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Pathogenic bacteria are a major cause of foodborne diseases, which not only seriously threaten human safety but also cause significant losses for the national economy. Therefore, it is very important and urgent to develop a method for the detection of pathogenic bacteria with high accuracy, high sensitivity, and easy interpretation for use in food safety and medical hygiene. Herein, based upon the sensitive color changes induced by the dispersion and aggregation states of gold nanoparticles (AuNPs), a point-of-care (POCT) colorimetric assay was constructed for the rapid and sensitive visual detection of pathogenic bacteria. The POCT visual sensing system is composed of two individual elements: (1) an alkaline phosphatase/graphene oxide (GO@PEI-ALP) nanoconjugate that can release free ALP molecules in the presence of pathogenic bacteria; (2) D-glucose-6-phosphate (pGlu) and 3-aminobenzene boric acid (AMBA)-functionalized AuNPs (pGlu/AMBA-AuNPs) that are cross-linked upon the digestion of pGlu by free ALP molecules, resulting in a significant color change. Under optimized conditions, the detection limit of this sensing system for target bacteria was as low as 24 CFU mL

Hypertension is a prevalent pathology that increases risk for numerous cardiovascular diseases. Because the etiology of hypertension varies across patients, specific and effective therapeutic approaches are needed. The role of renal sympathetic nerves is established in numerous forms of hypertension, but their contribution to salt sensitivity and interaction with factors such as endothelin-1 are poorly understood. Rats deficient of functional ETB receptors (ETB-def) on all tissues except sympathetic nerves are hypertensive and exhibit salt-sensitive increases in blood pressure. We hypothesized that renal sympathetic nerves contribute to hypertension and salt sensitivity in ETB-def rats. The hypothesis was tested through bilateral renal sympathetic nerve denervation and measuring blood pressure during normal salt (0.49% NaCl) and high-salt (4.0% NaCl) diets. Denervation reduced mean arterial pressure in ETB-def rats compared with sham-operated controls by 12 ± 3 (SE) mmHg; however, denervation did not affect the increase in blood pressure after 2 wk of high-salt diet (+19 ± 3 vs. +16 ± 3 mmHg relative to normal salt diet; denervated vs. sham, respectively). Denervation reduced cardiac sympathetic-to-parasympathetic tone [low frequency-high frequency (LF/HF)] during normal salt diet and vasomotor LF/HF tone during high-salt diet in ETB-def rats. We conclude that the renal sympathetic nerves contribute to the hypertension but not to salt sensitivity of ETB-def rats.

The main actions of the renin–angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT 1 Rs). The major murine AT 1 R isoform, AT 1A R, is expressed throughout the nephron, including the collecting duct in both principal and intercalated cells. Principal cells play the major role in sodium and water reabsorption. Although aldosterone is considered to be the dominant regulator of sodium reabsorption by principal cells, recent studies suggest a role for direct actions of AT 1 R. To specifically examine the contributions of AT 1A R in principal cells to BP regulation and the development of hypertension in vivo, we generated inbred 129/SvEv mice with deletion of AT 1A R from principal cells (PCKO). At baseline, we found that BPs measured by radiotelemetry were similar between PCKOs and controls. During 1-week of low-salt diet (P P 1A R on baseline BP and adaptation to changes in dietary salt are negligible, our studies suggest that direct actions of AT 1A R contribute to the initiation of hypertension and epithelial sodium channel activation.

In 2014, the American Heart Association (AHA) announced the funding of a Strategically Focused Research Network (SFRN) focused on hypertension. Hypertension was chosen for a SFRN because interdisciplinary hypertension research has great potential to impact the AHA’s mission and 2020 goals of improving the cardiovascular health of all Americans by 20% while reducing deaths from cardiovascular disease (CVD) and stroke by 20% by 2020. Four centers with highly integrated basic, clinical, and population science projects were selected for the AHA SFRN: Cincinnati Children’s Hospital, Medical College of Wisconsin (MCW), University of Alabama at Birmingham (UAB), and University of Iowa (UI). The Cincinnati center is studying ambulatory blood pressure (BP) and hypertensive target organ damage in adolescents. The MCW center is studying epigenetics of hypertension by conducting studies of genome-wide DNA methylation patterns. The UAB center is studying racial differences in the prevalence and determinants of abnormal diurnal BP patterns. The UI center is studying copeptin, a possible early biomarker for preeclampsia and vascular endothelial function, and arterial stiffness changes occurring before and during preeclampsia. In this article, we provide an overview of the science being conducted at each center and describe the innovative training program incorporated into the SFRN. A goal of the Hypertension SFRN is to facilitate collaborative research. Therefore, we describe opportunities and resources available to researchers interested in collaborating with SFRN investigators. The AHA Hypertension SFRN has the potential to identify new approaches for the prevention and treatment of hypertension and, ultimately, improve the cardiovascular health of Americans. The SFRN is a mechanism initiated by the AHA to address key strategic issues as determined by the AHA Board of Directors. In 2014, hypertension was selected for a SFRN because improvement in its prevention and treatment has the potential to impact the AHA’s mission and 2020 goals: to …

Crab paste is regularly consumed by people in the coastal area of China. The fermentation time plays a key role on the quality of crab paste. Here, we investigated the dynamic evolution of metabolite profile of crab paste during fermentation by combined use of NMR spectroscopy and multivariate data analysis. Our results showed that crab paste quality was significantly affected by fermentation. The quality change was manifested in the decline of lactate, betaine, taurine, trimethylamine-N-oxide, trigonelline, inosine, adenosine diphosphate, and 2-pyridinemethanol, and in the fluctuation of a range of amino acids as well as in the accumulation of glutamate, sucrose, formate, acetate, trimethylamine, and hypoxanthine. Trimethylamine production and its increased level with fermentation could be considered as a freshness index of crab paste. These results contribute to quality assessment of crab paste and confirm the metabolomics technique as a useful tool to provide important information on the crab paste quality.

Healthy individuals display a diurnal blood pressure (BP) rhythm and salt-sensitivity increases the susceptibility for dysfunctional BP rhythms. Previously, bayesian modeling found that Rfwd2 gene was associated with circadian clock gene pathways in the Dahl Salt-sensitive rats (SS). SS rats lacking Rfwd2 were generated to test the hypothesis that Rfwd2 alters the circadian BP rhythm and salt-sensitivity in SS rats. Rfwd2 total knockout rats on the SS background are embryonic lethal, thus experiments utilized male Rfwd2 heterozygous SS ( Rfwd2 +/- ; n=6) and SS rats (n=8). All rats were maintained on Teklad (NIH-31) normal salt diet (NS; 0.4% NaCl) and then fed high salt diet (HS; 4% NaCl). BP was recorded via telemetry and systolic BP analyzed on days 4-7 of diet. On NS, the amplitude (difference between peak and mean) and mesor (rhythm-adjusted mean) were similar between genotypes. However, HS diet increased the amplitude (SS: 5.2 ± 0.4 to 9.3 ± 0.5, P+/- : 3.7 ± 0.5 to 9.0 ± 0.5, P+/- : 150 ± 1.6 mmHg to 177 ± 3.1 mmHg, P+/- rats compared to SS had greater mesor (178 ± 3.1 mmHg vs. 166 ± 1.7 mmHg, P=0.003, respectively) and similar amplitude. Rfwd2 +/- had significant 1.4hr delay in trough (lowest value) SBP timing compared to SS within both salt diets (NS: P=0.005; HS: P=0.008). In addition, HS caused a delay in trough SBP timing in both genotypes. These results (figure) show that Rfwd2 has a role in trough BP timing, and the lack of this gene results in an exaggerated mesor on HS diet and a 1.4hr delay on NS and HS diets, which could potentially be coupled with renal injury.

Angiotensin type 1 (AT1) receptors are key effector elements of the renin-angiotensin system, mediating virtually all of the classical physiological actions of angiotensin II. Pharmacological blockade of the AT1 receptor effectively lowers blood pressure in a substantial proportion of patients with hypertension, indicating the pivotal role of these receptors in human hypertension. AT1 receptors are expressed in many different organ systems where they have myriad cellular actions. However, several lines of evidence have suggested that direct actions of AT1 receptors in kidney have a major role in regulation of blood pressure and in the pathogenesis of hypertension. Here we review recent studies suggesting that renal epithelium and vasculature may be key cellular targets, where AT1 receptor activation has powerful physiological impact. We will also examine novel regulatory mechanisms by peptides associated with the C-terminus of the AT1 receptor.

The renal collecting duct (CD), as the terminal segment of the nephron, is responsible for the final adjustments to the amount of sodium excreted in urine. While angiotensin II modulates reabsorptive functions of the CD, the contribution of these actions to physiological homeostasis is not clear. To examine this question, we generated mice with cell-specific deletion of AT1A receptors from the CD. Elimination of AT1A receptors from both principal and intercalated cells (CDKO mice) had no effect on blood pressures at baseline or during successive feeding of low- or high-salt diets. In contrast, the severity of hypertension caused by chronic infusion of angiotensin II was paradoxically exaggerated in CDKO mice compared with controls. In wild-type mice, angiotensin II induced robust expression of cyclooxygenase-2 (COX-2) in renal medulla, primarily localized to intercalated cells. Upregulation of COX-2 was diminished in CDKO mice, resulting in reduced generation of vasodilator prostanoids. This impaired expression of COX-2 has physiological consequences, since administration of a specific COX-2 inhibitor to CDKO and control mice during angiotensin II infusion equalized their blood pressures. Stimulation of COX-2 was also triggered by exposure of isolated preparations of medullary CDs to angiotensin II. Deletion of AT1A receptors from principal cells alone did not affect angiotensin II-dependent COX2 stimulation, implicating intercalated cells as the main source of COX2 in this setting. These findings suggest a novel paracrine role for the intercalated cell to attenuate the severity of hypertension. Strategies for preserving or augmenting this pathway may have value for improving the management of hypertension.

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor–deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor–deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Hypertension contributes to multiple forms of cardiovascular disease and thus morbidity and mortality. The mechanisms inducing hypertension remain unclear although the involvement of homeostatic systems, such as the renin–angiotensin and sympathetic nervous systems, is established. A pivotal role of the angiotensin type 1 receptor in the proximal tubule of the kidney for the development of experimental hypertension is established. Yet, other systems are involved. This study tests whether the expression of angiotensin type 1A receptors in catecholaminergic cells contributes to hypertension development. Using a Cre-lox approach, we deleted the angiotensin type 1A receptor from all catecholaminergic cells. This deletion did not alter basal metabolism or blood pressure but delayed the onset of angiotensin-dependent hypertension and reduced the maximal response. Cardiac hypertrophy was also reduced. The knockout mice showed attenuated activation of the sympathetic nervous system during angiotensin II infusion as measured by spectral analysis of the blood pressure. Increased reactive oxygen species production was observed in forebrain regions, including the subfornical organ, of the knockout mouse but was markedly reduced in the rostral ventrolateral medulla. These studies demonstrate that stimulation of the angiotensin type 1A receptor on catecholaminergic cells is required for the full development of angiotensin-dependent hypertension and support an important role for the sympathetic nervous system in this model.

Vinasse pike eel (Muraenesox cinereus) is a traditional Chinese food with a characteristic flavour, taste, and nutritional composition. Its flavour is closely related to the molecular nutritional composition of this food pickled product. In this study, we characterised the changes in the nutritional composition of pike eel during vinasse pickling. Nuclear magnetic resonance spectroscopy revealed 33 components in eel, e.g. a range of organic acids, amino acids, alcohols, and sugars. Multivariate data analysis further revealed that the nutritional composition of eel undergoes major changes during pickling, which were highlighted by the consumption of sucrose and creatine, the accumulation of a range of organic acids, alcohols, glucose, and creatinine, as well as in the fluctuation of some amino acids. The abundant sucrose, glutamate, creatine, and lactate could take an active part in the flavour formation of vinasse eel. This work provides insight into the nutritional characteristics of vinasse eel during pickling.

Circadian rhythms are controlled by an endogenous time-keeping system oscillating approximately on a 24-h cycle under constant conditions. These rhythms depend on a network of interacting genes and proteins, including transcriptional activators such as CLOCK, NPAS2, and ARNTL (BMAL1), which induce transcription of the clock genes Period ( Per1 , Per2 , and Per3 ) and Cryptochrome ( Cry1 and Cry2 ). Human salivary cortisol and melatonin follow a clear circadian rhythm as well. Disruption of the circadian rhythm and sleep-wake cycles are considered risk factors for a variety of health problems, especially hypertension and other cardiovascular and metabolic diseases. Here we put together practical methods for assessing circadian rhythms in adult subjects conducted by each individual. This method is non-invasive, inexpensive and provides a predictive profile of an individual’s circadian rhythm related to clock-controlled gene expression in buccal cells, salivary cortisol, salivary melatonin, and subject’s activity or sleep. Subjects are instructed on how to obtain buccal cells using swabs (Whatman OmniSwab) from the inside of their cheeks and collect saliva using salivettes (Sarstedt) every 4 hours starting at 6am, for 2 consecutive days. Subjects also wear actigraphy watches (Phillips Respironics) during the 2 days, to record their activity, light exposure and estimates of sleep times. To monitor adherence to correct time point collections, each subject is given an electronic vial called eCAP (Information Mediary Corp) that records the exact time the container is opened to place samples once collected. We demonstrate feasibility to extract up to 150ng/μl of RNA (Ambion RNAqueous-Micro Total RNA Isolation Kit) from buccal cells swabs. Salivary melatonin and cortisol are measured by radioimmunoassay (Buhlmann Lab) with melatonin peak levels ranging from 14 to 23 pg/ml and cortisol peak levels ranging from 10 to 24 ng/ml. We suggest that buccal cell expression of clock-controlled genes, salivary melatonin, salivary cortisol, and actigraphy data are valuable in providing reliable assessment of human circadian rhythm profiles under a variety of conditions.

The pathogenesis of hypertension is multi-factorial, involving many of the systems contributing to blood pressure homeostasis including the vasculature, kidneys, central, and sympathetic nervous systems, along with various hormonal regulators. However, over the years, many studies have indicated a predominant importance of the kidney in blood pressure homeostasis and hypertension. This work has established the notion that the kidney is a key determinant of the chronic level of intra-arterial pressure playing a major role in the pathogenesis of hypertension. Therefore, this review will focus on recent work using genetically modified mouse models addressing the role of the kidney in hypertension. In particular, human genetic studies of Mendelian disorders with major impact on blood pressure homeostasis have provided powerful evidence for a role of the kidney in hypertension. Of the approximately 20 genes identified as causal in these disorders, virtually all have an effect on the control of solute transport in the kidney. As such, we have especially focused on generation of mouse models addressing the nature of these specific molecular defects in nephron function that produce an alteration in blood pressure.

Vasoconstriction is a signature physiological action of angiotensin II (AngII) acting via AT1 receptors (AT1R). In order to define the contribution of AT1R in vascular smooth muscle cells (VSMCs) to BP control, we generated mice with cell-specific deletion of AT1AR from VSMCs (SMKOs) using Cre-loxp technology. Baseline BP was reduced by ~7 mmHg and responses to AngII-induced hypertension were significantly blunted by in SMKO mice compared to controls (16 vs. 30 mm Hg change in BP from baseline after 4 wks AngII, P

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1*±0.8 vs. 1.4*±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2.

Left ventricular hypertrophy (LVH) is a common manifestation of end-organ injury in HTN. ACEi and ARBs attenuate the development of LVH through actions suggested to depend on direct interruption of AT1R in cardiac myocytes, rather than their effects to lower BP. In order to define the contribution of AT1R in cardiac myocytes to LVH, we made 2 mouse lines with cell-specific deletion of AT1AR from the heart using a floxed Agtr1a allele. In the 1st line, loss of AT1AR was achieved using a heterologous Cre transgene driven by the Sm22 promoter (Tagln-Cre) expressing Cre in VSMC and cardiac myocytes. There was virtually complete loss of AT1AR from cardiac myocytes and VSMCs in aorta, but not resistance vessels. Thus, with chronic AngII infusion, Tagln-KOs develop HTN to the same level as controls (MAP: 157±6 vs. 153±6 mm Hg, P=NS). In AngII-infused controls, there was cardiac oxidative stress indicated by enhanced nitrated tyrosine, but this was reduced by ≈75% in Tagln-KOs (P

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)–dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II–dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%–10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II–dependent vascular responses in the kidney to effect natriuresis and BP control.

The main actions of the renin angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT 1 R). The major murine AT 1 R isoform, AT 1A is highly expressed in epithelial cells throughout the nephron, including the collecting duct (CD). The CD consists of two cell types, principal (PC) and intercalated cells (IC) with distinct functions. Activation of AT 1 R in PC stimulates solute reabsorption in the CD by increasing the activity of epithelial sodium channels. To examine the role of the AT 1A R in the PC to BP regulation and the development of hypertension in vivo , we generated inbred 129SvEv mice with cell-specific deletion of AT 1A R in PC (PC-KO, n=6). AQP2-Cre transgenic mouse line was used to excise the floxed Agtr1a allele in PC. mTmG reporter mouse line was used to confirm specific expression of AQP2-Cre transgene in PC, and found the pattern of expression to be predominantly in medullary CD. Using RT-PCR, mRNA levels for AT 1A R were reduced by ≈50% in the inner medulla of PC-KO mice ( P 1A R in PC of the medullary CD have little influence on BP, adaptation to changes in dietary salt intake or development of hypertension. We suggest that other mediators, such as aldosterone, may have a more important role to influence sodium handling in this nephron segment.

Aims The caudal ventrolateral medulla (CVLM) is important for autonomic regulation and is rich in angiotensin II type 1A receptors (AT1AR). To determine their function, we examined whether the expression of AT1AR in the CVLM of mice lacking AT1AR (AT1A−/−) alters baroreflex sensitivity and cardiovascular responses to stress. Methods and results Bilateral microinjections into the CVLM of AT1A−/− mice of lentivirus with the phox-2 selective promoter (PRSx8) were made to express either AT1AR (Lv-PRSx8-AT1A) or green fluorescent protein (Lv-PRSx8-GFP) as a control. Radiotelemetry was used to record mean arterial pressure (MAP), heart rate (HR), and locomotor activity. Following injection of Lv-PRSx8-GFP, robust neuronal expression of GFP was observed with ∼60% of the GFP-positive cells also expressing the catecholamine-synthetic enzyme tyrosine hydroxylase. After 5 weeks, there were no differences in MAP or HR between groups, but the Lv-PRSx8-AT1A- injected mice showed reduced baroreflex sensitivity (−25%, P = 0.003) and attenuated pressor responses to cage-switch and restraint stress compared with the Lv-PRSx8-GFP-injected mice. Reduced MAP mid-frequency power during cage-switch stress reflected attenuated sympathetic activation (Pgroup × stress = 0.04). Fos-immunohistochemistry indicated greater activation of forebrain and hypothalamic neurons in the Lv-PRSx8-AT1A mice compared with the control. Conclusion The expression of AT1AR in CVLM neurons, including A1 neurons, while having little influence on the basal blood pressure or HR, may play a tonic role in inhibiting cardiac vagal baroreflex sensitivity. However, they strongly facilitate the forebrain response to aversive stress, yet reduce the pressor response presumably through greater sympatho-inhibition. These findings outline novel and specific roles for angiotensin II in the CVLM in autonomic regulation.

The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT1ARs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT1ARs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT1AR-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT1AR promoter. The overall distribution correlated with that of the AT1ARs mapped by other methods and demonstrated that the majority of C1 neurons express the AT1AR. Cre-recombinase expression in C1 neurons of AT1AR-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT1AR in these neurons. Lentiviral-induced expression of wild-type AT1ARs in C1 neurons of global AT1AR knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT1ARs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 ± 4 mmHg for AT1AR vs 10 ± 1 mmHg for GFP;p< 0.001), which was restored toward that of the wild-type group (28 ± 2 mmHg). This study demonstrates that AT1AR expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress.

In adult mice we determined whether expression of angiotensin II (Ang II) type 1A receptors (AT 1A Rs) in C1 neurons mediates the excitation of the rostral ventrolateral medulla (RVLM) by Ang II. Blood pressure, heart rate, and sympathetic nerve activity were measured in anesthetized, artificially ventilated wild-type (n=15) and AT 1A R knockout (AT 1A −/− ; n=9) mice. Microinjection of Ang II (50 nL of 0.1 to 1.0 mmol/L) into the RVLM induced a dose-related, sympathetically mediated pressor response (maximum of 17±2 mm Hg) in wild-type mice. These microinjections had no effect in AT 1A −/− mice. Endogenous AT 1 Rs occur on catecholaminergic C1 neurons in the RVLM. We induced AT 1A R or green fluorescent protein expression in C1 neurons of AT 1A −/− mice through bilateral microinjection of replication-deficient lentiviruses, with transgene expression under the control of a phox2 transcription factor binding promoter (PRSx8) (Lv-PRSx8-AT 1A , n=10, and Lv-PRSx8-GFP, n=5). Transgene expression was observed in a significant proportion of RVLM C1 neurons. In anesthetized Lv-PRSx8-AT 1A injected mice, unilateral RVLM microinjection of Ang II (50 nL of 1 mmol/L) increased blood pressure (17±4 mm Hg) and sympathetic nerve activity (155±32%). No response to Ang II occurred in Lv-PRSx8-GFP microinjected mice. These results show that Ang II–mediated excitation of RVLM neurons in adult mice depends on the AT 1A R with little or no effect of type 1B or 2 receptors. Expression of the AT 1A R predominantly in C1 catecholamine neurons restores the response to Ang II in the AT 1A −/− mouse and demonstrates that these neurons are sympathoexcitatory in the mouse.

BACKGROUND Abnormal circadian variation of blood pressure (BP) increases cardiovascular risk. In this study, we examined the influence of angiotensin AT(1A) receptors on circadian BP variation, and specifically on its behavioral activity-related and -unrelated components. METHODS BP and locomotor activity were recorded by radiotelemetry in AT(1A)-receptor knockout mice (AT(1A)(-/-)) and their wild-type controls (AT(1A)(+/+)) placed on a normal-salt diet (NSD) or high-salt diet (HSD, 3.1% Na). RESULTS The 24-h BP was lower in AT(1A)(-/-) than AT(1A)(+/+) mice on a NSD (92 +/- 2 and 118 +/- 2 mm Hg, respectively), whereas the day-night BP difference (DeltaDNBP) was similar between groups (11 +/- 2 and 12 +/- 1 mm Hg, respectively). HSD increased BP by 20 +/- 2 mm Hg and DeltaDNBP by 7 +/- 1 mm Hg in AT(1A)(-/-) mice, without affecting these parameters much in AT(1A)(+/+) mice. The DeltaDNBP increase in AT(1A)(-/-) mice was caused by nondipping BP during the inactive late-dark period. Conversely, BP rise associated with circadian behavioral activation during the early dark period was not altered by HSD in AT(1A)(-/-) mice. The BP change associated with spontaneous ultradian activity-inactivity bouts was also similar between strains on HSD as was the BP rise associated with induced (cage-switch) behavioral activity. Ganglionic or alpha(1)-adrenergic blockade decreased BP in both strains; HSD did not affect this response in AT(1A)(-/-), but abolished it in AT(1A)(+/+) mice. CONCLUSIONS AT(1A)-receptor deficiency, when combined with HSD, can increase circadian BP difference in mice. This increase is mediated principally by activity-unrelated factors, such as the nonsuppressibility of basal resting sympathetic tone by HSD, thus suggesting a form of salt-/volume-dependent hypertension.

ANG II, the main circulating effector hormone of the renin-angiotensin system, is produced by enzymatic cleavage of angiotensinogen. The present study aimed to examine whether targeted deletion of the angiotensinogen gene ( Agt) altered brain ANG II receptor density or responsiveness to ANG II. In vitro autoradiography was used to examine the distribution and density of angiotensin type 1 (AT1) and type 2 receptors. In most brain regions, the distribution and density of angiotensin receptors were similar in brains of Agt knockout mice ( Agt−/−) and wild-type mice. In Agt−/−mice, a small increase in AT1receptor binding was observed in the rostral ventrolateral medulla (RVLM), a region that plays a critical role in blood pressure regulation. To examine whether Agt−/−mice showed altered responses to ANG II, blood pressure responses to intravenous injection (0.01–0.1 μg/kg) or RVLM microinjection (50 pmol in 50 nl) of ANG II were recorded in anesthetized Agt−/−and wild-type mice. Intravenous injections of phenylephrine (4 μg/kg and 2 μg/kg) were also made in both groups. The magnitude of the pressor response to intravenous injections of ANG II or phenylephrine was not different between Agt−/−and wild-type mice. Microinjection of ANG II into the RVLM induced a pressor response, which was significantly smaller in Agt−/−compared with wild-type mice (+10 ± 1 vs. +23 ± 4 mmHg, respectively, P = 0.004). Microinjection of glutamate into the RVLM (100 pmol in 10 nl) produced a robust pressor response, which was not different between Agt−/−and wild-type mice. A diminished response to ANG II microinjection in the RVLM of Agt−/−mice, despite an increased density of AT1receptors suggests that signal transduction pathways may be altered in RVLM neurons of Agt−/−mice, resulting in attenuated cellular excitation.

We determined whether genetic deficiency of angiotensin II Type 1A (AT 1A ) receptors in mice results in altered neuronal responsiveness and reduced cardiovascular reactivity to stress. Telemetry devices were used to measure mean arterial pressure, heart rate, and activity. Before stress, lower resting mean arterial pressure was recorded in AT 1A −/− (85±2 mm Hg) than in AT 1A +/+ (112±2 mm Hg) mice; heart rate was not different between groups. Cage-switch stress for 90 minutes elevated blood pressure by +24±2 mm Hg in AT 1A +/+ and +17±2 mm Hg in AT 1A −/− mice ( P 1A +/+ and +121±9 bpm in AT 1A −/− mice ( P 1A −/− (3.0±0.4 U) than in AT 1A +/+ animals (6.0±0.4 U), but differences in blood pressure and heart rate persisted during nonactive periods. In contrast to wild-type mice, spontaneous baroreflex sensitivity was not inhibited by stress in AT 1A −/− mice. After cage-switch stress, c-Fos immunoreactivity was less in the paraventricular ( P P =0.001) nuclei of the hypothalamus and rostral ventrolateral medulla ( P 1A −/− compared with AT 1A +/+ mice. Conversely, greater c-Fos immunoreactivity was observed in the medial nucleus of the amygdala, caudal ventrolateral medulla, and nucleus of the solitary tract ( P 1A −/− compared with AT 1A +/+ mice. Greater activation of the amygdala suggests that AT 1A receptors normally inhibit the degree of stress-induced anxiety, whereas the lesser activation of the hypothalamus and rostral ventrolateral medulla suggests that AT 1A receptors play a key role in autonomic cardiovascular reactions to acute aversive stress, as well as for stress-induced inhibition of the baroreflex.

Pharmacological evidence suggests that angiotensin II type 1 (AT(1)) receptors are involved in the regulation of cardiovascular response to emotional stress and reinforcing effect of dietary salt on this response. In this study, we examined the effect of genetic deletion of AT(1A) receptors on the cardiovascular effects of stress and salt in mice. AT(1A) receptor knockout (AT(1A)(-/-)) and wild-type (AT(1A)(+/+)) mice were implanted with telemetry devices and placed on a normal (0.4%) or high (3.1%) salt diet (HSD). Resting blood pressure (BP) in AT(1A)(-/-) mice (84+/-3 mm Hg) was lower than in AT(1A)(+/+) mice (107+/-2 mm Hg). Negative emotional (restraint) stress increased BP by 33+/-3 mm Hg in AT(1A)(+/+) mice. This response was attenuated by 40% in AT(1A)(-/-) mice (18+/-3 mm Hg). Conversely, the BP increase caused by food presentation and feeding was similar in AT(1A)(-/-) (25+/-3 mm Hg) and AT(1A)(+/+) mice (26+/-3 mm Hg). HSD increased resting BP by 14+/-4 mm Hg in AT(1A)(-/-) mice without affecting it significantly in AT(1A)(+/+) mice. Under these conditions, the pressor response to restraint stress in AT(1A)(-/-) mice (30+/-3 mm Hg) was no longer different from that in wild-type animals (28+/-3 mm Hg). The BP response to feeding was not altered by HSD in either AT(1A)(-/-) or AT(1A)(+/+) mice (25+/-2 and 27+/-3 mm Hg, respectively). These results indicate that AT(1A) receptor deficiency leads to a reduction in BP reactivity to negative emotional stress, but not feeding. HSD can selectively reinforce the cardiovascular response to negative stress in AT(1A)(-/-) mice. However, there is little interaction between AT(1A) receptors, excess dietary sodium and feeding-induced cardiovascular arousal.

Angiotensin II acts through specific receptors to alter several brain functions including fluid and electrolyte control, neuroendocrine function and autonomic efferent activity. This review discusses one brain site, the rostral ventrolateral medulla, where the actions of angiotensin II have been intensively studied. The rostral ventrolateral medulla plays a critical role in the generation and regulation of sympathetic activity to the cardiovascular system and hence is important for blood pressure control. Current evidence indicates that angiotensin II activates neurons in the rostral ventrolateral medulla via the AT1A receptor. In some models of cardiovascular disease, blockade of AT1 receptors in the rostral ventrolateral medulla reduces sympathetic nerve activity and blood pressure suggesting that overactivity of the angiotensin system in this nucleus may play a role in the maintenance of high blood pressure.

head-up tilt test, referred for immune evaluation to the AllergyImmunology clinic at our institution (2007–2014). Their clinical history and immune laboratory studies (e.g. immunoglobulin levels, specific antibody responses, ANA, anti-SSA/SSO, dysautonomia autoimmune profile, serum cytokines) were reviewed. Results: Twenty patients (3 males, 17 females), mean age of 19.9 years (15–34), were evaluated. Symptoms suggestive of immune dysfunction included arthralgia (7), joint swelling (3), backache (2), dermatitis (5), alopecia (2), sicca (4), Raynaud’s (1), and recurrent infections (5). Five patients (25%) had autoantibodies (i.e. anti-SSA, ANA, anti-GAD65, folate receptor autoantibodies), and 9 of 14 patients (64%), had elevated serum cytokines. Severe dysautonomia was present in one patient with IDDM and 4 patients with B-cell immunodeficiency, all of which had other coexisting autoimmune problems. Conclusions: Young patients with dysautonomia may suffer from immune dysregulation and the severity of their disease is associated with severe dysautonomia. It remains to be determined if this immune dysregulation is a primary or secondary process.