Brainstem Of Spontaneously Hypertensive Rats Research Articles

Targeted Metabolic Profiling and PRM Analysis of Proteins Revealed Impaired Polyunsaturated Fatty Acid Metabolism and GTP Metabolism in the Brainstem of Spontaneously Hypertensive Rats.

An untargeted multi-omics study implicated the potential dysregulation of fatty acid, nucleotide, and energy metabolism in the brainstems of spontaneously hypertensive rats (SHRs). A further quantitative exploration of the alterations in the metabolic pathways is necessary for a deep understanding of the central nervous system in SHRs. Targeted metabolic profiling of 40 fatty acids (PeptideAtlas: PASS01671) and 32 metabolites of nucleotides and energy metabolism (PeptideAtlas: PASS01672) and parallel reaction monitoring analysis of 5 proteins (PeptideAtlas: PASS01673) were performed on the brainstems of SHRs (n = 8, 11 weeks old) and normotensive Wistar rats (n = 8, age-matched) using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem MS. The targeted profiling results of metabolites and proteins revealed decreased polyunsaturated fatty acid (PUFA) synthesis with a significant downregulation of cis-11,14-eicosadienoic acid, cis-13,16-docosadienoic acid, and docosatetraenoate and impaired PUFA oxidation with the accumulation of γ-linolenate induced by the significantly downregulated expression of 2,4-dienoyl-CoA reductase (p < 0.05). Dysregulated GTP and ATP metabolism was observed, with significantly decreased GDP and ADP (p < 0.05) correlated with reduced GTPases of guanine nucleotide-binding protein subunit beta-1 (GNB1), transforming protein RhoA (RHOA), and Rho-related GTP-binding protein RhoB (RHOB) in the brainstem of SHRs. In addition, protein-arginine deiminase type-2 was significantly reduced in the brainstems of SHRs (p < 0.05). The aberrant PUFA and energy metabolism might help to explain the alterations in the brainstem of SHRs. The findings on both metabolites and proteins could provide systemic insights into the pathology basis of altered PUFA and energy metabolism in hypertension, especially in the central nervous system.

Integrative Metabolic and Proteomic Profiling of the Brainstem in Spontaneously Hypertensive Rats.

The brainstem, the core of the central nervous system, plays a vital role in controlling arterial blood pressure and its elevation of hypertension subtypes, especially essential hypertension. Integrative metabolic and proteomic profiling was performed on the brainstem samples of 11 week old spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar rats, using hydrophilic interaction liquid chromatography quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOFMS) (PeptideAtlas: PASS01621) and nano-liquid chromatography-high-resolution-MS (nano-LC-high-resolution) combined with quantitative tandem mass tags (ProteomeXchange: PXD021210). The results showed a potentially significant measure of metabolic disorders in the brainstem of SHRs, including purine and pyrimidine metabolism and carnitine and acylcarnitine deficiency. By integrating the differential metabolites (VIP > 1 and p < 0.1) with the differentially expressed proteins (>1.2-fold and p < 0.05), the results revealed aberrant insulin signaling in the brainstem of SHRs, including reduced carnitine and acetylcarnitine; increased arginine; and increased flotillin-1 (FLOT1), hemoglobin subunit alpha-1/2, and hemoglobin subunit beta-2 proteins verified by the parallel reaction monitoring analysis (PeptideAtlas: PASS01622). The aberrant insulin signaling pathway in the brainstem of SHRs might help explain the correlation between essential hypertension and insulin resistance. These findings on the brainstem of SHRs could provide new insights into the dysregulation of the central nervous system in hypertension, especially as it relates to metabolite and protein levels.

Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.

Brain nitric oxide synthase activity in spontaneously hypertensive rats during the development of hypertension.

Blockade of neuronal nitric oxide synthase (nNOS) in the brain induced an increase in mean arterial pressure of spontaneously hypertensive rats (SHR). We hypothesize that increased nitric oxide (NO) synthesis in the brain compensates for hypertension. Therefore, we measured NOS activity in different brain regions in SHR at prehypertensive, onset and established hypertension, and compared with age-matched Wistar-Kyoto (WKY) rats. NOS activity was measured by the ability of tissue homogenate to convert [3H]l-arginine to [3H]l-citrulline in a Ca2+- and NADPH-dependent manner. NOS activity was impaired in the cerebral cortex and brainstem of prehypertensive SHR. At established hypertension, SHR showed an augmentation in NOS activity in hypothalamus and brainstem. Chronic treatment of SHR with the angiotensin-1 converting enzyme (ACE)-inhibitor, enalapril, and the AT(1) receptor antagonist, losartan, normalized NOS activity in the hypothalamus but not in the brainstem. Treatment with a peripheral vasodilator, hydralazine, did not affect NOS activity. Attenuated NOS activity in the cortex and brainstem of prehypertensive SHR may play a role in the pathogenesis of hypertension. The upregulated NOS activity in the hypothalamus and brainstem of SHR possibly serves to compensate for hypertension. Hypothalamic, but not brainstem, NO is involved in antihypertensive effects of ACE inhibition and AT(1) receptor blockade. Since a blood pressure decrease per se had no effect on NOS activity, it appears that central sympathetic activity influenced by endogenous angiotensin II, rather than blood pressure, represents the stimulus for the increased NOS activity in the hypothalamus of SHR.

Electroacupuncture-related changes of NADPH-diaphorase and neuronal nitric oxide synthase in the brainstem of spontaneously hypertensive rats

The aim of this study was to investigate the electroacupuncture-related changes of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) in the brainstem of spontaneously hypertensive rats (SHR). We evaluated the changes of NADPH-d-positive neurons using a histochemical method and the changes of nNOS-positive neurons using an immunohistochemical method. The staining intensities of NADPH-d-positive neurons and nNOS-positive neurons were assessed in a quantitative fashion using a microdensitometrical method based on optical density by means of an image analyzer. The optical density of NADPH-d-positive neurons and nNOS-positive neurons of the Shinsu (BL23) and Choksamni (ST36) electroacupuncture groups were significantly decreased in most brainstem areas as compared to the normal and arbitrary groups, with the exception of the optical density of NADPH-d positive neurons in the prepositus nucleus as compared to the arbitrary group. The present results demonstrated that electroacupuncture changes the activity in the NO system in the brainstem of SHR and the site where electroacupuncture is administered is of importance for this effect.

Genetic analysis of renin gene expression in the central nervous system of spontaneously hypertensive rats

Renin expression in the central nervous system in spontaneously hypertensive rats (SHR) has been reported to be higher than that in age-matched Wistar–Kyoto (WKY) rats. In the present study, we examined the mechanisms of enhancement of renin mRNA concentration in the brainstem of SHR using F 2 hybrid rats derived from cross-breeding SHR with WKY. The SHR allele of the renin gene was significantly associated with higher brain renin mRNA concentration in both 10- and 24-week-old F 2 populations. However, neither the genotype of the renin gene nor the brain renin mRNA concentration was associated with blood pressure or urinary volume. Thus, the higher renin mRNA concentration in the brainstem of SHR was indicated to be genetically determined by the genotype of the renin gene. Further study is required to clarify the pathophysiological meaning of this increased renin expression in the brain of SHR.

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Alterations in Vasopressin Mechanisms in Captopril-Treated Spontaneously Hypertensive Rats

The effects of lifetime captopril treatment on vasopressin (VP) were assessed in spontaneously hypertensive rats (SHR). Pregnant and nursing dams were treated with oral Captopril (100 mg/kg/day). After weaning, the pups were maintained on Captopril (50/kg/day) for 19-20 wks. Blood pressures of Captopril-treated SHR were in the normotensive range and significantly lower (p less than .001) than SHR control rats. Control and Captopril-treated SHR were perfused and brains were sectioned for immunohistochemical staining with a polyclonal antibody directed against vasopressin (VP). Compared to control SHR, Captopril-treated rats displayed decreased VP-like immunoreactivity in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Captopril treatment also selectively decreased the number of brightly labeled cell bodies in the SON and PVN and reduced VP-like labeling in the axons of the neurons in these nuclei. Concurrent with a decrease in VP-like immunoreactivity, Captopril treatment reduced plasma VP levels (RIA) (p less than 0.01, Captopril, 5.6 +/- 0.5 pg/ml; control, 11.8 +/- 2.2 pg/ml). Scatchard analysis of 3H-VP binding indicated that Captopril treatment increased the number but not the affinity of VP receptors in the hypothalamus and brain stem of SHR. These results suggest that in SHR oral Captopril treatment attenuates the synthesis and release of VP, an effect that may contribute to the blood pressure lowering effect of converting enzyme inhibitors.

Genetically Altered Brain Amino Acid Metabolism in Spontaneously Hypertensive Rats: A Study by Using Young Spontaneously Hypertensive Rats and Renal Hypertensive Rats

Previously we demonstrated altered amino acid levels in brainstem regions of adult spontaneously hypertensive rats (SHR). For comparison, in this study, we determined amino acid concentrations in discrete brainstem regions in young prehypertensive SHR and renal hypertensive rats. In prehypertensive SHR, the content of glutamate was increased in the rostral ventrolateral medulla and the caudal ventrolateral medulla, and the content of beta-alanine was decreased in the nucleus tractus solitarii. In renal hypertensive rats, there was no change in glutamate and beta-alanine contents in all the regions. The profiles of contents of glutamate and beta-alanine in the brainstem regions in young SHR but not in renal hypertensive rats are the same as those found previously in adult SHR. Thus, the results of the present study suggest that the altered amino acid metabolism in the brainstem of SHR may be genetically inherent.

Evidence that the cultured multipolar neurons of the lower brainstem of SHR and WKY rats differ significantly in the mode of responses to catecholamines

By using cultures of 6 types of dissociated neurons from the lower brainstem of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, we analyzed differences in the mode of response of the cultured neurons to 9 kinds of peptides and 7 kinds of neurotransmitters, and obtained evidence that there is a significant difference in the mode of response of multipolar neurons to catecholamines; dominantly facilitation in SHR, but dominantly inhibition in WKY rats.

Levels of Tyrosine and Tryptophan in the Plasma and Brain of Spontaneously Hypertensive Rats

Tyrosine and tryptophan contents in the plasma and brain, and noradrenaline and serotonin contents in the brain were measured in spontaneously hypertensive rats (SHR) and two normotensive strains, Wistar/Kyoto rats (WKR) and Wistar/Carworth rats (WCR) to investigate the quantitative relationship between monoamines and their precursor amino acids in the brain of SHR. WKR is the parent strain of SHR. Tyrosine level in the plasma and brain of SHR was significantly lower than that in WCR, whereas tryptophan level in the plasma and brain of SHR was higher than that of WCR. The levels of these amino acids were the same in SHR and WKR. Noradrenaline and serotonin contents in the brain stem of SHR and WKR were lower than the respective contents of WCR, while no difference was found in noradrenaline and serotonin contents in the telencephalon of these 3 strains. In SHR, 1.0% L-tyrosine ingestion for 5 days produced a slight but significant fall of the blood pressure accompanied by an increase of tyrosine in the plasma and brain. However, noradrenaline in the brain stem did not change following the L-tyrosine feeding. These results suggest that differences in contents of the monoamines and their precursor amino acids in SHR and WKR could be due to genetic factors in a specific closed colony.