Comparative characterization of ß-adrenoceptors in the bladder, heart, and lungs of rats: Alterations in spontaneously hypertensive rats.

The present study aimed to characterize and compare ß-adrenoceptors in the rat bladder with those in the heart and lungs of SD rats (8-10 weeks old) using subtype-selective agonists and antagonists in a radioligand binding assay with (-)-[125I]cyanopindolol ([125I]CYP), and also to clarify alterations in ß-adrenoceptors in the bladder of spontaneously hypertensive rats (SHR) at 14 weeks old, from those of Wistar-Kyoto rats (WKY) and Wistar rats at the same age. A radioligand binding assay with [125I]CYP was used to measure ß-adrenoceptor binding activity in rat tissues. Metoprolol exhibited the highest affinity to specific binding sites of [125I]CYP in the rat heart, indicating the dominance of ß1-adrenoceptors. ß3-selective agonists (BRL37344 and CL316243) and antagonist (SR59230A) exhibited higher affinity to specific binding sites of [125I]CYP in the bladder than in the heart and lungs. Furthermore, the binding affinity of the ß2-selective antagonist, ICI118551 was the highest in the bladder. The Bmax of specific [125]CYP binding in the bladder was significantly lower in WKY and SHR than in Wistar rats. The present study provides further evidence for the coexistence of ß2-and ß3-adrenoceptors in the rat bladder, and indicates that ß-adrenoceptor density is lower in the bladders of WKY and SHR.

Aortic butyrylcholinesterase is reduced in spontaneously hypertensive rats.

Despite the fact that vessels have sparse cholinergic innervation, acetylcholine (ACh), the primary neurotransmitter of parasympathetic nervous system, has been commonly used in physiological experiments to assess vascular function. ACh is hydrolyzed by two cholinesterases (ChE), namely acetylcholin-esterase and butyrylcholinesterase (BChE). However, little is known about these enzymes in blood vessels. The aim of the project was to characterize the expression and activity of ChE in rat aorta. As the effect of ACh on vascular tone depends on the presence of endothelium, Wistar rats were used as a model with intact endothelium and spontaneously hypertensive rats as a model of impaired endothelial function. Relative expressions of both ChE in different parts of the aorta were determined using RT-qPCR. Enzyme activities were assessed in tissue homogenates by Ellman's assay. Here we showed that both ChE are present in each part of rat aorta, while mRNA is more abundant for BChE than for AChE, irrespective of aortic compartment or genotype. Normotensive Wistar rats possess higher aortic mRNA expression and activity of BChE compared to SHR. We concluded that BChE is the dominant type of ChE in rat aorta and it might play an important role in the regulation of vascular tone.

Changes in the mesocorticolimbic pathway after low dose reserpine-treatment in Wistar and Spontaneously Hypertensive Rats (SHR): Implications for cognitive deficits in a progressive animal model for Parkinson's disease.

Reserpine (RES) is an irreversible inhibitor of VMAT2 used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low dose of reserpine was proposed as a model capable of emulating progressive neurochemical, motor and non-motor impairments in PD. Conversely, compared to Wistar rats, Spontaneously Hypertensive Rats (SHR) are resistant to motor changes induced by repeated treatment with a low dose of RES. However, such resistance has not yet been investigated for RES-induced non-motor impairments. We aimed to assess whether SHR would have differential susceptibility to the object recognition deficit induced by repeated low-dose reserpine treatment. We submitted male Wistar and SHR rats to repeated RES treatment (15 s.c. injections of 0.1 mg/kg, every other day) and assessed object memory acquisition and retrieval 48 h after the 6th RES injection (immediately before the appearance of motor impairments). Only RES Wistar rats displayed memory impairment after reserpine treatment. On the other hand, untreated SHR rats displayed object recognition memory deficit, but RES treatment restored such deficits. We also performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last RES injection. In a different set of animals submitted to the same treatment, we quantified DA, 5-HT and products of lipid peroxidation in the prefrontal cortex (PFC) and hippocampus (HPC). SHR presented increased constitutive levels of DA in the PFC and reduced immunoreactivity to TH in the medial PFC and dorsal HPC. Corroborating the behavioral findings, RES treatment restored those constitutive alterations in SHR. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potentially relevant targets to the study of susceptibility to diseases related to dopaminergic alterations.

Involvement of sex hormones, oxidative stress, ACE and ACE2 activity in the impairment of renal function and remodelling in SHR.

AIMS: Hypertension is a relevant sex and sex hormones-dependent risk factor where the cardiovascular and renal health of the population are concerned. Men experience greater losses of renal function (RF) than women, but the mechanisms remain somewhat unclear. Our goal was to evaluate the relationship between oxidative stress (OS), angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activities and RF in male and female SHR. MAIN METHODS: Twelve-week-old spontaneously hypertensive rats (SHR) were submitted to either castration or SHAM surgery and divided into 4 groups, SHAM or Castrated (CAST) males or females. After 51 days we evaluated RF (inulin and sodium para-aminohippurate), ACE and ACE2 activities (fluorimetry), OS (flow cytometry), collagen deposition (picrosirius red) and protein expression (western blot). KEY FINDINGS: Males presented lower RF than females and castration impaired this parameter in both groups. Sexual dimorphism was not observed regarding OS and inflammation; however, castration increased this parameter more severely in males than in females. SHAM males exhibited higher collagen deposition than females, though castration increased it in both sexes, eliminating the difference. We found sexual dimorphism regarding renal ACE and ACE2 activities, which were lower in males than in females. Although castration did not alter ACE activity, it reduced ACE2 activity in females and increased it in males. SIGNIFICANCE: These results indicate that sex hormones affect RF in SHR. As alterations in the oxidative system were capable of promoting podocyte injury, inflammation, and collagen deposition, we put forward that these effects are differently modulated by ACE and ACE2.

Overexpression of FNTB and the activation of Ras induce hypertrophy and promote apoptosis and autophagic cell death in cardiomyocytes.

Farnesyltransferase (FTase) is an important enzyme that catalyses the modification of protein isoprene downstream of the mevalonate pathway. Previous studies have shown that the tissue of the heart in the suprarenal abdominal aortic coarctation (AAC) group showed overexpression of FTaseß (FNTB) and the activation of the downstream protein Ras was enhanced. FTase inhibitor (FTI) can alleviate myocardial fibrosis and partly improve cardiac remodelling in spontaneously hypertensive rats. However, the exact role and mechanism of FTase in myocardial hypertrophy and remodelling are not fully understood. Here, we used recombinant adenovirus to transfect neonatal rat ventricular cardiomyocytes to study the effect of FNTB overexpression on myocardial remodelling and explore potential mechanisms. The results showed that overexpression of FNTB induces neonatal rat ventricular myocyte hypertrophy and reduces the survival rate of cardiomyocytes. FNTB overexpression induced a decrease in mitochondrial membrane potential and increased apoptosis in cardiomyocytes. FNTB overexpression also promotes autophagosome formation and the accumulation of autophagy substrate protein, LC3II. Transmission electron microscopy (TEM) and mCherry-GFP tandem fluorescent-tagged LC3 (tfLC3) showed that FNTB overexpression can activate autophagy flux by enhancing autophagosome conversion to autophagolysosome. Overactivated autophagy flux can be blocked by bafilomycin A1. In addition, salirasib (a Ras farnesylcysteine mimetic) can alleviate the hypertrophic phenotype of cardiomyocytes and inhibit the up-regulation of apoptosis and autophagy flux induced by FNTB overexpression. These results suggest that FTase may have a potential role in future treatment strategies to limit the adverse consequences of cardiac hypertrophy, cardiac dysfunction and heart failure.

Sex differences and the role of ovarian hormones in site-specific nociception of SHR.

Accurate diagnosis and treatment of pain is dependent on knowledge of the variables that might alter this response. Some of these variables are the locality of the noxious stimulus, the sex of the individual, and the presence of chronic diseases. Among these chronic diseases, hypertension is considered a serious and silent disease that has been associated with hypoalgesia. The main goal of this study was to evaluate the potential nociceptive differences in spontaneously hypertensive rats (SHR) regarding the locality of the stimulus, i.e., the temporomandibular joint or paw, the sex, and the role of ovarian hormones in a model of mechanical nociception (Von Frey test) or formalin-induced inflammatory nociception. Our results indicate that SHR had lower orofacial mechanical nociception beyond the lower mechanical nociception in the paw compared with WKY rats. In a model of formalin-induced inflammatory nociception, SHR also had decreased nociception compared with normotensive rats. We also sought to evaluate the influence of sex and ovarian hormones on orofacial mechanical nociception in SHR. We observed that female SHR had higher mechanical nociception than male SHR only in the paw, but it had higher formalin-induced orofacial nociception than male SHR. Moreover, the absence of ovarian hormones caused an increase in mean arterial pressure and a decrease in paw nociception in female SHR.

High-intensity intermittent training is as effective as moderate continuous training, and not deleterious, in cardiomyocyte remodeling of hypertensive rats.

Exercise training offers possible nonpharmacological therapy for cardiovascular diseases including hypertension. High-intensity intermittent exercise (HIIE) training has been shown to have as much or even more beneficial cardiovascular effect in patients with cardiovascular diseases than moderate-intensity continuous exercise (CMIE) training. The aim of this study was to investigate the effects of the two types of training on cardiac remodeling of spontaneously hypertensive rats (SHR) induced by hypertension. Eight-week-old male SHR and normotensive Wistar-Kyoto rats (WKY) were divided into four groups: normotensive and hypertensive control (WKY and SHR-C) and hypertensive trained with CMIE (SHR-T CMIE) or HIIE (SHR-T HIIE). After 8 wk of training or inactivity, maximal running speed (MRS), arterial pressure, and heart weight were all assessed. CMIE or HIIE protocols not only increased final MRS and left ventricular weight/body weight ratio but also reduced mean arterial pressure compared with sedentary group. Then, left ventricular tissue was enzymatically dissociated, and isolated cardiomyocytes were used to highlight the changes induced by physical activity at morphological, mechanical, and molecular levels. Both types of training induced restoration of transverse tubule regularity, decrease in spark site density, and reduction in half-relaxation time of calcium transients. HIIE training, in particular, decreased spark amplitude and width, and increased cardiomyocyte contractility and the expression of sarco(endo)plasmic reticulum Ca2+-ATPase and phospholamban phosphorylated on serine 16. NEW & NOTEWORTHY High-intensity intermittent exercise training induces beneficial remodeling of the left ventricular cardiomyocytes of spontaneously hypertensive rats at the morphological, mechanical, and molecular levels. Results also confirm, at the cellular level, that this type of training, as it appears not to be deleterious, could be applied in rehabilitation of hypertensive patients.

Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Regulates Cholinergic Signaling and Cardiovascular and Sympathetic Responses in Hypertensive Rats.

The rostral ventrolateral medulla (RVLM) is a key region in cardiovascular regulation. It has been demonstrated that cholinergic synaptic transmission in the RVLM is enhanced in hypertensive rats. Angiotensin-converting enzyme 2 (ACE2) in the brain plays beneficial roles in cardiovascular function in hypertension. The purpose of this study was to determine the effect of ACE2 overexpression in the RVLM on cholinergic synaptic transmission in spontaneously hypertensive rats (SHRs). Four weeks after injecting lentiviral particles containing enhanced green fluorescent protein and ACE2 bilaterally into the RVLM, the blood pressure and heart rate were notably decreased. ACE2 overexpression significantly reduced the concentration of acetylcholine in microdialysis fluid from the RVLM and blunted the decrease in blood pressure evoked by bilateral injection of atropine into the RVLM in SHRs. In conclusion, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced cholinergic synaptic transmission in SHRs.

Icariside II prevents hypertensive heart disease by alleviating endoplasmic reticulum stress via the PERK/ATF-4/CHOP signalling pathway in spontaneously hypertensive rats.

OBJECTIVES: Reducing endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis is a key strategy for preventing hypertensive heart disease. In our previous study, Icariside II can improve left ventricular remodelling in spontaneously hypertensive rats (SHRs). This study aims to determine whether Icariside II can exert its effect by inhibiting ERS-induced cardiomyocyte apoptosis via the PERK/ATF-4/CHOP signalling pathway. METHODS: Spontaneously hypertensive rats were randomly divided into model group and Icariside II groups. The rats in the Icariside II groups were intragastrically administrated with Icariside II 4, 8 and 16 mg/kg from 14 to 26 week-age, respectively. The left ventricular function was measured at the 18, 22 and 26 week-age by small animal ultrasound. At the end of the 26th week, cardiomyocyte apoptosis was analysed and the levels of GRP78, PERK, ATF-4 and CHOP gene and protein were detected. KEY FINDINGS: The function of left ventricular became declined with age in SHRs, but improved in Icariside II groups. Myocardial apoptosis was aggravated in SHRs, but alleviated in Icariside II groups. Icariside II could reduce the levels of GRP78, PERK, ATF-4, CHOP gene and protein that increased in SHRs. CONCLUSIONS: Icariside II prevents hypertensive heart disease by alleviating ERS-induced cardiomyocyte apoptosis, and its mechanism is related to the impediment of the PERK/ATF-4/CHOP signalling pathway.

The Effect of Chronic NO Synthase Inhibition on the Vasoactive and Structural Properties of Thoracic Aorta, NO Synthase Activity, and Oxidative Stress Biomarkers in Young SHR.

Although the role of nitric oxide (NO) in essential hypertension is still unclear, the effects of long-term NO deficiency have not yet been investigated during the critical juvenile period in spontaneously hypertensive rats (SHR). We aimed to analyze the effects of chronic NO synthase (NOS) inhibition on systolic blood pressure (sBP), vasoactivity, morphological changes and superoxide level in the thoracic aorta (TA), NOS activity in different tissues, and general biomarkers of oxidative stress in plasma of young SHR. Four-week-old SHR were treated with NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day, p.o.) for 4-5 weeks. L-NAME treatment induced a transient sBP increase only, and surprisingly, slightly inhibited endothelium-dependent relaxation of TA. Hereby, the inhibition of NOS activity varied from tissue to tissue, ranging from the lowest in the TA and the kidney to the highest in the brain stem. In spite of an increased sensitivity of adrenergic receptors, the maximal adrenergic contraction of TA was unchanged, which was associated with changes in elastin arrangement and an increase in wall thickness. The production of reactive oxygen species in the TA was increased; however, the level of selected biomarkers of oxidative stress did not change. Our findings proved that the TA of young SHR responded to chronic NO deficiency by the development of adaptive mechanisms on the functional (preserved NO-derived vasorelaxation, unincreased contraction) and molecular (preserved NOS activity) level.

Connexin 43 in splenic lymphocytes is involved in the regulation of CD4+CD25+ T lymphocyte proliferation and cytokine production in hypertensive inflammation.

Chronic inflammation promotes the development of hypertension and is associated with increased T cell infiltration and cytokine production in impaired organs. Gap junction protein connexin 43 (Cx43), is ubiquitously expressed in immune cells and plays an important role in T cell proliferation and activation, and cytokine production. However, the correlation between Cx43 in T cells and the hypertensive inflammatory response remains unknown. Thus, in this study, we wished to examine this correlation. First, our results revealed that hypertension caused significant thickening of the vascular wall, inflammatory cell infiltration into part of the renal interstitium and glomerular atrophy, and it increased the tubular damage scores in the kidneys of spontaneously hypertensive rats (SHRs). Moreover, the SHRs exhibited stenosis in the central artery wall ofthe spleen with increased serum levels of interleukin (IL)-2 and IL-6 compared with normotensive Wistar-Kyoto (WKY) rats. The spleens of the SHRs exhibited a significantly decreased percentage of CD4+CD25+ (Treg) T cells. However, the percentages of CD3+, CD4+ and CD8+ T cell and the levels of CD4+Cx43 and CD8+Cx43 did not differ significantly between the SHRs and WKY rats. In cultured lymphocytes from the SHRs and WKY rats, low percentages of Treg cells and reduced cytokine (IL-2 and IL-6) mRNA expression levels were observed in the lymphocytes obtained from the SHRs and WKY rats treated with the connexin blocker, Gap27, or concanavalin A (ConA) plus Gap27. The effects of ConA and Gap27 differed between the SHRs and WKY rats. On the whole, our findings demonstrate that the splenic Treg cell-mediated suppression in SHRs may be involved in hypertensive inflammatory responses. Cx43 in the gap junctional channel may regulate lymphocyte activation and inflammatory cytokine production.

NLRP3 inflammasome activation contributes to VSMC phenotypic transformation and proliferation in hypertension.

Inflammation is involved in pathogenesis of hypertension. NLRP3 inflammasome activation is a powerful mediator of inflammatory response via caspase-1 activation. The present study was designed to determine the roles and mechanisms of NLRP3 inflammasome in phenotypic modulation and proliferation of vascular smooth muscle cells (VSMCs) in hypertension. Experiments were conducted in spontaneously hypertensive rats (SHR) and primary aortic VSMCs. NLRP3 inflammasome activation was observed in the media of aorta in SHR and in the VSMCs from SHR. Knockdown of NLRP3 inhibited inflammasome activation, VSMC phenotypic transformation and proliferation in SHR-derived VSMCs. Increased NFκB activation, histone acetylation and histone acetyltransferase expression were observed in SHR-derived VSMCs and in media of aorta in SHR. Chromatin immunoprecipitation analysis revealed the increased histone acetylation, p65-NFκB and Pol II occupancy at the NLRP3 promoter in vivo and in vitro. Inhibition of NFκB with BAY11-7082 or inhibition of histone acetyltransferase with curcumin prevented the NLRP3 inflammasome activation, VSMC phenotype switching and proliferation in VSMCs from SHR. Moreover, curcumin repressed NFκB activation. Silencing of NLRP3 gene ameliorated hypertension, vascular remodeling, NLRP3 inflammasome activation and phenotype switching in the aorta of SHR. These results indicate that NLRP3 inflammasome activation response to histone acetylation and NFκB activation contributes to VSMC phenotype switching and proliferation and vascular remodeling in hypertension.

The Quinpirole Hypolocomotive Effects are Strain and Route of Administration Dependent in SHR and SLA16 Isogenic Rats.

The SHR and SLA16 inbred strains present behavioral differences in anxiety/emotionality that could be under the influence of dopaminergic neurotransmission. In order to investigate the role of D2 receptors in modulating such differences, an agonist (quinpirole) and an antagonist (haloperidol) of this receptor were administered, either via systemic injection (IP), or microinjected into the ventral area of the hippocampus (vHIP). Quinpirole and haloperidol IP decreased locomotor activity, only in SLA16 rats in the open-field (OF), and in both strains in the elevated plus-maze (EPM). Quinpirole also increased the preference for the aversive areas of the EPM. Quinpirole vHIP decreased locomotor activity in both strains. Haloperidol vHIP did not elicit behavioural changes and no differences in the levels of D2 receptors and of dopamine transporter in the hippocampus were found. Results indicate that systemic activation/blocking of D2 receptors caused a strain-dependent hypolocomotion, whereas activation of D2 receptors in the vHIP, but not D2 receptor antagonism, regardless of dose, decreased general locomotor activity in the two strains. Therefore, we suggest that genomic differences in the chromosome 4 can influence the locomotor activity regulated by the D2 dopaminergic receptor, especially in the vHIP.

Methylphenidate and Atomoxetine-Responsive Prefrontal Cortical Genetic Overlaps in "Impulsive" SHR/NCrl and Wistar Rats.

Impulsivity, the predisposition to act prematurely without foresight, is associated with a number of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Identifying genetic underpinnings of impulsive behavior may help decipher the complex etiology and neurobiological factors of disorders marked by impulsivity. To identify potential genetic factors of impulsivity, we examined common differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of adolescent SHR/NCrl and Wistar rats, which showed marked decrease in preference for the large but delayed reward, compared with WKY/NCrl rats, in the delay discounting task. Of these DEGs, we examined drug-responsive transcripts whose mRNA levels were altered following treatment (in SHR/NCrl and Wistar rats) with drugs that alleviate impulsivity, namely, the ADHD medications methylphenidate and atomoxetine. Prefrontal cortical genetic overlaps between SHR/NCrl and Wistar rats in comparison with WKY/NCrl included genes associated with transcription (e.g., Btg2, Fos, Nr4a2), synaptic plasticity (e.g., Arc, Homer2), and neuron apoptosis (Grik2, Nmnat1). Treatment with methylphenidate and/or atomoxetine increased choice of the large, delayed reward in SHR/NCrl and Wistar rats and changed, in varying degrees, mRNA levels of Nr4a2, Btg2, and Homer2, genes with previously described roles in neuropsychiatric disorders characterized by impulsivity. While further studies are required, we dissected potential genetic factors that may influence impulsivity by identifying genetic overlaps in the PFC of "impulsive" SHR/NCrl and Wistar rats. Notably, these are also drug-responsive transcripts which may be studied further as biomarkers to predict response to ADHD drugs, and as potential targets for the development of treatments to improve impulsivity.

Pathological characterization and morphometric analysis of hepatic lesions in SHRSP5/Dmcr, an experimental non-alcoholic steatohepatitis model, induced by high-fat and high-cholesterol diet.

SHRSP5/Dmcr is a newly established substrain of stroke-prone spontaneously hypertensive rat (SHRSP). Recently, high-fat and high-cholesterol (HFC) diet-fed SHRSP5/Dmcr has been reported as a novel rat model of developing hepatic lesions similar to human non-alcoholic steatohepatitis (NASH). The aim of this study was to investigate the detailed pathological conditions induced by HFC diet in SHRSP5/Dmcr rats using molecular biological methods and morphometric analysis. SHRSP5/Dmcr rats at 6 weeks of age were fed on either HFC diet or stroke-prone (SP) diet for 2, 4, 6, 8 and 16 weeks and histopathological changes in the liver, blood chemistry and mRNA expression levels in the liver were investigated. As evidenced by the histopathological examination of the liver of the SHRSP5/Dmcr rats, hepatic steatosis and lobular inflammation were present, with gradual increasing severity from 2 weeks after the introduction of the HFC diet. Partial hepatic fibrosis was detected at 6 weeks and spread over the entire region of the liver with more severe bridging formation by 16 weeks. The degrees of NASH-like hepatic lesions such as steatosis (the size distribution of lipid droplets), inflammation and fibrosis were quantified by morphometric analysis. Eosinophilic inclusion bodies encountered in the hepatocytes had immunoreactivity with Cox-4 and double-membrane walls, identified as mega-mitochondria. Serum ALT and bilirubins, and the mRNA expression levels related to fibrosis were closely correlated with hepatic histopathological changes. The clear feeding time-dependent progression of NASH-like hepatic lesion in HFC diet-fed SHRSP5/Dmcr rats reinforced the conclusion that this strain might be a useful model of NASH and of inflammatory fibrotic liver disease.

Isolation of a Genomic Region Affecting Most Components of Metabolic Syndrome in a Chromosome-16 Congenic Rat Model.

Metabolic syndrome is a highly prevalent human disease with substantial genomic and environmental components. Previous studies indicate the presence of significant genetic determinants of several features of metabolic syndrome on rat chromosome 16 (RNO16) and the syntenic regions of human genome. We derived the SHR.BN16 congenic strain by introgression of a limited RNO16 region from the Brown Norway congenic strain (BN-Lx) into the genomic background of the spontaneously hypertensive rat (SHR) strain. We compared the morphometric, metabolic, and hemodynamic profiles of adult male SHR and SHR.BN16 rats. We also compared in silico the DNA sequences for the differential segment in the BN-Lx and SHR parental strains. SHR.BN16 congenic rats had significantly lower weight, decreased concentrations of total triglycerides and cholesterol, and improved glucose tolerance compared with SHR rats. The concentrations of insulin, free fatty acids, and adiponectin were comparable between the two strains. SHR.BN16 rats had significantly lower systolic (18-28 mmHg difference) and diastolic (10-15 mmHg difference) blood pressure throughout the experiment (repeated-measures ANOVA, P < 0.001). The differential segment spans approximately 22 Mb of the telomeric part of the short arm of RNO16. The in silico analyses revealed over 1200 DNA variants between the BN-Lx and SHR genomes in the SHR.BN16 differential segment, 44 of which lead to missense mutations, and only eight of which (in Asb14, Il17rd, Itih1, Syt15, Ercc6, RGD1564958, Tmem161a, and Gatad2a genes) are predicted to be damaging to the protein product. Furthermore, a number of genes within the RNO16 differential segment associated with metabolic syndrome components in human studies showed polymorphisms between SHR and BN-Lx (including Lpl, Nrg3, Pbx4, Cilp2, and Stab1). Our novel congenic rat model demonstrates that a limited genomic region on RNO16 in the SHR significantly affects many of the features of metabolic syndrome.

Genetically determined differences in noradrenergic function: The spontaneously hypertensive rat model.

While genetic predisposition is a major factor, it is not known how development of attention-deficit/hyperactivity disorder (ADHD) is modulated by early life stress. The spontaneously hypertensive rat (SHR) displays the behavioral characteristics of ADHD (poorly sustained attention, impulsivity, hyperactivity) and is the most widely studied genetic model of ADHD. We have previously shown that SHR have disturbances in the noradrenergic system and that the early life stress of maternal separation failed to produce anxiety-like behavior in SHR, contrary to control Sprague-Dawley and Wistar-Kyoto (WKY) who showed typical anxiety-like behavior in later life. In the present study we investigated the effect of maternal separation on approach behavior (response to a novel object in a familiar environment) in preadolescent SHR and WKY. We also investigated whether maternal separation altered GABAA and NMDA receptor-mediated regulation of norepinephrine release in preadolescent SHR and WKY hippocampus. We found that female SHR, similar to male SHR, exhibited greater exploratory activity than WKY. Maternal separation significantly increased GABAA receptor-mediated inhibition of glutamate-stimulated release of norepinephrine in male and female SHR hippocampus but had no significant effect in WKY. Maternal separation had opposite effects on NMDA receptor-mediated inhibition of norepinephrine release in SHR and WKY hippocampus, as it increased inhibition of both glutamate-stimulated and depolarization-evoked release in SHR hippocampus but not in WKY. The results of the present study show that noradrenergic function is similarly altered by the early life stress of maternal separation in male and female SHR, while GABA- and glutamate-regulation of norepinephrine release remained unaffected by maternal separation in the control, WKY, rat strain. This article is part of a Special Issue entitled SI: Noradrenergic System.

Ultrastructural Correlates of Enhanced Norepinephrine and Neuropeptide Y Cotransmission in the Spontaneously Hypertensive Rat Brain.

The spontaneously hypertensive rat (SHR) replicates many clinically relevant features of human essential hypertension and also exhibits behavioral symptoms of attention-deficit/hyperactivity disorder and dementia. The SHR phenotype is highly complex and cannot be explained by a single genetic or physiological mechanism. Nevertheless, numerous studies including our own work have revealed striking differences in central catecholaminergic transmission in SHR such as increased vesicular catecholamine content in the ventral brainstem. Here, we used immunolabeling followed by confocal microscopy and electron microscopy to quantify vesicle sizes and populations across three catecholaminergic brain areas-nucleus tractus solitarius and rostral ventrolateral medulla, both key regions for cardiovascular control, and the locus coeruleus. We also studied colocalization of neuropeptide Y (NPY) in norepinephrine and epinephrine-containing neurons as NPY is a common cotransmitter with central and peripheral catecholamines. We found significantly increased expression and coexpression of NPY in norepinephrine and epinephrine-positive neurons of locus coeruleus in SHR compared with Wistar rats. Ultrastructural analysis revealed immunolabeled vesicles of 150 to 650 nm in diameter (means ranging from 250 to 300 nm), which is much larger than previously reported. In locus coeruleus and rostral ventrolateral medulla, but not in nucleus tractus solitarius, of SHR, noradrenergic and adrenergic vesicles were significantly larger and showed increased NPY colocalization when compared with Wistar rats. Our morphological evidence underpins the hypothesis of hyperactivity of the noradrenergic and adrenergic system and increased norepinephrine and epinephrine and NPY cotransmission in specific brain areas in SHR. It further strengthens the argument for a prohypertensive role of C1 neurons in the rostral ventrolateral medulla as a potential causative factor for essential hypertension.

Blood pressure, sex, and female sex hormones influence renal inner medullary nitric oxide synthase activity and expression in spontaneously hypertensive rats.

BACKGROUND: We previously reported that sexually mature female spontaneously hypertensive rats (SHRs) have greater nitric oxide (NO) synthase (NOS) enzymatic activity in the renal inner medulla (IM), compared to age-matched males. However, the mechanisms responsible for this sexual dimorphism are unknown. The current study tested the hypothesis that sex differences in renal IM NOS activity and NOS1 expression in adult SHRs develop with sexual maturation and increases in blood pressure (BP) in a female sex hormone-dependent manner. METHODS AND RESULTS: Renal IM were isolated from sexually immature 5-week-old and sexually mature 13-week-old male and female SHRs. Whereas NOS activity and NOS1 expression were comparable in 5- and 13-week-old male SHRs and 5-week-old female SHRs, 13-week-old females had greater NOS activity and NOS1 expression, compared to 5-week-old female SHRs and age-matched males. NOS3 expression was greater in 5-week-old than 13-week-old SHRs regardless of sex. Treatment with antihypertensive therapy (hydrochlorothiazide and reserpine) from 6 to 12 weeks of age to attenuate age-related increases in BP abolished the sex difference in NOS activity and NOS1 expression between sexually mature SHR males and females. To assess the role of female sex hormones in age-related increases in NOS, additional females were ovariectomized (OVX), and NOS activity was studied 8 weeks post-OVX. OVX decreased NOS activity and NOS1 expression. CONCLUSIONS: The sex difference in renal IM NOS in SHR is mediated by a sex hormone- and BP-dependent increase in NOS1 expression and NOS activity exclusively in females.

Overexpression of ANO1/TMEM16A, an arterial Ca2+-activated Cl- channel, contributes to spontaneous hypertension.

Calcium-activated chloride channels (CaCCs) have been implicated in hypertension; however, the mechanism underlying their involvement is unknown. The aim of this study was to determine whether the CaCC ANO1 is involved in the pathogenesis of spontaneous hypertension. Arterial ANO1 expression and the effects on blood pressure (BP) of inhibiting ANO1 with an ANO1 inhibitor, T16(Ainh)-A01, and in vivo RNAi, were examined in spontaneously hypertensive rats (SHRs). Knockdown of ANO1 by siRNA prevented hypertensive development, and attenuation of ANO1 channel activity reduced BP in SHRs. Angiotensin II upregulated ANO1 expression in primary cultures of vascular smooth muscle cells (VSMCs). The protein level and activity of cellular ANO1 positively correlated with VSMC proliferation. Our data indicate an important role of increased ANO1 expression and activity in inducing hypertension in SHRs. It may mediate angiotensin II-dependent vascular remodeling. Our results increase the mechanistic understanding of hypertension and suggest ANO1 as a possible therapeutic target for hypertension.