Cerebrospinal Fluid Changes in the Renin-Angiotensin System in Alzheimer's Disease.

Observations in autopsied brain tissue indicate that overactivation of the classical renin-angiotensin system (cRAS) and underactivity within regulatory RAS pathways (rRAS) are associated with pathology in Alzheimer's disease (AD). The primary aim of this study was to investigate whether cerebrospinal fluid (CSF) markers of RAS are altered in AD in relation to established CSF markers of disease pathology (lower Aß42 and elevated tau) and CSF markers of capillary dysfunction. We studied 40 controls and 40 AD cases grouped according to a biomarker profile (i.e., AD cases t-tau>400 pg/mL, pTau >60 pg/mL, and Aß42 <550 pg/mL). Angiotensin-II converting enyme-1 (ACE1) and ACE2 enzyme activity was measured using fluorogenic peptide substrates; sPDGFRß and albumin level by sandwich ELISA; and angiotensin-I (Ang-I), Ang-II, and Ang-(1-7) by direct ELISA. CSF Aß42, total, and phosphorylated tau levels were previously measured by INNOTEST sandwich ELISA. CSF ACE1 activity was significantly elevated in AD (p = 0.008) and positively correlated with ACE2 in AD (r = 0.420, p = 0.007). CSF ACE1 weakly correlated with t-tau (r = 0.294, p = 0.066) and p-tau (r = 0.329, p = 0.038) but not with Aß42 in the controls but not in AD. ACE1 correlated positively with sPDGFRß (r = 0.426, p = 0.007), a marker of pericyte injury, and ACE2 correlated positively with albumin (r = 0.422, p = 0.008), a marker of blood-brain barrier integrity. CSF Ang-I, Ang-II, and Ang-(1-7) levels were unchanged in AD. This cross-sectional CSF study indicates RAS dysfunction in relation to capillary damage in AD.

Characterization by high performance liquid chromatography of angiotensin peptides in the plasma and cerebrospinal fluid of the dog.

A high performance liquid chromatography (HPLC) method is described for the separation of angiotensin (Ang) peptides and their subsequent quantification by radioimmunoassay in plasma and cerebrospinal fluid (CSF). The use of the ion-pair solvent heptafluorobutyric acid in gradient HPLC achieves baseline resolution of Ang I, Ang II, and the C-terminal fragments des-[Asp1]-Ang I, des-[Asp1]-Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in approximately 25 min. Recovery of synthetic Ang standards after phenylsilica extraction and HPLC separation was greater than 70% for each peptide in both plasma and CSF. Ang I and Ang II were shown to be the major immunoreactive Ang components in plasma, and Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in CSF.

Cerebroventricular and intravascular metabolism of [125I]angiotensins in rat.

This study compared the metabolism of [125I]angiotensin II (AII), [125I]angiotensin III (AIII), and [125I]Sar1,Ile8-AII (SI-AII) in the vascular and cerebroventricular compartments. Using HPLC methods to monitor degradation the following t1/2 values were established in the vascular compartment: AII, 12.7 +/- 1.4 s; AIII, 16.3 +/- 0.7 s; and SI-AII, 100.7 +/- 7.3 s. HPLC analysis also revealed that [125I]AII is converted in an obligatory manner to [125I]AIII during its degradation sequence. Cerebrospinal fluid contained no degradative capacity for [125I]AII but exhibited a significant capacity to degrade [125I]AIII. A technique that combined the intra-cerebroventricular injection of [125I]angiotensins followed by focused microwave fixation to stop all peptidase activity was used to determine the half-life of [125I]angiotensins in the ventricular space. Results indicated very rapid metabolism of angiotensins with the following t1/2 values: AII, 23.0 s; and AIII, 7.7 s. This extremely rapid, differential, and sequential metabolism of AII and AIII in two relevant body fluid compartments underscores the need for caution when interpreting data derived from intravascular and intracerebroventricular application of angiotensins. In addition the faster metabolism of AIII than AII in the ventricular space indicates that the actual potency of AIII at central angiotensin receptors is being underestimated.

Angiotensinogen levels in the brain and cerebrospinal fluid of the genetically hypertensive rat.

The present experiments were designed to document changes in the regional distribution of angiotensinogen in the rat brain with the development of hypertension in spontaneously hypertensive rats (SHR) relative to age-matched normotensive Wistar-Kyoto rats (WKY). Levels of angiotensinogen were measured in discrete brain nuclei and cerebrospinal fluid from rats at 4, 7, and 16 weeks of age and in cerebrospinal fluid obtained by cisternal puncture at 7 and 16 weeks. Age-dependent changes in angiotensinogen were found, with levels higher in both strains at 4 weeks of age compared with 7 or 16 weeks. In contrast, plasma levels of angiotensinogen were essentially the inverse of the brain levels, low at 4 weeks and higher at 7 and 16 weeks. Levels in a number of regions adjacent to the rostral third ventricle from the 4-week-old SHR (prehypertensive phase) were significantly elevated relative to the WKY (p less than 0.05), while levels in the amygdala and posterior hypothalamus were significantly lower in the SHR (p less than 0.05). In 7-week-old rats (evolving phase), levels in nine brain regions were significantly elevated in the SHR relative to the WKY and included the nucleus tractus solitarii (p less than 0.01). Unlike the prehypertensive and evolving phases, in 16-week-old rats (maintenance phase) only two brain areas, the nucleus of the diagonal band and the lateral hypothalamus, had significantly elevated levels in the SHR (p less than 0.05). Cerebrospinal fluid levels of angiotensinogen did not correlate well with brain levels of angiotensinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats.

We report here on the extraction and characterization of angiotensin I (ANG I) and angiotensin II (ANG II) from the brain of rats. High pressure liquid chromatography (HPLC) with different mobile phases combined with specific radioimmunoassays (RIA) proved to be a powerful tool for peptide characterization in biological samples; (Ile5)-ANG I, (Ile5)-ANG II and (Ile5)-ANG III could clearly be identified in cerebrospinal fluid (CSF), incubated in vivo and in vitro with renin, in total brain extracts, as well as in hypothalamus (HT), medulla oblongata (MO), cerebellum (CER) and cortex (CO). Angiotensin cleaved from CSF angiotensinogen and angiotensin extracted from brain showed retention times identical to those of plasma angiotensin and synthetic standard peptides, indicating that their amino acid sequence is probably identical. ANG I and ANG II were highest in the HT and lowest in the CO. Following bilateral nephrectomy (NX) both ANG I and ANG II persisted at control levels. Young 10 week old spontaneously hypertensive rats (SHRSP) showed significantly lower ANG I and ANG II concentrations in the HT compared with Wistar Kyoto rats (WKY). Intracerebroventricular (i.c.v.) administration of the converting enzyme inhibitor captopril caused a significant increase in ANG 1 in nephrectomized SHRSP but not in WKY. These differences were not found in 40 week old SHRSP. The data show that ANG I and ANG II are synthetized in the brain of rats. The lower concentrations and the enhanced accumulation of ANG I after converting enzyme blockade in nephrectomized young SHRSP indicate an increased turnover of angiotensin in hypertensive rats.

Angiotensinogen concentration in the cerebrospinal fluid in different experimental conditions in the rat.

Angiotensinogen is the most important component of the renin-angiotensin system present in the cerebrospinal fluid (CSF) of the rat. Its physiological significance as well as its origin have not been clearly elucidated. In this experiment we have examined plasma renin activity (PRA) and plasma and CSF angiotensinogen concentration under the following experimental conditions in male rats of the Wistar strain: 1) adrenalectomy (Adx) 4 days prior to sample collection; controls were sham Adx animals; 2) nephrectomy (Nx) 48 hours before blood and CSF collection; controls were sham Nx rats; 3) DOC-salt treatment (Cortexon depot, 50 mg/kg.s.c. twice a week) plus saline to drink was given during 4 weeks; controls were intact rats; 4) DOC-salt plus captopril: captopril (100 mg/kg/day) in the drinking fluid was added to the treatment of experimental and control animals of Group 3; 5) two-kidney, two clip hypertension: silver clips placed in both renal arteries 8 weeks before samples collection; control: sham-operated rats; 6) water deprivation: rats deprived of water for 5 days; controls: intact rats; 7) peripheral sympathectomy: 6-hydroxydopamine (6-HODA) injected s.c. from birth until 16 weeks of age, adrenodemedullectomy and adrenal denervation performed at 8 weeks; controls were vehicle-injected animals. Determination of angiotensinogen concentration in plasma and CSF was accomplished by incubation of the samples with excess hog renin. The angiotensin I released as well as PRA were evaluated using an specific radioimmunoassay technique. PRA was significantly increased by Adx, captopril treatment, and water deprivation, and was almost suppressed by Nx, DOC-salt, and DOC-salt plus captopril treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of angiotensinogen in cerebrospinal fluid and plasma of rats.

The origin and regulation of angiotensinogen in cerebrospinal fluid (CSF) was investigated in rats by measuring renin substrate in plasma and CSF under different experimental conditions. Nephrectomy (NX) increased the circulating and the central angiotensinogen levels. There was no correlation between the individual values of plasma and CSF. Adrenalectomy (ADX) diminished and hydrocortisone treatment augmented the angiotensinogen levels in plasma and CSF. The combination of ADX and NX caused a dissociation between peripheral and central angiotensinogen, since the values were elevated in plasma but unchanged in CSF. After the application of the converting-enzyme inhibitor captopril a significant decrease of angiotensinogen was observed in plasma only. A specific radioimmunoassay for renin substrate of rat plasma also recognized CSF angiotensinogen. There was a linear correlation between the CSF substrate levels obtained by direct and indirect measurement. In conclusion, CSF angiotensinogen appears to be immunologically similar to the plasma molecule. The angiotensinogen levels in CSF and plasma may be affected in parallel but can nevertheless be dissociated from each other.

Clearance of rabbit plasma angiotensinogen and relationship to CSF angiotensinogen.

Rabbit plasma angiotensinogen was purified 1,390-fold by classical purification procedures. Analytical polyacrylamide gel electrophoresis and direct activity assay revealed that the purified preparations were greater than 90% native angiotensinogen. The purified angiotensinogen was radiolabeled with 125I-Na using the immobilized lactoperoxidase-Sepharose method and injected into awake, conscious rabbits. Complex clearance kinetics were observed that were resolved by a three-component model; half of the protein was cleared rapidly (t1/2 = 6 and 54 min), presumably reflecting mixing and redistribution, whereas half exhibited slow clearance kinetics (t1/2 = 8.58 h). The clearance kinetics were independent of the method of iodination and the isoelectric-point microheterogeneity of the protein. With knowledge of clearance kinetics we tested whether cerebrospinal fluid angiotensinogen could derive from the plasma pool. After injection of approximately 100 microCi 125I-angiotensinogen into the rabbit circulation, little 125I-angiotensinogen was detected in cerebrospinal fluid. Further, the brain space of 125I-angiotensinogen was identical to that of 125I-albumin, a protein that does not partition from plasma into the central nervous system. We conclude that the brain prohormone does not appear to be derived from the plasma pool.

Peptides in the cerebrospinal fluid of neuropsychiatric patients: an approach to central nervous system peptide function.

This review highlights that essentially all of the recently discovered putative central nervous system (CNS) peptides and other peptide substances are measurable in human cerebrospinal fluid (CSF). Preliminary evidence also suggests that peptides in CSF may have an active regulatory role in relation to CNS function and behavior. Even if this is not the case, CSF peptides may prove to be a useful indirect marker of CNS peptide function and metabolism. Alterations in peptides have been reported in neurological and psychiatric illness, pain symptoms and their treatment, symptoms such as anxiety, and following treatment with CNS active drugs such as carbamazepine. CSF methodologies provide a strategy for the study of the interaction of classical neurotransmitters and peptide substances and their relationship to neural function and behavior in man. Assessment of peptides in CSF may supplement post mortem studies of peptide levels and receptor distribution and help lead to new diagnostic and treatment approaches in neuropsychiatric disorders.

In vivo enzyme activity of purified human brain renin.

Cerebrospinal fluid (CSF) of rats contains high angiotensinogen concentrations. When 3500-fold purified renin from human brain was injected into the brain ventricles of rats, angiotensin I concentrations increased from undetectable levels to 147.9 +/- 18.8 fMol per ml CSF. In parallel, mean arterial blood pressure increased from 93 +/- 2.4 mm Hg to 107 +/- 3.7 mm Hg. The increase in blood pressure could be abolished by intraventricular administration of saralasin, a blocker of angiotensin II receptors. Intraventricular injection of cathepsin D had no effect on arterial blood pressure and the agiotensin I concentration in CSF remained below detection limits of the radioimmunoassay. We conclude that brain renin acts on endogenous brain angiotensinogen under physioloical in vivo conditions to form angiotensin I. The latter is converted to angiotensin II and leads to biological effects, i.e. increase of blood pressure.