extracellular compartments of the brain. This model (Figure 2) allows independently coordinated gene expression and synthesis of renin (#1R), angiotensinogen (#1A), and angiotensin-converting enzyme (# 1C) in the same or different cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The renin-angiotensin system in the rat brain

Summary The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem.Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

Physiological genomic analysis of the brain renin-angiotensin system

The brain renin-angiotensin system (RAS) has long been considered pivotal in cardiovascular regulation and important in the pathogenesis of hypertension and heart failure. However, despite more than 30 years of study, the brain RAS continues to defy explanation. Our lack of understanding of how the brain RAS is organized at the cellular and regional levels has made it difficult to resolve long-sought questions of how ANG II is produced in the brain and the precise mechanisms by which it exerts its actions. A major reason for this is the difficulty in experimentally dissecting the brain RAS at the regional, cellular, and whole organism levels. Recently, we and others developed a series of molecular tools for selective manipulation of the murine brain RAS, in parallel with technologies for integrative analysis of cardiovascular and volume homeostasis in the conscious mouse. This review, based in part on a lecture given in conjunction with the American Physiological Society Young Investigator Award in Regulatory and Integrative Physiology (Water and Electrolyte Homeostasis Section), outlines the physiological genomics strategy that we have taken in an effort to unravel some of the complexities of this system. It also summarizes the principles, progress, and prospects for a better understanding of the brain RAS in health and disease.     

Evidence against an essential role of endogenous brain dopamine in methamphetamine-induced dopaminergic neurotoxicity

The present studies examined the role of endogenous dopamine (DA) in methamphetamine (METH)-induced dopaminergic neurotoxicity while controlling for temperature-related neuroprotective effects of the test compounds, reserpine and alpha-methyl-p-tyrosine (AMPT). To determine if the vesicular pool of DA was essential for the expression of METH-induced DA neurotoxicity, reserpine (3 mg/kg, given iintraperitoneally 24-26 h prior to METH) was given prior to a toxic dose regimen of METH. Despite severe striatal DA deficits during the period of METH exposure, mice treated with reserpine prior to METH developed long-term reductions in striatal DA axonal markers, suggesting that vesicular DA stores were not crucial for the development of METH neurotoxicity, but leaving open the possibility that cytoplasmic DA might be involved. To evaluate this possibility, cytoplasmic DA stores were depleted with AMPT prior to METH administration. When this study was carried out at 28 degrees C, complete neuroprotection was observed, likely due to lingering effects on core temperature because when the same study was repeated at 33 degrees C (to eliminate AMPT's hypothermic effect in METH-treated animals), the previously observed neuroprotection was no longer evident. In the third and final set of experiments, mice were pretreated with a combination of reserpine and AMPT, to deplete both vesicular and cytoplasmic DA pools, and to reduce striatal DA levels to negligible values during the period of METH administration (< 0.05%). When core temperature differences were eliminated by raising ambient temperature, METH-induced DA neurotoxic changes were evident in mice pretreated with reserpine and AMPT. Collectively, these findings bring into question the view that endogenous DA plays an essential role in METH-induced DA neurotoxicity.     

Fusicoccin receptors. Evidence for endogenous ligand

The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H⁺ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H⁺/K⁺ exchange system in higher plants.Abbreviation FC Fusicoccin     

Evidence for an endogenous factor involved in maintenance of pirenzepine high-affinity binding in rat brain stem

Sucrose gradient centrifugation was used to isolate membranes enriched in muscarinic receptors from bovine brain stem. Unlike the receptors in crude synaptosomal preparations of this tissue, the enriched preparations displayed only low-affinity pirenzepine binding. Similar results were obtained when purified preparations were preincubated for 1 hr in pH 7.0 buffer at 37 degrees C; however, preincubation in a pH 5.0 buffer partially restored the high-affinity pirenzepine binding. These results suggest that an endogenous factor, which is present in the crude synaptosomes of the brain stem and is removed by sucrose gradient centrifugation, is involved in maintenance of the high-affinity pirenzepine binding of the muscarinic receptors.     

The brain renin-angiotensin system: location and physiological roles

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.     

Evidence for an endogenous parathyroid hormone-sensitive adenylate cyclase activator

The middle medullary membrane of canine renal tissue was completely insensitive to parathyroid hormone (PTH) stimulation in the absence of GTP or tissue supernatant. In contrast, removal of endogenous GTP did not eliminate the PTH stimulation of cAMP formation in the renal cortical membrane preparation (P₃). Addition of boiled cortical P₃ to native cortical P₃ enhanced PTH stimulation in a dose-dependent manner. The boiled cortical P₃ was not active in middle medullary tissue. The minimum concentration of GTP which was required to cause stimulation in both cortical and middle medullary preparations was similar. The results suggest that (1) there are two classes of PTH-sensitive adenylate cyclase; one class is GTP dependent, while the other is GTP independent. (2) A new factor, as yet unidentified, in addition to GTP, is important in the regulation of adenylate cyclase by PTH in the renal cortex.     

Evidence for an endogenous peptide ligand for the phencyclidine receptor

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.     

Evidence for an endogenous ATPase inhibitor protein in plant mitochondria. Purification and characterization

An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria. The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity. The isolated inhibitor is a heat-stable, trypsin-sensitive, basic protein, with a molecular mass approximately 8.3 kDa. Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline; threonine and leucine. The interaction of the inhibitor with F1-ATPase requires the presence of Mg2(+)-ATP in the incubation medium. The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 micrograms inhibitor/mg F1. A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value. The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1. The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, epsilon subunit, of chloroplast ATPase.     

Diabetes-induced stimulation of the renin-angiotensin system in the rat brain cortex

Cerebrovascular disease is a threat to people with diabetes and hypertension. Diabetes can damage the brain by stimulating the renin-angiotensin system (RAS), leading to neurological deficits and brain strokes. Diabetes-induced components of the RAS, including angiotensin-converting enzyme (ACE), angiotensin-II (Ang-II), and angiotensin type 1 receptor (AT1R), have been linked to various neurological disorders in the brain. In this study, we investigated how diabetes and high blood pressure affected the regulation of these major RAS components in the frontal cortex of the rat brain. We dissected, homogenized, and processed the brain cortex tissues of control, streptozotocin-induced diabetic, spontaneously hypertensive (SHR), and streptozotocin-induced SHR rats for biochemical and Western blot analyses. We found that systolic blood pressure was elevated in SHR rats, but there was no significant difference between SHR and diabetic-SHR rats. In contrast to SHR rats, the heartbeat of diabetic SHR rats was low. Western blot analysis showed that the frontal cortexes of the brain expressed angiotensinogen, AT1R, and MAS receptor. There were no significant differences in angiotensinogen levels across the rat groups. However, the AT1R level was increased in diabetic and hypertensive rats compared to controls, whereas the MAS receptor was downregulated (p < 0.05). These findings suggest that RAS overactivation caused by diabetes may have negative consequences for the brain's cortex, leading to neurodegeneration and cognitive impairment.     

Antisense inhibition of the renin-angiotensin system in brain and peripheral organs

Antisense inhibition is a method of attenuating the target at the gene expression level. There are two main groups of molecular tools for this goal. The first includes the use of short synthetic stretches of DNA-antisense oligodeoxynucleotides. The second tool is the use of vectors (plasmids or viruses) containing the gene of interest subcloned in the antisense orientation, which in the cells produces the antisense RNA. Both antisense DNA and RNA can bind to the complementary sense mRNA and interfere with its translation. Effects are usually short lasting (days) for oligodeoxynucleotides and longer lasting (weeks or months) for vectors. In this article we briefly describe techniques of antisense inhibition in the context of the renin-angiotensin system.     

The renin-angiotensin system and the central nervous system.

One of several factors affecting the secretion of renin by the kidneys is the sympathetic nervous system. The sympathetic input is excitatory and is mediated by beta-adrenergic receptors, which are probably located on the membranes of the juxtaglomerular cells. Stimulation of sympathetic areas in the medulla, midbrain and hypothalamus raises blood pressure and increases renin secretion, whereas stimulation of other parts of the hypothalamus decreases blood pressure and renin output. The centrally active alpha-adrenergic agonist clonidine decreases renin secretion, lowers blood pressure, inhibits ACTH and vasopressin secretion, and increases growth hormone secretion in dogs. The effects on ACTH and growth hormone are abolished by administration of phenoxybenzamine into the third ventricle, whereas the effect on blood pressure is abolished by administration of phenoxybenzamine in the fourth ventricle without any effect on the ACTH and growth hormone responses. Fourth ventricular phenoxybenzamine decreases but does not abolish the inhibitory effect of clonidine on renin secretion. Circulating angiotensin II acts on the brain via the area postrema to raise blood pressure and via the subfornical organ to increase water intake. Its effect on vasopressin secretion is debated. The brain contains a renin-like enzyme, converting enzyme, renin substrate, and angiotensin. There is debate about the nature and physiological significance of the angiotensin II-generating enzyme in the brain, and about the nature of the angiotensin I and angiotensin II that have been reported to be present in the central nervous system. However, injection of angiotensin II into the cerebral ventricles produces drinking, increased secretion of vasopressin and ACTH, and increased blood pressure. The same responses are produced by intraventricular renin. Angiotensin II also facilitates sympathetic discharge in the periphery, and the possibility that it exerts a similar action on the adrenergic neurons in the brain merits investigation.     

Comparative physiology of the renin-angiotensin system.

Renin or a renin-like substance is found in the kidneys of many vertebrate species. It is absent from the kidneys of cyclostomes and elasmobranchs and first appears in holosteans and the bony fishes as well as in all higher vertebrate species. Juxtaglomerular cell granules also appear first in holosteans and the bony fishes while the macula densa first appears in amphibians. In telecost fishes, the corpuscles of Stannius contain Bowie-stainable granules and a renin-like pressor substance. Among classes and, in some cases, species of vertebrates, specificity in the reaction of renin with a substrate has been demonstrated. There is also some species and class variation in the angiotensin molecule since angiotensins of fishes, amphibians, reptiles and birds have chemical characteristics different from each other and from those of ammmals. A role for renin in stimulating interrenal gland steroid biosynthesis and in influencing water and ion regulation in nonmammalian vertebrates is discussed.     

Divergent mechanism regulating fluid intake and metabolism by the brain renin-angiotensin system

The purpose of this review is two-fold. First, I will highlight recent advances in our understanding of the mechanisms regulating angiotensin II (ANG II) synthesis in the brain, focusing on evidence that renin is expressed in the brain and is expressed in two forms: a secreted form, which may catalyze extracellular ANG I generation from glial or neuronal angiotensinogen (AGT), and an intracellular form, which may generate intracellular ANG in neurons that may act as a neurotransmitter. Second, I will discuss recent studies that advance the concept that the renin-angiotensin system (RAS) in the brain not only is a potent regulator of blood pressure and fluid intake but may also regulate metabolism. The efferent pathways regulating the blood pressure/dipsogenic effects and the metabolic effects of elevated central RAS activity appear different, with the former being dependent upon the hypothalamic-pituitary-adrenal axis, and the latter being dependent upon an interaction between the brain and the systemic (or adipose) RAS.