bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of P4504a Genes for Hypertension and Renal Disease Using Chromosome 5 Congenic Strains of Dahl S Rats.
Authors: M Hoagland Kimberly , Alonso-Galicia Magdalena , Maier Kristopher G , Flasch Averia K , Wang Xiaohu , Jacob Howard J , Nobrega Marcelo , Rapp John P , Garrett Michael R , Roman Richard J
Published: March 2022

53

P4504A1 and 4A2 genes are differentially expressed in the kidney and cosegregate with blood pressure in an F
2
cross of Dahl S and Lewis rats; however, it is unclear whether this pathway plays a causal or secondary role in the development of hypertension. The present study examined whether transfer of overlapping segments of chromosome 5 from D5Rat84 to D5Rat34 that excludes (4A- strain) or includes all four P4504A genes(4A+ strain),or just the 4A3 and 4A8 genes (4A3,8+ strain), of a Lewis rat alters blood pressure, proteinuria, and renal injury in 12 week old congenic strains of Dahl S rats fed a high salt diet (8% NaCl) for 3 weeks. MAP measured in conscious rats using a chronic catheter averaged 188 ± 3 mm Hg in male Dahl S rats (n=24), 190 ± 3 mm Hg in the 4A- congenic strain (n=24), 169 ± 4 mm Hg in the 4A+ congenic strain (n=30), and 172 ± 3 mm Hg in the 4A3,8+ strain (n=33). Transfer of the P4504A region also significantly reduced proteinuria, renal hypertrophy, and the degree of glomerular and tubulointerstitial injury. Protein excretion averaged 246 ± 22 mg/day in male Dahl S rats and 255 ± 25 mg/day, 129 ± 8 mg/day, and 169 ± 12 mg/day in the 4A-, 4A+, and 4A3,8+ strains, respectively. The antihypertensive and renoprotective effects of transfer of the P4504A region were associated with signficant increases in the expression of P4504A mRNA, P4504A protein levels, and the renal formation of 20-HETE. Interestingly, transfer of the P4504A region had no effect on blood pressure in female rats of the 4A+ or 4A3,8+ congenic strains, but it still reduced the degree of proteinuria and renal injury. These studies exclude the P4504A1 and 4A2 loci as the causal genes in this region in the development of hypertension in Dahl S rats. However, they indicate that the P4504A3 and 4A8 genes still lie in the interval between D5Rat108 and D5Rat54 that contains a locus that reduces blood pressure, is renoprotective, and alters the expression of P4504A genes in Dahl S rats.
Central Orexin-A Augments Sympathoadrenal Outflow in Conscious Rabbits
Authors: Matsumura Kiyoshi , Tsuchihashi Takuya , Abe Isao
Published: March 2022

63

Factors regulating food intake, such as leptin and neuropeptide Y, have been shown to participate in central cardiovascular and sympathetic regulations. Orexin-A is a newly discovered hypothalamic peptide that stimulates food intake. The aim of the present study was to determine the neurohormonal and cardiovascular responses to intracerebroventricular administration of orexin-A in conscious rabbits with chronically instrumented electrodes on the renal sympathetic nerve. The experiments were carried out at least 3 days after the surgical procedures. Intracerebroventricular injection of orexin-A elicited dose-related increases in mean arterial pressure and renal sympathetic nerve activity (RSNA). Peak values of mean arterial pressure and RSNA induced by intracerebroventricular injection of 100 pmol of orexin-A (+14.0±0.7 mmHg and +55.4±14.9 %) were obtained at 40 and 25 minutes after injection, respectively. Plasma epinephrine and glucose concentrations significantly increased at 60 and 90 min after intracerebroventricular injection of orexin-A (control vs. 90 min; epinephrine: 38.0±12.8 vs. 167.5±42.5 pg/ml, p<0.01; glucose: 6.66±0.18 vs. 7.75±0.14 mmol/l, p<0.01). Plasma norepinephrine and insulin concentrations increased at 60 and 90 min, but did not reach the significant values. Intracerebroventricular injection of orexin-A also caused significant increases in plasma vasopressin concentrations. However, pretreatment with intravenous injection of pentolinium (5 mg/kg), a ganglion blocking agent, abolished these cardiovascular and neurohormonal responses. On the other hand, intravenous injection of the same dose of orexin-A (100 pmol) as that used in the intracerebroventricular experiment failed to cause any cardiovascular and renal sympathetic nerve responses. These results suggest that intracerebroventricular orexin-A acts in the central nervous system and activates sympathoadrenal outflow, resulting in increases in arterial pressure and plasma glucose levels in conscious rabbits.
Role of Nitric Oxide in Modulating the Chronic Cardiovascular and Renal Actions of Leptin
Authors: Kuo Jay J , Jones Oscar B , Hall John E
Published: March 2022

64

Acute studies suggest that leptin has pressor and depressor actions, including stimulation of sympathetic activity as well as increased release of nitric oxide (NO)from the vascular endothelium. The goal of this study was to examine the role of NO in modulating the chronic blood pressure, heart rate, and renal responses to hyperleptinemia, comparable to that found in obesity hypertension. Male Sprague-Dawley rats were implanted with arterial and venous catheters and mean arterial pressure (MAP) and heart rate (HR) were monitored continuously 24h/d. After a 4 day control period, rats were treated with vehicle (n=6) or L-NAME (n=7) for 7 days. After 7 days of vehicle or L-NAME (10 μg/kg/min) treatment to inhibit NO synthesis, rats received a low rate (0.5 μg/kg/min) leptin infusion for 7 days followed by a moderate rate (1.0 μg/kg/min) leptin infusion for 7 days, along with vehicle or L-NAME. A 21 day infusion of L-NAME alone (n=6) served as a control for the leptin+L-NAME rats. The low and moderate leptin infusions reduced food intake by 53±2%and 51±7%, respectively, but L-NAME did not alter the anorectic effect of leptin. Although the low dose of leptin alone did not significantly elevate MAP, it raised HR by 18±3 bpm. The moderate dose of leptin raised MAP from 96±3 to 104±3, but did not increase HR further. In rats pre-treated with L-NAME, the moderate dose of leptin raised MAP by 40±5 mmHg and HR by 73±17 bpm compared to pre-treatment levels. In control L-NAME rats, MAP increased by 31±4 mmHg, while HR was not altered significantly, compared to pre-treatment levels. Chronic leptin infusion alone did not alter glomerular filtration rate (GFR) or renal plasma flow (RPF). However, Leptin+L-NAME reduced GFR by 47±8% and RPF 58±7%; L-NAME alone decreased GFR by 36±11% and RPF 36±17%. Thus, NO does not appear to modulate the anorectic effect of leptin. However, impaired NO synthesis enhances the chronic renal hemodynamic and hypertensive effects of leptin, and greatly amplifies the tachycardia caused by hyperleptinemia, at levels comparable to those found in obesity hypertension.
Dual Action of Leptin on Renal Sodium Handling
Authors: Ise Takuyuki , Shimizu Kazuaki , Kato Tamayo , Izumiya Yoshiaki , Takakuwa Hiroshi , Yokoyama Hitoshi , Kobayashi Kenichi
Published: March 2022

66

BACKGROUND: Leptin exerts a natriuretic effect when infused directly into renal artery. However, in the conditions like obesity where circulating level of leptin is increased, this local effect may be opposed by centrally mediated stimulation of renal sympathetic nerve activity (RSNA). To investigate the interrelationship of the two factors, we made renal clearance study during intravenous leptin infusion in male Sprague-Dawley rats pretreated with unilateral renal denervation. METHODS: Rats were anesthetized with 50 mg/kg body weight (BW) intraperitoneal pentobarbital and supplementation with intravenous α-chloralose. Left renal vessels were isolated from surrounding tissue and applied with 10% phenol solution. Catheters were inserted into bladder and the proximal cut end of left ureter. Recombinant murine leptin was infused intravenously at 1 mg/kg BW over 3 hours. Renal function was assessed by timed collection of urine from each kidney and arterial blood under constant infusion of inulin. In another series of experiment, RSNA was recorded from left renal nerve and expressed as percent change from baseline. RESULTS: Mean blood pressure and heart rate were not affected by leptin. RSNA increased gradually by 45% at 105 min after leptin infusion and remained elevated. Glomerular filtration rates were 0.49±0.04 and 0.44±0.03 ml/min/100 gBW in intact and denervated kidney and both did not change significantly after leptin infusion. Urinary sodium excretion (UNaV) from the intact kidney was 527±42 nEq/min at baseline, increased to 1098±173 at 45 min, and returned to 606±82 nEq/min at 135 min after leptin infusion. Concomitant values from the denervated kidney were 898±105, 1822±272, and 1305±159 nEq/min, respectively. Thus, the ratio of UNaV from denervated to intact kidney increased significantly from 1.71±0.14 and 1.68±0.16 at baseline and 45 min to 2.27±0.30 at 135 min after leptin infusion. CONCLUSIONS: These results suggest that sodium balance after exogenous leptin infusion is determined by tubular effect and renal sympathetic nerve activity. Derangement of these factors may facilitate hypertension in obese subjects.
Familial Isolated Growth Hormone Deficiency Is Associated with Hypertension, Central Obesity and Dyslipidemia But not the Classical Syndrome X
Authors: Filho Jose Augusto Barreto , Alcantara Marta Regina , Freitas Maria Erenildes , Bastos Walquiria , Souza Anita Herminia , Aguiar-Oliveira Manuel Herminio
Published: March 2022

69

We have described a splice mutation in the GHRH receptor gene (G to A at position +1 , intron 1) in Itabaianinha county, Sergipe, Brazil. These genetically homogeneous individuals provide a unique opportunity to investigate the effects of isolated growth hormone deficiency [GHD] on cardiovascular risks. Therefore, we studied 16 dwarfs with GHD and no past of growth hormone therapy (8M:8F,49±14 yrs) and 31 age-matched normal controls [C] (12M:19F,44±12 yrs). We have examined insulin-like growth factor 1(IGF-1), arterial blood pressure, body mass index (BMI), waist-to-hip ratio (WHR), body composition by near infra-red interactance,leptin,serum lipids, uric acid, fasting glucose and insulin. IGF-1 in GHD was markedly lower (2.7±1.8 vs 154.9±82 ng/mL; p<0.001). Systolic blood pressure was higher in GHD (147±37 vs 129±24 mmHg; p=0.046) but diastolic blood pressure was similar (84±16 vs 80±16 mmHg; p=NS). BMI (22.9±4.1 vs 24.4±3.8 Kg/m
2
;p=NS) and leptin (11±8.9 vs 10.2±11.3 ng/mL;p=NS) were similar (GHD vs C), but WHR (0.97±0.08 vs 0.90±0.08; p=0.01) and % of fat mass (34±7 vs 21±10 %;p<0.001)were higher in GHD. Total cholesterol (236±53 vs 195±38 mg/dL;P=0.003) and LDL-C (165 ±47 vs 133±32 mg/dL;p=0.009) were higher in GHD but no differences were observed in HDL-C (40±9 vs 38±7 mg/dL;p=NS), apo B (1.16±0.36 vs 1.33±0.96 g/L; p=NS) and triglycerides (158±117 vs 115±62 mg/dL;p=NS). Uric acid was higher in GHD (4.0±1.2 vs 3.3±0.8; p=0.02). Despite central obesity and hypertension, fasting glucose (86±11 vs 87±14 mg/dL;p=NS),fasting insulin (2.9±2.4 vs 4.3±3.4 μU/mL;p=NS) and insulin/glucose ratio (0.03±0.03 vs 0.05±0.03μU/mL;p=NS) were smaller but not significantly in GHD. We conclude that GHD due to this GHRH receptor mutation is associated with systolic hypertension, central obesity and dyslipidemia with some features different from the classical syndrome X. These data suggest that in GHD another mechanism different from insulin resistance participate in the pathogenesis of hypertension and metabolic alterations.
An Antisense β1-Reducer: Repeated Administration Reduces Hypertension and Cardiac Hypertrophy Persistently Without Toxicity or Immune Response
Authors: Zhang Y. Clare , Shen Leping , Phillips M. Ian
Published: March 2022

72

In order to overcome the adverse effects associated with conventional β-blockers in treating hypertension, we have developed a novel β1-reducer by specifically targeting β1-adrenoceptor mRNA with an antisense oligonucleotide (β1-AS-ODN). A single intravenous injection of β1-AS-ODN deliverd with cationic liposomes caused a profound (25-35 mmHg) and prolonged (20-30 days) reduction in blood pressure in spontaneously hypertensive rats (SHR) without central effects. To be clinically useful the β1-AS-ODN would have to be effective with repeated injections and non-toxic. In this study, we have examined the effects of repeated administration of β1-AS-ODN on blood pressure and cardiac hypertrophy. Toxicity and immune response were assessed. Injections of 1mg/kg β1-AS-ODN (n=9) i.v. at 15, 20 and 26 days maintained systolic blood pressure of SHR at 20-30 mmHg lower than controls for more than 2 months (P<0.05). There was no loss in the antihypertensive efficacy with repeated injections. Instead, the intensity and duration of blood pressure reduction were greater with the second and third treatments. Control animals treated with control inverted-ODN (n=7) or saline (n=7) had no change in blood pressure with repeated injections. Left ventricular hypertrophy normalized by body weight was significantly attenuated 2 months after β1-AS-ODN treatment (P<0.05). Analysis of serum following each antisense delivery revealed no abnormalities in liver transaminase levels (ALT, AST) or hematology parameters (WBC, platelets, hematocrit). No antibodies to ODN/liposome complex were detected in gel immunodiffusion assay. Histological examination of heart, liver, kidney and spleen revealed no immunopathology or organ damage. These results demonstrate that β1-AS-ODN i.v. can be given repeatedly to achieve a sustained reduction of hypertension and reduces cardiac hypertrophy without causing toxic effects or immune stimulation. These data, together with results of our previous studies, show β1-AS-ODN is viable as a potentially new prolonged antihypertensive agent with benefits on cardiac remodeling.
Human Adrenomedullin Gene Delivery Protects Against Cardiac Hypertrophy, Fibrosis and Renal Damage in Hypertensive Dahl Salt-Sensitive Rats
Authors: Zhang Jenny J , Yoshida Hideaki , Chao Julie , Chao Lee
Published: March 2022

73

Adrenomedullin (AM) is a potent vasodilator expressed in tissues relevant to cardiac and renal functions. Our previous study showed that delivery of the human AM gene in the form of naked DNA caused a prolonged reduction of blood pressure in genetically hypertensive rats. In this study, we evaluated potential protective effects of adenovirus-mediated AM gene delivery in salt-induced cardiorenal lesions in hypertensive Dahl salt-sensitive (DSS) rats. Adenovirus carrying the human AM cDNA under the control of cytomegalovirus promoter/enhancer (Ad.CMV-hAM) was generated by homologous recombination. Expression of recombinant human AM was detected in the media of HEK 293A cells transfected with Ad.CMV-hAM. A single intravenous injection of Ad.CMV-hAM caused a significant reduction of systolic blood pressure for 4 weeks in DSS rats compared to control rats with or without injection of adenovirus carrying the green fluorescent protein gene. AM gene delivery significantly reduced left ventricular mass, urinary protein, and increased cAMP levels, as well as enhanced renal function as evidenced by increases in glomerular filtration rate and renal blood flow. Morphological investigations showed that AM gene transfer reduced cardiomyocyte diameter and interstitial fibrosis in the heart as well as reduced glomerular sclerosis, tubular disruption and protein cast accumulation in the kidney. Expression of human AM mRNA was identified in rat heart, kidney, lung, liver and aorta, and immunoreactive human AM levels was measured in rat plasma and urine. These results indicate that human AM gene delivery protects against salt-induced hypertension, cardiac and renal lesions in DSS rats via activation of cAMP as a second messenger. These findings provide new insights into the role of AM in salt-induced hypertension and may have implications in therapeutic applications to salt-related cardiovascular and renal diseases.
Human Tissue Kallikrein Gene Transfer Induces Angiogenesis
Authors: Madeddu Paolo Roberto , Salis Bonaria Maria , Chao Julie , Chao Lee , Costanza Emanueli
Published: March 2022

74

Angiogenesis therapy is emerging as a new strategy for treatment of vascular disease, not susceptible of revascularization. As kinins are implicated in vascular protection and repair, we hypothesized that targeted potentiation of kinin-nitric oxide (NO) pathway may stimulate neo-vascularization. CD1 mice were IM injected with 3.6x10
8
plaque forming units of adenovirus (Ad) harboring human tissue kallikrein (HK) or β-galactosidase (LacZ) gene under the control of cytomegalovirus enhancer/promoter (CMV). Successful transduction of injected adductor skeletal muscle was documented at mRNA and protein level. HK expression reached a peak between 3 and 7 d and then declined to become undetectable at 28 d. Contralateral muscles and liver were not transduced. Immunohistochemical identification of endothelial cell antigen factor VIII combined with blinded morphometric analysis of capillary density revealed a marked angiogenic effect in HK-transduced muscle (1013±18 vs 518±15 n
cap
/mm
2
in LacZ-injected muscle at 28 d, P<.001), whereas no angiogenic effect was seen in ipsilateral gastrocnemious or contralateral muscles. HK increased muscular kinin (60±12 vs 20±6 pg/g tissue in controls at 3 d, P<.05), cAMP (1.02±0.06 vs 0.48±0.04 ng/mg prot, P<.01) and cGMP levels (99±5 vs 68±5 fmol/mg prot, P<.01). HK-induced angiogenic response was completely prevented by pharmacologic blockade of kinin B
1
or B
2
receptors, or inhibition of NO synthase, but not by cycloxygenase inhibition. Neovascularization was associated with a transient increase in muscular blood flow as determined by laser Doppler flowmetry. HK-induced neo-vascularization was stable and functionally utilitarian, as surgical induction of hindlimb ischemia 28 d after gene tranfer was followed by accelerated hemodynamic recovery of HK-injected muscle (perfusion ratio: 0.84±0.05 vs 0.68±0.03 in LacZ-injected muscle at 14 d, P<.05). We conclude that (a) HK acts as an angiogenic factor via kinin-mediated stimulation of NO release, (b) neo-vascularization can preserve tissue from supervening ischemia. The discovery that HK has angiogenic activity may represent an important advancement in vascular medicine for therapeutic benefit.
Cardiac Protective Effect of Aldosterone Blockade Is Mediated Through Ap-1 and Nf-κB Suppression in Angiotensin Ii-Induced Cardiac Damage
Authors: Fiebeler Anette , Schmidt Folke , Muller Dominik N , Park Joon-Keun , Dechend Ralf , Shagdarsuren Erdenechimeg , Bieringer Markus , Breu Volker , Ganten Detlev , Luft Friedrich C , Haller Hermann
Published: March 2022

77

Aldosterone is a multifunctional mineralocorticoid affecting homeostasis of water and electrolytes, sympathetic activity, and tissue fibrosis. Aldosterone synthesis is induced by activation of the renin-angiotensin system. We tested whether or not the inhibition of aldosterone signaling can prevent fibrotic tissue remodeling in vivo. In a transgenic rat model overexpressing the human renin and angiotensinogen gens (dTGR), we investigated the effect of spironolactone (SPIRO 20 mg/kg/d) and the AT1 receptor blocker valsartan (VAL; 10 mg/kg/d) on growth factors and transcription factors leading chronic inflammation and tissue fibrosis. Untreated dTGR develop severe hypertension, cardiac hypertrophy, vasculopathy, and perivascular fibrosis. The hearts also show chronic inflammation and the animals have a 50% mortality at 7 weeks. VAL completely reversed these pathological features. SPIRO and VAL both prevented mortality, while only VAL normalized systolic blood pressure (VAL 121±9, SPIRO 161±11, dTGR 182±8, SD 109±2 mm Hg). Both reduced cardiac hypertrophy (SPIRO 4.2±0.1, VAL 3.6±0.1, dTGR 5.7±0.2, SD 3.6±0.1 mg/g) and reduced vasculopathy. Quantitative bFGF RT-PCR showed a complete mRNA reduction in the left ventricle in both treatment groups (SPIRO 11±4, VAL 5±1 dTGR 43±4, SD 4±1 arbitrary units). In contrast, RT-PCR of TGF beta and PDGF showed no upregulation in dTGR compared to non-transgenic hearts. Gel shift assay of the heart demonstrated a suppression of AP-1 and NF-κB DNA binding activity after SPIRO and VAL treatment. Immunohistology revealed reduced bFGF expression and less collagen I, fibronectin, and laminin in the interstitium of SPIRO and VAL-treated rats. These findings show that aldosterone promotes hypertrophy, cardiac remodeling, and fibrosis. In dTGR hearts this effect is at least partly mediated through the transcription factors AP-1 and NF-κB as well as bFGF. Blocking the aldosterone receptor downregulates these effector molecules and reduces angiotensin II-induced cardiac damage.
Nf-Kb Inhibition Lowers Blood Pressure in Mineralocorticoid Hypertensive Rats.
Authors: Beswick Richard Anthony , Dorrance Anne N , Rajagopalan Sanjay , Webb R. Clinton
Published: March 2022

78

Hypertension is a major risk factor for cardiac and renal damage; however, the exact mechanism for this damage is not completely understood. It has been reported that hypertension causes increased reactive oxygen species (ROS) accumulation, which may result in nuclear factor-kB (NF-kB) activation; leading to activation of many inflammation mediating genes as well as iNOS expression. We have previously reported increased iNOS mRNA expression in aorta from DOCA-salt rats. We therefore tested the hypothesis that DOCA-salt hypertension causes oxidative stress and generation of ROS which act as a signal tranduction messenger for NF-kB activation. O2- concentration within aorta was measured by lucigenin chemiluminescence. Aorta from DOCA-salt rats showed increased O2- counts (9814.18 counts +/- 552.4 vs. 6334.89 counts +/-27.38, p<0.005) in comparison to sham rats. Chronic treatment with pyrrolidine dithiocarbamate (PDTC) (200mg kg-1 day-1 IP), a NF-kB antagonist, substantially decreased blood pressure (DOCA =187.5 +/-3.4 vs. DOCA/PDTC=130 +/-1.9 mm Hg, p<0.005) in DOCA treated animals compared with DOCA alone and sham animals. Preliminary analysis observed kidney hypertrophy (.0075 +/- .0001 vs .0052 and .0047 +/- .0002; kidney wt/Animal wt) in DOCA treated rats compared to DOCA/PDTC and sham treated rats, respectively. These results suggest a role for ROS and NF-kB activation in the vascular changes characteristic of mineralocorticoid hypertension. (NIH-
HL018575
)
Increased Epidermal Growth Factor Receptor Expression in Rat Aorta in Deoxycorticosterone Acetate-Salt Induced Hypertension/ATL>
Authors: Manteuffel Jacob J , Florian Jennifer A , Dorrance Anne N , Watts Stephanie W
Published: March 2022

79

Receptors and signal transduction

Epidermal growth factor (EGF) causes contraction in arteries from Deoxycorticosterone Acetate-Salt (DOCA) hypertensive rats but not in normotensive sham rats. We hypothesized that an increase in the number of EGF receptors (EGFR) in arteries from DOCA-salt rats enables the observed contraction to EGF to occur. All DOCA-salt rats had a systolic blood pressure (SBP) > 170 mm Hg, while all sham rats had a SBP < 135 mm Hg. Thoracic aorta were removed for measurement of isometric force, EGFR mRNA levels, and EGFR protein levels. EGF caused a significant contraction in endothelium-denuded aorta from DOCA-salt rats [(35 +/- 3% of maximal phenylephrine (PE, 10uM)-induced contraction)] as compared to aorta from sham rats (4+/-2%). EGFR tyrosine kinase specific inhibitors, 4,5-Dianilinophthalimide (10uM) and AG1478 (250 nM), reduced the contraction in aorta from DOCA-salt rats by 85 +/- 14% and 65+/- 10% respectively. The mRNA for EGFR in DOCA-salt aorta was increased 4.2 fold as compared to sham aorta. EGFR protein levels were examined using immunohistochemistry where specific binding was defined as binding which was quenched by a 10x concentration of neutralizing peptide. In 7 of 8 quantitative comparisons between aorta from DOCA-salt and sham rats, DOCA-salt aorta sections demonstrated greater specific binding than in sections of sham aorta. In the 7 positive comparisons, the difference in specific binding between DOCA-salt and sham sections ranged from 2.31 to 20.67 gray scale units (gsu) with an average of 9.18 gsu (figure). An increase in specific binding in aorta from DOCA-salt rats suggests the vessels have more EGFR that could facilitate the enhanced contractility to EGF in DOCA-salt aorta.
Dehydration Increases Angiotensin At1a But not At1b Mrna in Mouse Brain
Authors: Chen Yanfang , Morris Mariana
Published: March 2022

P3

Previous study showed that dehydration in mice produced an increase in brain ANG AT1 receptors. Because the receptor-binding assay can not distinguish the two AT1 receptors, AT1a and AT1b, we usedin situ hybridization methods to distinguish the ANG receptor mRNAs. Specific oligonucleotides probes from the 5’ or 3’ untranslated region of cDNA sequences were used. The objective was to determine the effect of 48-hr dehydration on AT1 mRNA expression in mouse brain. Plasma osmolality and hematocrit were increased after dehydration (349.6±6.1 vs 314.3±4.4 mOsm, p<0.01; 57.7±1.7 vs 46.9±1.4%, p<0.01). Paraformaldehyde fixed brains sections were processed for in situ hybridization using
35
S labeled probes. Analysis was conducted using a phosphor-imaging system and emulsion coated slides. There was specific labeling of areas known to contain Ang AT1 mRNA in rats, anteroventral third ventricle(AV3V), subfornical organ (SFO) and parvicellular paraventricular nucleus (PVN). The pattern of expression was similar for AT1a and AT1b. Dehydration produced an increase in AT1a mRNA in SFO, AV3V and PVN with no changes noted in lateral septal and anterodorsal nucleus. Dehydration produced no effect on AT1b expression. Results show 1) the localization pattern of Ang receptor subtypes in mouse brain with evidence for both AT1a and AT1b and 2) the specific effect of dehydration on AT1a mRNA.
Angotensin II Type 2 Receptors Participate in Reducing Blood Pressure During Angiotensin II Type 1 Receptor Blockade But Do not Affect Cardiovascular Mass and Coronary Hemodynamics
Authors: Frohlich Edward D
Published: March 2022

P7

This study was designed to determine the role of AT
2
receptors in the beneficial cardiovascular effects of chronic AT
1
antagonism in spontaneously hypertensive rats (SHR). A selective AT
1
receptor antagonist, candesartan (10mg/kg/day) alone (CAND), or in combination with the selective AT
2
receptor antagonist PD 123319 (50g/kg/day) (CAND+PD) was given for 12 weeks to adult male SHR. Control SHR received placebo (PLACEBO) for the same period. Left ventricular coronary blood flow (CBF), flow reserve (CFR) and minimal coronary vascular resistance (MCVR) were measured using radiomicrospheres in 35-week old rats. Mean arterial pressure (MAP), peripheral resistance (TPR), left ventricular weight (LVI), and aortic weight (AWI)were also measured. The grater MAP in CAND+PD group compared with CAND suggests that AT
2
receptor stimulation was responsible, in part for the hypotensive effect of AT
1
receptor antagonism, although they did not contribute to improved coronary hemodynamics and reduced LV and aortic masses after selective AT
1
receptor inhibition.
Angiotensin II Suppression in Humans by the Orally Active Renin Inhibitor SPP100; Comparison with Enalapril
Authors: Nussberger Juerg , Wuerzner Gregoire
Published: March 2022

P11

Renin is the main determinant of Ang II levels. It therefore always appeared desirable to reduce Ang II levels by direct inhibition of renin. So far, all specific renin inhibitors lacked potency or were clinically ineffective after oral administration. We tested the new orally active non-peptidic renin inhibitor SPP100 in 18 healthy male volunteers on a constant sodium diet (100mmol/day) using a double-blind, threeway crossover protocol. In 3 periods of 8 days, separated by wash-outs of 1 week, each volunteer received 2 dosage levels of SPP100 once a day (40,80,160 or 640mg) and placebo or 20mg enalapril. SPP100 was well tolerated. Not surprisingly, blood pressure and heart rate remained unchanged in these normal volunteers. SPP100 plasma levels showed that steady state was reached after 8 days of dosing. The table below summarizes median plasma levels at peak (P, 0.5-6h) and trough (T, 24h after dosing) on Day 8: In conclusion, the renin inhibitor SPP100 dose-dependently decreases Ang II levels in humans following oral administration. The effect is long-lasting and at 160mg at least equivalent to that of 20mg enalapril. SPP100 has the clear potential to become the first renin inhibitor that provides a true alternative to ACE-inhibitors and Ang II receptor antagonists in the therapy of hypertension, cardiovascular and renal disease.
Renal Expression of Angiotensinogen Protein in Angiotensin II-Infused Hypertensive Rats Maintained on High Salt Diet
Authors: Kobori Hiroyuki , Harrison-Bernard Lisa M , Navar L. Gabriel
Published: March 2022

P12

Chronic infusion of angiotensin (Ang) II leads to the development of hypertension and enhances intrarenal AngII content to levels greater than can be explained from the circulating concentrations. We previously reported that angiotensinogen mRNA is enhanced in AngII-dependent hypertension and may contribute to augmented intrarenal AngII levels. It has been shown that high salt diet suppresses renal expression of angiotensinogen mRNA. The purpose of the present study was to test the hypothesis that AngII augments renal angiotensinogen protein synthesis under condition of suppressed renal angiotensinogen production by high salt diet. We examined the effect of chronic infusion of AngII on angiotensinogen protein levels in male Sprague-Dawley rats (BW 158±10 g, n=12) maintained on an 8% salt diet. AngII was administered via osmotic minipumps (40 ng/min) to one group (n=6) while the remaining rats were sham-operated (n=6). High salt diet alone did not alter systolic BP in sham animals (109±6 mmHg at day 12); however, combination of AngII infusion and high salt diet significantly increased systolic BP (167±7 at day 12) and intrarenal AngII content (459±107 fmol/g vs. 270±42) in spite of a marked suppression of plasma renin activity (0.88±0.22 ng AngI/mL/h vs. 2.79±1.31). Western blot analysis of protein extracts from kidney (15 μg) showed an immunoreactive band at ∼52 kDa, while plasma protein (1.25 μg) showed two bands at ∼52 kDa and ∼64 kDa using a specific angiotensinogen polyclonal antibody (1:5000). Densitometric analysis of the immunoreactive bands showed that the combination of AngII infusion and high salt diet significantly increased kidney (88%) and plasma (61% for 52 kDa and 4.0 fold for 64 kDa) angiotensinogen protein compared with sham animals. Thus, AngII infusion to rats fed high salt diet elicited progressive hypertension and elevated intrarenal AngII through enhanced kidney and plasma angiotensinogen protein expression despite suppressed plasma renin activity. These data suggest that the augmented intrarenal AngII in AngII-dependent hypertension occurs, in part, by activated renal expression of angiotensinogen protein.
Vasorelaxation to Angiotensin I After Angiotensin Converting Enzyme Inhibition
Authors: Baltatu Ovidiu , Todiras Mihai , Bader Michael
Published: March 2022

P14

We aimed at studying the vasoactive function of angiotensin I (Ang I) degradation products in the absence of tissue ACE (angiotensin-converting enzyme) activity. The effect of Ang I on captopril (10
-5
M)-treated rat aortic rings pre-contracted with phenylephrine (Phe, 10
-7
M) was studied. Mechanism of action of Ang I degradation products were analyzed using Ang-receptor antagonists [D-Ala
7
- Ang-(1-7), A779 - Ang-(1-7)-selective; PD123319, PD - AT
2
receptor specific; Sar
1
-Thr
8
-Ang II, sarthran - nonspecific), bradykinin B
2
receptor antagonist (HOE140) and inhibitors of neutral endopeptidase (thiorphan, NEPi) or prolyl endopeptidase (Z-Pro-Pro-aldehyde dimethylacetal, PEPi). Ang I induced an evident dose-dependent relaxation of the pre-contracted aortic rings treated with captopril, with a maximum relaxation at 10
-7
M (10
-9
M: -18.64 ± 1.63; 10
-8
M: -27.6 ± 1.9; 10
-7
M: -34.3 ± 2.1 % of Phe contraction). Additional treatment with A779 (10
-6
or 10
-5
M), HOE140 (10
-6
M), high dose of PD (10
-5
M) or PEPi significantly inhibited the relaxation to Ang I. Sarthran, PD (10
-6
M) or NEPi did not significantly affect the relaxation to Ang I. Endothelium removal prevented the relaxation to Ang I. Our results indicate that in the absence of tissue ACE activity Ang I is metabolized, predominantly by prolyl endopeptidase present on endothelium, to Ang-(1-7) that induces vasorelaxation with the participation of putative Ang-(1-7) receptors, AT
2
and B
2
receptors. This may constitute an additional mechanism for the effects of ACE inhibitors.
Is the Serotonin1b Receptor Involved in Arterial Serotonergic Hypersensitivity in Deoxycorticosterone Acetate-Salt Hypertension?
Authors: Banes Amy K , Watts Stephanie W
Published: March 2022

P27

The 5-HT1B receptor has been recently implicated in mediating arterial contraction to serotonin (5-hydroxytryptamine, 5-HT).The 5-HT1B receptor is unmasked/enabled by depolarizing stimuli. It is possible that 5-HT1B receptors in arteries from hypertensive animals, arteries reported to have a depolarized resting potential in smooth muscle cells, are enabled and participate in the supersensitivity observed to 5-HT in hypertension. To test this hypothesis, we utilized an isolated tissue bath apparatus and examined the response of thoracic aorta from normotensive sham rats [systolic blood pressure (SBP) 123 +2 mmHg] and hypertensive deoxycorticosterone acetate (DOCA)-salt rats [SBP 194 + 10 mmHg]. The 5-HT1B agonist RU24969 and the 5-HT1B/1D receptor agonist sumatriptan (10-9 -10-5 M) caused a maximal contraction [50 % + 12 of phenylephrine (10-5 M) contraction] in tissues from DOCA-salt rats; no contraction was observed in normotensive arteries. The 5-HT1B antagonist
SB216641
(30 nM) was unable to block the RU24969-induced contraction as well as the 5-HT (10-9 -10-4 M)-induced contraction in aorta from both DOCA-salt and normotensive rats. Tissues from normotensive rats were incubated with 15mM KCl as a depolarizing stimulus, and then curves were performed to 5-HT, RU24969, and sumatriptan. These studies were performed in the presence of ketanserin (10 nM, 5-HT 2A receptor antagonist) to remove the contribution of the 5-HT2A receptor. The 5-HT-induced contraction was not changed in the presence of the KCl stimulus. High concentrations (10-5 M) of RU24969 did result in a 30% + 9 contraction. However, the presence of a depolarizing stimulus was unable to produce a leftward shift of the response to resemble that of aorta from DOCA-salt rats. These data support the conclusions that 5-HT1B receptors do not contribute significantly to the 5-HT-induced contraction in the rat aorta or to the hypersensitivity to 5-HT observed in DOCA-salt hypertension. In addition, these data support the conclusion that the arterial hypersensitivity to 5-HT in hypertension is not due to membrane depolarization alone.
A High Salt Diet Increases Renal Nitrotyrosine Formation and Urinary Excretion of F
2
-Isoprostanes and Protein in Dahl Salt-Sensitive Rats
Authors: Meng Shumei , Roberts L. Jackson , Cason Garrick W , Manning R. Davis
Published: March 2022

P28

Superoxide formation causes tissue injury in many disease processes, and interaction between superoxide and nitric oxide forms peroxynitrite, a very strong oxidant. An index of peroxynitrite damage to tissues is nitrotyrosine formation. Also, F
2
-isoprostanes have been shown to be a reliable marker of oxidative stress in vivo. Recently, oxidative stress was shown to contribute to the maintenance of hypertension in the SHR; however, whether oxidative stress is involved in the renal damage that occurs in Dahl salt-sensitive (S) hypertension is not clear. Our goal was to determine the role of oxidative stress in the renal damage that occurs in Dahl S/Rapp rats on high Na intake by determining the urinary excretion of F
2
-isoprostanes and renal nitrotyrosine formation and determining urinary protein excretion as an index of renal damage. Studies were conducted in S rats on a low salt (0.3% NaCl) or a high salt (8% NaCl) diet for 3 weeks. After 3 weeks urine was collected continuously for 24 hours, and urinary F
2
-isoprostane concentration was measured with a gas chromatography/mass spectrometric assay. Renal nitrotyrosine formation was determined by immunohistochemistry and urinary protein excretion by the Bradford method. We found that high Na intake significantly increased urinary F
2
-isoprostane excretion to 24.2±1.7 ng/day compared with 9.7±0.8 ng/day in rats on a low Na diet, P<0.05. Renal glomerular nitrotyrosine was prevalent in the high Na group compared to the low Na group. Urinary protein excretion was 274±32 mg/day in high Na rats and 55±11 mg/day in low Na rats, P<0.05. The results suggest that oxidative stress is significantly increased in Dahl S rats on a high salt diet, which possibly contributes to the progression of renal damage.
Release of Prostanoids from Paired Venules Regulates Functional Vasodilation of 3rd Order But NOT 4th Order ARterioles.
Authors: Hammer Leah W , Ligon Alison L , Hester Robert L
Published: March 2022

P32

Previous studies from our laboratory suggest that functional arteriolar dilation of larger diameter “feed” arterioles paired with venules is mediated by arachidonic acid metabolites released from the venular endothelium. The aim of the present study was to test the hypothesis that the release of prostanoids from venules is important in the functional arteriolar dilation of 3rd and 4th order arterioles. The microcirculation of the hamster cremaster muscle was visualized using
in vivo
video microscopy. We measured the diameter responses of arterioles paired and unpaired with venules in response to 2 minutes of field stimulation (40 μs, 10V, 1 Hz). Indomethacin (28 μM) superfused on the preparation was used to inhibit prostanoid release. For the unpaired vessels field stimulation resulted in an increase in diameter of 3rd order vessels from 14 ± 1 to 26 ± 2 μm (n=6) and 4th order vessels from 11 ± 2 to 21 ± 2 μm (n=6). This response was not affected by indomethacin. 3rd order arterioles dilated 14 ± 2 to 26 ± 3 μm, 4th order arterioles dilated 10 ± 1 to 19 ± 2 μm. For the paired vessels there were differences in the responses between 3rd and 4th order arterioles. The diameter of 3rd-order arterioles averaged 32 ± 3 μm (n=6) and increased to 57 ± 5 μm following 2 minutes of field stimulation. During indomethacin treatment an inhibition of the functional vasodilation was observed as the arteriolar diameter increased from 29 ± 4 to only 38 ± 5 μm (p<0.05). The paired 4th order arterioles averaged 18 ± 4 μm and increased to 35 ± 5 μm with field stimulation (n=4). This response was not affected by indomethacin treatment (17 ± 5 to 32 ± 7 μm following stimulation). In conjunction with previous studies from our laboratory, these results support the hypothesis that cyclooxygenase products are released from the venular endothelium and are an important regulator of functional vasodilation of 3rd order arterioles but not 4th order arterioles.
Disruption of Caveolae Results in Attenuated Endothelium Dependent Relaxation
Authors: Weber David S , Webb R. Clinton
Published: March 2022

P33

A significant pathology associated with hypertension is endothelial dysfunction caused by reduced availability of endothelial nitric oxide. There are several potential contributing mechanisms to the decreased nitric oxide availability including changes in membrane receptor function, alterations in second messenger systems, or decreases in crucial cofactors associated with enzyme regulation. Recently it has been demonstrated that endothelial nitric oxide synthase is concentrated in the caveolae of endothelial cells, presumably by formation of a heteromeric complex. The current studies hypothesize that disruption of caveolar membranes by the sterol binding agent filipin will result in the attenuation of endothelium dependent vasodilator responses. Aorta from male Sprague-Dawleys rats were removed, cleaned of adhering fat, and cut into rings for the measurement of isometric force development. Rings were incubated in the presence of filipin complex (5 ug/ml) or vehicle for one hour. To determine vasodilation all vessels were precontracted with an EC50 concentration of phenylephrine. Filipin treatment significantly attenuated endothelium dependent relaxation in response to both cholinergic activation with acetylcholine (maximum relaxation, filipin 40±7% vs. vehicle 95±2%; EC50 -5.8±.20 vs. -6.6±.03) and the Ca
2+
ionophore A-23187 (maximum relaxation, filipin 24±5% vs. vehicle 82±3%; EC50 -4.2±.19 vs. -6.5±.09). Maximal relaxation in response to 10
-5
M sodium nitroprusside was unchanged between groups. These results implicate the caveolae in endothelium dependent relaxation and suggest that the damage to the endothelium such as in hypertension may result in diminished caveolar function. This impaired caveolar function may provide a novel mechanism contributing to the reduction in nitric oxide available in hypertension.
Attenuated Development of Hypertension by Antioxidant Vitamins in Stroke-Prone Shr Is Associated with Increased Vascular Superoxide Dismutase Activity and Decreased Oxidative Stress.
Authors: Touyz Rhian M , Chen Xin , Park Jeong Bae , Schiffrin Ernesto L
Published: March 2022

P34

Oxidative stress has been implicated in the pathophysiology of cardiovascular diseases. We investigated effects of antioxidant vitamins and tempol, superoxide dismutase (SOD) mimetic, on vascular superoxide anion (×O
2
-
) concentration, NADPH oxidase activity, SOD activity, plasma antioxidant levels and blood pressure in stroke-prone SHR (SHRSP). 16 week-old SHRSP on a high salt diet (4%NaCl) were divided into 4 groups: control (C), Vit C (1000mg/day), Vit E (1000 U/day) and tempol (1 mmol/d). Systolic blood pressure (SBP) and antioxidant status were measured weekly for 6 weeks. Aortic ×O
2
-

concentration and NADPH oxidase activity were measured by luminometry using lucigenin (5 μmol/L) and aortic SOD activity and total plasma antioxidant status were measured using a spectrophotometric assay system. SBP and plasma antioxidant levels were similar in all groups prior to treatment. Table demonstrates results after 6 weeks treatment. Data are means ± SEM. Data from this study demonstrate that antioxidant vitamins and tempol attenuate the development of hypertension in SHRSP. This is associated with decreased vascular ×O2- production, increased activity of SOD and increased plasma antioxidant concentrations. We demonstrate the novel findings that Vits E and C induce their antioxidant effects, in part, by stimulating SOD activity. Thus increased oxidative stress is an important factor underlying vascular changes associated with severe hypertension in SHRSP.
Impaired Norepinephrine Reuptake in Skeletal Muscle But not in Adipose Tissue After the Development of Hypertension in Rats: an
in Vivo
Microdialysis Study
Authors: Cabassi Aderville , Vinci Simonetta , Quartieri Fabio , Moschini Luigi , Bruschi Giacomo , Borghetti Alberico
Published: March 2022

P50

Presynaptic reuptake of norepinephrine (NE) represents the main mechanism to end its effects, once released from sympathetic nerves. This study evaluated the effect of locally administered neuronal uptake inhibitor (desipramine, 10 pmol/min through microdialysis probes) on interstitial NE concentration in the skeletal muscle and subcutaneous adipose tissue from awake and freely moving Wistar-Kyoto (WKY)and spontaneously hypertensive rats (SHR) before and after the development of arterial hypertension. Systolic arterial pressure and plasma NE levels in SHR were similar to WKY at 5 weeks of age, but significantly increased at the age of 16 weeks. Basal interstitial NE was increased in both tissues of young and old SHR as compared to age-matched WKY. Desipramine perfusion induced a higher rise of interstitial NE dialysate from skeletal muscle and adipose tissue of SHR than WKY with a difference in the skeletal muscle between young and old SHR (see graphic). These results indicates an increased interstitial NE turnover in pre-hypertensive SHR, not shown by plasma NE level. Once hypertension is established, NE turnover is always increased in SHR as compared to WKY in both tissues. In addition in the skeletal muscle, where sympathetic efferences are baroreflex-dependent, old SHRs show a reduced interstitial NE reuptake contributing to a higher availability of interstitial NE for postsynaptic vascular effects.
Disruption of Subtype 2 Dopamine Receptor (D2-R) Causes Hypotension Independently of Its Action on Sympatetic Nerve System
Authors: Ozono Ryoji , Ueda Atsushi , Oshima Tetsuya , Matsuura Hideo , Teranishi Yasuhiro , Kajiyama Goro , Kambe Masayuki
Published: March 2022

P51

D
2
-R, known to be involved in the pre-synaptic sympathetic inhibition, is also postulated to be present in the cardiovascular system and to modulate blood pressure via local dopaminergic system. In the present study, we investigated the localization of D
2
-R and the effects of the targeted disruption of both D
2
-
long
and D
2
-
short
receptor genes on sympathetic activity and blood pressure. The male homozygous littermates at ages of 6-16 week (KO mice) and the age-matched wild type (WT) control mice were used. We assessed systemic sympathetic activity by 24-h urine norepinephrine (NE) excretion and local sympathetic activity by tissue content of NE in the heart and kidney. RT-PCR revealed that the D
2
-R mRNA was expressed in the heart, the aorta and the kidney as well as in the brain only in WT mice. Urinary NE excretion (KO vs. WT: 8.67± 1.71 vs. 7.78± 0.64 micro-g/day)was not different between KO and WT mice. Renal (KO vs. WT: 390±42 vs.535±155 pg/mg protein,p<0.05)but not cardiac (718±75 vs.697± 193pg/mg protein) content of NE was significantly decreased in KO than in WT mice, reflecting the depletion of stored NE in the renal sympathetic neurons. Conscious systolic tail-cuff blood pressure (KO vs. WT: 102±3.3 vs. 106.6±5.1 mmHg at 16 week, n= 10, p<0.001) and direct mean arterial blood pressure under anesthesia(86±13 vs. 101.4±10, n=5, p<0.05) were significantly lower in KO mice than in WT mice. Heart rate (563±31 vs.549±41), food and water consumption, and 24-hour urine sodium excretion (368±44 vs.359±36 microEq/day)were not different between the KO and WT mice. These results indicate that (1) D
2
-R is expressed in the cardiovascular system, (2) D
2
-R in the kidney is involved in the renal sympathetic control, but (3) disruption of D
2
-R causes hypotension independently of its effects on sympathetic activity.