bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Generation of Vascular Reactive Oxygen Species by Ang II Is Mediated Via Src-Dependent Pathways in Essential Hypertension.
Authors: Touyz Rhian M , Schiffrin Ernesto L
Published: July 2021

42

We tested the hypothesis that augmented Ang II-induced vascular smooth muscle cell (VSMC) growth in human hypertension is mediated via Src-dependent pathways that generate reactive oxygen species (ROS). VSMCs from arteries of normotensive and hypertensive subjects were studied. Production of ROS was measured by fluorescence digital imaging using dichlorofluorescin diacetate (6 μM). The roles of Src and NADH/NADPH oxidase were assessed with the specific inhibitors, PP2 (10 μM) and diphenylene iodinium (DPI) (10 μM) respectively. c-Src phosphorylation was determined by western blot and kinase activity was assessed by measuring enolase phosphorylation. Ang II increased DCFDA fluorescence. This effect was inhibited by catalase, indicating that the signal was derived predominantly from H
2
O
2
. Ang II increased H
2
O
2
production within 40 minutes. Responses were greater (p<0.05) in cells from hypertensive patients (E
max
=82±nM) than normotensive subjects (E
max
= 67±nM). DPI and PP2, but not PP3 (inactive analogue) attenuated (p<0.05) Ang II-induced H
2
O
2
production. PP2 effects were greater in cells from hypertensive patients (delta H
2
O
2
, 28±5nM) vs controls (delta H
2
O
2
, 16±2nM). Ang II increased c-Src phosphorylation and activity, with responses 3-4 fold higher in hypertensives. DPI and PP2 (p<0.01) attenuated Ang II-induced DNA and protein synthesis, as measured by
3
H-thymidine and
3
H-leucine incorporation respectively. Growth responses in hypertensive patients were normalized by PP2. In VSMCs from hypertensive patients, Ang II-induced generation of ROS and growth are augmented. These effects are mediated, in part, by Src-dependent, NADH/NADPH oxidase-dependent cascades. Thus increased Src activity may be an upstream modulator of redox-sensitive pathways that regulate vascular growth and remodeling in essential hypertension.
Genetic Screening of F2 WKY/WKHA Crosses Reveals That the Traits of High Ventricular Mass and Low Ventricular ANF Concentration Are Linked to a Locus on Chromosome 5 That Overlaps with the Locus of the Natriuretic Peptide Precusor A (
Nppa
) Gene.
Authors: Deschepper Christian F , Masciotra Sandro , Zahabi Ahmad , Boutin-Ganache Isabelle , Picard Sylvie , Reudelhuber Timothy L
Published: July 2021

55

The cardiac ventricular mass of WKHA inbred rats is higher than that of WKY rats, yet both strains exhibit normal and identical values of blood pressure (as verified by telemetry). We have shown previously in the F2 male progeny of WKY/WKHA crosses that low ventricular concentration of atrial natriuretic factor (ANF) co-segregated significantly with high cardiac ventricular mass (Circ. Res. 84:1453-1458, 1999). To extend these findings, we have performed a scan of the whole genome of 158 rats originating from the male F2 progeny of the WKY/WKHA cross. By linkage analysis, we have identified on chromosome 5 one QTL that was linked to left ventricular ANF concentration (QTL
ANF
) with a LOD score of 17, and one QTL that was was linked to left ventricular mass (QTL
LVM
) with a LOD score of 3.5. The 2-LOD confidence interval for the location of QTL
ANF
ranged from positions 94 to 104 cM, which overlaps partly with the 2-LOD confidence interval for the location of QTL
LVM
between positions 80 to 103 cM. The locus of the natriuretic peptide precursor A (
Nppa
) gene (at about 100 cM) is located within an interval covered by both QTL’s. Sequencing of a 600 bp fragment of the promoter regions of the
Nppa
genes from WKY and WKHA revealed that there were single nucleotide polymorphisms within two different regions of the promoters. These mutations lie within regions known to contain response elements to transcription factors, but the results of ongoing experiments are needed to determine whether they can affect the ability of the respective promoters to drive the expression of reporter genes in transfected cardiomyocytes. Altogether, these data suggest that the
Nppa
gene may be considered as a candidate gene to explain, at least in part, the variance of left ventricular ANF concentration and/or left ventricular mass in WKY and WKHA rats. They also define the region that will be studied in congenic lines to obtain a better spatial resolution of the QTL’s, and in particular to determine whether QTL
ANF
and

QTL
LVM
are identical or adjacent.
Reactive Oxygen Species-Mediated Homologous Downregulation of Angiotensin II Type 1 Receptor Mrna by Angiotensin II
Authors: Ichiki Toshihiro , Takeda Kotaro , Takeshita Akira
Published: July 2021

58

Recent studies suggest a crucial role of reactive oxygen species (ROS) for the signaling of Angiotensin II (Ang II) through type 1 Ang II receptor (AT1-R). However, the role of ROS in the regulation of AT1-R expression has not been explored. In this study, we examined the effect of an antioxidant on the homologous downregulation of AT1-R by Ang II. Ang II (10
-6
mol/L) decreased AT1-R mRNA with a peak suppression at 6 hours of stimulation in rat aortic vascular smooth muscle cells (VSMC). Ang II dose-dependently (10
-8
-10
-6
) suppressed AT1-R mRNA at 6 hours of stimulation. Preincubation of VSMC with N-acetylcysteine (NAC), a potent antioxidant, almost completely inhibited the Ang II-induced downregulation of AT1-R mRNA. The effect of NAC was due to stabilization of the AT1-R mRNA that was destabilized by Ang II. Ang II did not affect the promoter activity of AT1-R gene. Diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase failed to inhibit the Ang II-induced AT1-R mRNA downregulation. The Ang II-induced AT1-R mRNA downregulation was also blocked by PD98059, an extracellular signal-regulated protein kinase (ERK) kinase inhibitor. Ang II-induced ERK activation was inhibited by NAC as well as PD98059 whereas DPI did not inhibit it. To confirm the role of ROS in the regulation of AT1-R mRNA expression, VSMC were stimulated with H
2
O
2
. H
2
O
2
suppressed the AT1-R mRNA expression and activated ERK. These results suggest that production of ROS and activation of ERK are critical for downregulation of AT1-R mRNA. The differential effect of NAC and DPI on the downregulation of AT1-R mRNA may suggest the presence of other sources than NADH/NADPH oxidase pathway for ROS in Ang II signaling. Generation of ROS through stimulation of AT1-R not only mediates signaling of Ang II but may play a crucial role in the adaptation process of AT1-R to the sustained stimulation of Ang II.
Endothelial Dysfunction and Subclincal Atherosclerosis in Insulin Resistance Syndrome
Authors: Satoh Akira , Matsuoka Hidehiro , Iida Shuji , Fukami Kei , Okuda Seiya , Imaizumi Tsutomu
Published: July 2021

68

Insulin resistance syndrome, which constitutes obesity, hypertension, impaired glucose tolerance, and low high density lipoprotein is closely associated with cardiovascular events. Impairment of endothelial function precedes and exacerbates atherosclerosis. Accordingly, the aim of this study was to investigate a possible link among endothelial dysfunction, subclinical atherosclerosis, and insulin resistance syndrome. First, to examine the association between functional changes of large artery and insulin resistance syndrome, flow-mediated vasodilation (FMD) of the brachial artery was examined by high resolution ultrasonography, as a non-invasive measure of endothelial function in subjects without cardiovascular diseases (n=75). Coronary risk factors, including factors constituting insulin resistance syndrome were evaluated as well. Univariate and multivariate analyses revealed that FMD was inversely correlated with body mass index (BMI; r=-.26, p<0.03), mean arterial pressure (r=-.27, p<0.03), HbA1C (r=-.39, p<0.01), triglyceride (r=-.37, p<0.01), and the number of risk factors (r=-.47, p<0.001). Second, to examine the association between structural changes of large artery and insulin resistance syndrome in apparently healthy subjects (n=545), duplex scanning of the carotid arteries was performed to measure the intima-media thickness (IMT), an index of subclinical atherosclerosis. Age-adjusted-IMT was positively correlated with BMI (r=.14, p<0.002), mean arterial pressure (r=.27, p<0.001), HbA1C (r=.40, p<0.001), triglyceride (r=.12, p<0.005), HOMA, an index of insulin resistance, (r=.17, p<0.007), and the number of risk factors (r=.37, p<0.001). Finally, FMD was inversely correlated with IMT (r=-.28, p<0.03). Thus, the factors constituting insulin resistant syndrome were closely associated with both functional and structural changes of arteries in apparently healthy subjects. Our results suggest that insulin resistance may play a key role in the early process of atherosclerosis.
Perinatal Delivery of Angiotensin Type 2 Receptor (AT2R) Antisense cDNA Increases Blood Pressure in Adult Normotensive Rats
Authors: Katovich Michael J , Wang Hongwei , Gallinat Stephan , Sumners Colin , Raizada Mohan K
Published: July 2021

71

Recent observations suggest that AT
2
R have a role in the counter-regulatory actions of angiotensin II (Ang II) in cardiovascular protection and angiogenesis. The objective of the current investigation was to provide evidence for this cardioprotective action of AT
2
R by utilizing antisense gene transfer technology in normotensive rats. A retroviral vector containing full length AT
2
receptor antisense cDNA (LNSV-AT
2
R-AS) or missense (LNSV-AT
2
R-MS) was constructed. The LNSV-AT
2
R-AS viral particles were highly efficient in the transduction of AT
2
R-AS
in vitro
. The efficacy and effectiveness of this transduction was demonstrated by the long-lasting expression of AT
2
R-AS transcript and a decrease in AT
2
R binding.
In vivo
administration of LNSV-AT
2
R-AS resulted in similar findings. Five-day old normotensive Sprague Dawley rats received a single intracardiac bolus (25 μl) administration of LNSV-AT
2
R-AS viral particles (1x10
9
cfu/ml), which resulted in a robust expression of AT
2
R-AS transcript in tissues such as heart, kidney, adrenals and brain as early as five days post-delivery. Control rats received either LNSV alone or LNSV-AT
2
R-MS under identical conditions. The expression of AT
2
R-AS was persistent through adulthood indicating a high degree of transgene transduction
in vivo
. Mean blood pressure (BP) was elevated in the adult LNSV-AT
2
R-AS-treated rats when compared to the age-matched LNSV-AT
2
R-MS or control rats (123±5 mmHg vs. 100±9 mmHg). In addition, the pressor responses produced by Ang I and Ang II were enhanced in the LNSV-AT
2
R-AS- treated rats. For example, administration of 0.1 μg/Kg Ang I elicited a maximal increase in BP of 35±6 mmHg in the LNSV-AT
2
R-AS-treated rats compared to an increase of 23±6 mmHg in the LNSV-AT
2
R-MS. These observations demonstrate for the first time, that persistant inhibition of AT
2
R in normotensive rats influence cardiovascular responsiveness. Collectively, these data suggest that use of the AT
1
receptor antagonist-based therapy, with the resultant increase in Ang II levels, might provide additional benefit to the hypertensive patient via increased AT
2
R stimulation.
Evaluation of Anemia in Angiotensin Converting Enzyme (ACE) Knockout Mice.
Authors: Cole Justin M , Corvol Pierre , Bernstein Kenneth E
Published: July 2021

P2

Using targeted homologous recombination, we made two lines of mice that lack ACE. ACE.1 mice are null for all ACE activity while ACE.2 mice lack tissue ACE but retain 1/3 normal plasma ACE activity. Both strains have a marked decrease in blood pressure and are unable to produce a concentrated urine. ACE.1 mice have renal failure, as measured by a reduced creatinine clearance and an elevated serum creatinine, when compared to wild type mice; ACE.2 mice have
no evidence of renal failure
as indicated by a normal serum creatinine and creatine clearance. Surprisingly, both ACE.1 and ACE.2 mice are anemic with a reduction in hematocrit (38.5 +/- 1.3% and 38.3 +/- 0.8% respectively vs 48.4 +/- 0.9% for wild type) and serum hemoglobin (12.0 +/- 0.3 mg/dl and 11.7 +/- 0.2 mg/dl vs 14.7 +/- 0.3 mg/dl for wild type). In both strains, serum erythropoietin is elevated. Neither strain differs from wild type in regard to MCV, MCH, or MCHC, nor does either strain show chemical evidence of iron deficiency or hemolysis. Both ACE.1 and ACE.2 mice have equally low serum angiotensin II levels with a significant elevation of serum angiotensin I and the stem cell inhibitory peptide AcSDKP. Due to the presence of renal failure in ACE.1 mice, we have characterized in detail the anemia of ACE.2 mice. These animals demonstrate a significant reduction in total circulating red cell mass (24.2 +/- 1.1 μl/g vs 31.7 +/- 1.8 μl/g for wild type mice) without a change in total blood volume (57.8 +/- 1.2 μl/g vs 56.0 +/- 5.0 μl/g for wild type mice) as measured by the infusion of
51
Cr labeled erythrocytes. ACE.2 mice also have an increase in total plasma volume as determined by Evans Blue Dye infusion. The administration of angiotensin II to ACE.2 knockouts significantly increases their hematocrit and serum hemoglobin. Conclusion: The finding of significant anemia in ACE.2 mice, animals without renal failure, suggests a previously unappreciated role of the renin-angiotensin system in regulating erythropoiesis.
Physiological Amounts of Angiotensinogen Increase Blood Pressure in Sprague Dawley Rats After I.V. Administration.
Authors: Klett Christoph P R , Granger Joey P
Published: July 2021

P9

The synthesis and secretion of hepatic angiotensinogen is controlled by a complex pattern of physiologic and pathophysiologic mediators including glucocorticoids, estrogens, thyroid hormones, cytokines, glucagon,insulin, and prostaglandins. Since plasma concentrations of angiotensinogen are close to the Michaelis Menten constant, it was hypothesized that changes in angiotensinogen plasma concentrations have an influence on the formation rate of angiotensin I and angiotensin II and, therefore, on blood pressure. To further test this hypothesis we injected purified rat angiotensinogen i.v. in Sprague Dawley rats via the femoral vein. Mean arterial blood pressure was measured after arterial cathederization. Control animals had a mean arterial pressure of 131 ± 2 mm Hg before and after the injection of vehicle (saline). The injection of 0.8, 1,2, and 2.9 mg/kg angiotensinogen caused a dose dependend increase in mean arterial blood pressure of 8 ± 0.4, 19.3 ± 2.1, and 32 ± 2.4 mm Hg, respectively. In contrast, the injection of a purified rabbit anti-rat-angiotensinogen antibody 1.4 mg/kg resulted in a significant decrease in blood pressure (-52 ± 3.2 mmHg). In an attempt to analyze how fast and efficient angiotensinogen production can sense regulatory input and convert into adaptation of secretion rate we determined the transit time (time needed for translation and post-translational modifications) for angiotensinogen in a pulse chase experiment employing
35
[S]-methionine as label in freshly isolated hepatocytes. During the chase periode, after quantitative immunoprecipitation, we determined the transit time for angiotensinogen with 2.5 h which is consistent with the constitutive type of angiotensinogen secretion and the time lag found for plasma concentrations to respond to regulatory mediators. In summary we conclude that variations in angiotensinogen plasma concentrations can result in changes in blood pressure. In contrast to renin known as a tonic regulator for the generation of angiotensin I, angiotensinogen seems to be a factor rather important for long-term control of the basal activity of the renin angiotensin system.
Potentiation of Bradykinin by Angiotensin-(1-7) on Resistance Vessels of Hypertensive Rats, Studied
in Vivo
.
Authors: Fernandes Liliam , Fortes Zuleica B , Nigro Dorothy , Scivoletto Regina , Santos Robson A S , Carvalho Maria Helena C
Published: July 2021

P19

Objective: To verify the Angiotensin-(1-7) [Ang-(1-7)]-activity on Bradykinin (BK)-induced vasodilation in SHR mesenteric arterioles,
in vivo-in situ.
Methods: Arteriolar diameter was measured by intravital microscopy before and after topical application of BK(1pmol), Acetylcholine(ACh 1.6nmol), Sodium nitroprusside (SNP 38pmol) or Histamine (5.4nmol) in the absence or presence of Ang-(1-7) (100pmol). To investigate the Ang-(1-7)/BK interaction, treatments were employed through topical application of antagonists of BK (HOE140,100pmol), Ang-(1-7)(A779,100pmol)and potassium channel (tetraethylammoniun - TEA,90pmol), with an inhibitor of NOSynthase (L-NAME 10nmol) and after cyclooxygenase blockade (indomethacin 5mg/Kg or diclofenac 2.5mg/Kg). To evaluate the effect of ACE- and/or AT
1
blockade on Ang-(1-7)/BK interaction, rats were treated for 21 days with enalapril, quinapril (10mg/Kg), losartan (15mg/Kg) or enalapril + losartan (10 and 15 mg/Kg, respectively). In those enalapril-treated rats the effect of BK (1pmol) was also analysed in the presence of A779 (100pmol). Results: BK-induced vasodilation, but not ACh, SNP or Histamine responses, was increased in the presence of Ang-(1-7) (4.96±0.7%
vs
9.07±1.0%
*
).This interaction was abolished by HOE (1.11±0.8%
*
), A779 (5.13±0.6%
*
), TEA (3.37±0.5%
*
), indomethacin (1.73±0.4%
*
)and diclofenac (3.63±0.5%
*
), whereas L-NAME did not modify the Ang-(1-7)-potentiating activity. The BK-potentiation by Ang-(1-7) was also observed after enalapril (10.57±0.5%
*
), quinapril (8.9±0.7%
*
), losartan (9.93±1.2%
*
) and enalapril + losartan (10.59±0.5%
*
). Enalapril increased the BK-vasodilation(8.21±0.7%
*
), but this effect was reversed in the presence of A779 (4.27±0.5%
*
).
*
p≤0.05 Conclusion: In the SHR microcirculation Ang-(1-7) potentiates BK through a specific receptor, probably releasing prostaglandins and EDHF. Our results indicate that the BK-potentiation by Ang-(1-7) may occur endogenously and contribute to the pharmacological effects of ACE inhibition. HOE 140 and Quinapril were gifts from HOECHST and Warner Lambert, respectively.
Structure-Activity Relationships for the Hypertensinogenic Activity of Ouabain: Role of the Sugar and Lactone Ring.
Authors: Hamlyn John M , Manunta Paolo , Hamilton Bruce P
Published: July 2021

P20

Elevated levels of an endogenous ouabain (EO) occur in many patients with essential hypertension (e.g., Manunta
et al
,
Hypertension
1999;34:450-456) and the prolonged infusion of ouabain induces hypertension (Manunta
et al J. Hypertension
1994;12:549-560). This study used a series of closely related structural analogs of ouabain to test the key hypothesis that sodium pump inhibition underlies ouabain-dependent hypertension. The functional roles of the sugar and lactone ring were tested for their effects on inhibition of the Na,K-ATPase, and on long term blood pressure. Normal male SD rats received infusions of vehicle (VEH), rhamnose (RHA), ouabain (OUA), ouabagenin (OGN), dihydroouabain (DHO), isoouabain (ISO), and ouabain in which the lactone ring was opened (ORO)at 30 μg/Kg/24 hr for 4-5 weeks via s.c. osmotic pumps. Cuff pressures were taken weekly. At the end of the study, blood and tissues were harvested, extracted by C-18 and analyzed by ouabain-RIA using appropriate standards. HPLC showed that plasma and tissues contained one main peak of immunoreactivity corresponding to the infused agent. Systolic pressures (SBP)in VEH, RHA, OUA, OGN, DHO, ISO and ORO were 132±2.5, 133±1.5, 159±2.6*, 154±4*, 167±4*†, 171±2.2*†, 169±2.4*†,respectively (*P<0.01 vs VEH and RHA, †P<0.05 vs OUA). The hypertensinogenic activity was significantly greater than ouabain in three of the analogs (DHO, ORO, ISO)in which the lactone was either saturated, conformationally restrained by linkage with C14, or opened. These compounds were weak or inactive as inhibitors of the Na,K-ATPase. Similarly, absence of RHA from ouabain affected SBP minimally. We conclude that the hypertensinogenic activity of ouabain is mediated exclusively by the steroid nucleus. The lactone ring is not required for ouabain to induce hypertension but is essential for inhibition of the Na,K-ATPase. A novel mechanism mediates ouabain-dependent hypertension.
Role of Endothelial K
+
Channels in NO-Dependent Vasorelaxant Responses to Acetylcholine in Rat Aorta.
Authors: Quilley John , Qiu Yue
Published: July 2021

P30

Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) in rat aorta are mediated solely by NO. Rings precontracted with U46619 were used to investigate the role of endothelial K
+
channels. Thus, any effect of K
+
channel inhibitors on Ach responses in the absence of an effect on those to nitroprusside (NP) can be attributed to interference with Ach-induced stimulation of NO. Vasorelaxant responses to Ach (log EC
50
-7.29M) were abolished by removal of the endothelium or inhibition of NO synthesis with nitroarginine (100μM) which potentiated responses to NP (log EC
50
-9.41M vs -8.47M for control). In the presence of TEA (10mM) to inhibit K
+
channels, the dose-response curve for Ach, but not NP, was shifted to the right (log EC
50
-6.06). Elevation of extracellular K
+
(25mM KCl)also shifted the dose-response curve for Ach to the right. Inhibitors of specific types of K
+
channels: BaCl
2
(30μM), apamin (100nM), glibenclamide (10μM), charybdotoxin (50nM) and iberiotoxin (100nM) were without effect on dose-response curves to either Ach or NP. However, the combination of apamin (100nM) and charybdotoxin (50nM) but not apamin plus iberiotoxin, reduced relaxant responses to Ach (log EC
50
-6.95M) without affecting those to NP.These results confirm that Ach-induced relaxation of rat aorta is mediated entirely by endothelium-derived NO, the release of which apparently involves hyperpolarization of the endothelium. This effect is dependent on activation of a K
+
channel that is blocked by a combination of apamin/charybdotoxin but neither agent alone, possibly indicating characteristics of both Ca
2+
- activated and voltage-dependent K
+
channels.
Carbon Monoxide Is an Inhibitory Regulator of Hypoxia-Induced Contraction of Small Pulmonary Arteries.
Authors: Zhang Fan , Kaide Jun-Ichi , Nasjletti Alberto
Published: July 2021

P36

Hypoxia elicits pulmonary vasoconstriction, endothelin production and expression of heme oxygenase (HO)-1. In small arteries and arterioles of the systemic circulation, HO catalyzes synthesis of carbon monoxide (CO) which attenuates vascular responsiveness to constrictor stimuli. This study examines the hypothesis that HO-derived CO inhibits constrictor responsiveness to hypoxia in rat pulmonary arterial vessels (PA). Experiments were conducted on rings of large PA (ID=1051±78 μm) and small PA (ID=153±12μm) bathed in Krebs buffer and mounted on a wire-myograph for measurement of isometric tension (IT). Exposure to hypooxygenated buffer (PO
2
15 mmHg) elicited minimal increase of IT in small PA (0.026±0.008 mN/mm) but in preparations bathed in buffer containing chromium mesoporphyrin (30 μM, CrMP), an inhibitor of HO-dependent CO synthesis, hypoxia elicited a much more intense increase of IT (0.77±0.06 mN/mm, P<0.05). Hypoxia-induced contraction of large PA also was magnified by pretreatment of the vessels with CrMP (from 0.030±0.007 to 0.077±0.016, P<0.05) but the intensity of the response was less than in similarly treated small PA. In small PA treated with CrMP, hypoxia-induced contractions were attenuated by exogenous CO (10 μM) (from 0.77±0.06 to 0.15±0.05 mN/mm); contraction also were reduced in preparations denuded of endothelium (0.27±0.09 versus 0.77±0.06 mN/mm). Hypoxia-induced contractions of endothelium-intact small PA bathed in buffer containing CrMP were greatly inhibited by pretreatment with L754,142 (10 μM), a blocker of endothelin receptors. These data suggest that endogenous CO is an inhibitory regulator of hypoxia-induced contractions in small PA. This action of CO may reflect inhibition of hypoxia-induced endothelin production or of endothelin-induced vascular contraction.
Potentiation of Bradykinin by Angiotensin-(1-7), Quinaprilat and Captopril Correlates Directly with Inhibition of the ACE C- and N-Terminal Domains.
Authors: Danser Alexander HJ , Vries Rene de , Tom Beril , Saxena Pramod R
Published: July 2021

P37

ACE inhibitors (ACEIs) block bradykinin type 2 (B
2
) desensitisation, thereby potentiating bradykinin (BK) beyond blocking its hydrolysis (Deddish et al.,
Hypertension
1998). Angiotensin-(1-7) (A(1-7)) also acts as an ACEI, with preference for the ACE C-domain (Deddish et al.), and causes relaxation in a BK- and AT
2
receptor-dependent manner (Gorelik et al.,
J Pharmacol Exp Ther
1998). Here, we compared the BK-potentiating effects of A(1-7) with those of quinaprilat (Q) and captopril (C). Porcine coronary arteries (PCAs), obtained from 24 pigs, were mounted in organ baths, preconstricted with PGF
2α
, and exposed to Q, C, BK or A(1-7) in the absence or presence of the AT
1
receptor antagonist irbesartan (1 μM) or the AT
2
receptor antagonist PD123319 (1 μM). BK induced complete relaxation (EC
50
7.9±0.1, mean±SEM), while Q, C and A(1-7) alone, at concentrations up to 10 μM, were without effect. Pretreatment with Q (0.1 pM-10 μM) shifted the BK curve to the left in a biphasic manner: a 5-fold shift at Q concentrations (0.1-1 nM) that are known to be specific for the ACE C-domain, and a 10-fold shift at Q concentrations (> 10 nM) that block both ACE domains. Pretreatment with C (0.1 pM-10 μM) or A(1-7) (1 nM-10 μM) monophasically shifted the BK curve to the left, by a factor of 10 and 5, respectively (both at concentrations of 1 μM and higher). Repeated exposure of PCAs to a BK concentration that causes maximal relaxation (0.1 μM) induced B
2
receptor desensitisation. The addition of 10 μM Q, C or A(1-7) to the bath, at a time when BK alone was no longer able to induce relaxation, fully restored the relaxant effects of BK. Irbesartan or PD123319 did not affect any of the observed effects of A(1-7). In conclusion, A(1-7), like Q and C, potentiates BK by acting as an ACE inhibitor and not through AT receptor stimulation. BK potentiation is maximal when both the ACE N- and C-terminal domain are inhibited. At physiological concentrations, the inhibitory effects of A(1-7) are limited to the ACE C-domain, raising the possibility that A(1-7) synergistically increases the blood pressure-lowering effects of N-domain-specific ACE inhibitors.
Effects of Chronic Eta-Receptor Blockade in Stroke-Prone Spontaneously Hypertensive Rats.
Authors: Barandier Christine Elise , Yang Zhihong , Luscher Thomas Felix
Published: July 2021

P38

It has been reported that some ET-receptor blockers prevent cerebral injury in stroke-prone spontaneously hypertensive rats (SPSHR), whereas chronic inhibition of nitric oxide synthase (NOS) precipitates stroke. We investigated the protective effects of chronic treatment with BSF208075, a selective ETA-receptor blocker in SPSHR treated with the NOS inhibitor, L-NAME, or untreated. 4 groups of male SPSHR, 10 week old, were studied: a control group and 3 groups treated with BSF208075 (30 mg/kg/day), L-NAME (10 mg/kg/day) or BSF208075+L-NAME (30 and 10 mg/kg/day, respectively). Systolic blood pressure, body weight and survival were recorded. In the control group, severe hypertension (292.3±1.0mmHg; p<0.01 vs baseline) developed within 4 weeks. In contrast, in the BSF208075-treated group, the development of hypertension (265.2±3.8mmHg; p<0.01 vs control) was limited and blood pressure started to decrease after 4 weeks of treatment. In both of these groups, the increase in body weight was normal and similar, and 90% of the animals were still alive after 50 days of treatment. In the L-NAME-treated group, the blood pressure immediately and drastically increased, the body weight was reduced by 50% within 6 weeks of treatment. 50% of mortality was observed after 22 days of treatment, and all rats died within 36 days. The mean survival was 25.9±2.4 days. In L-NAME+BSF208075-treated group, BSF208075, although it did not prevent the development of hypertension, it reduced the loss of body weight and increased mean survival to 36.7±1.4 days (p<0.01 vs L-NAME-treated group). In this group, 50% of mortality was observed after 37 days of treatment, and all rats died within 41 days. These results show that BSF208075 delays the development of hypertension in SPSHR, and increases survival of L-NAME-treated SPSHR, independent of hypertension.
Enhanced Sympathoexcitatory and Pressor Responses to Chronic Central Sodium Loading in Dahl Salt-Sensitive Vs. -Resistent Rats
Authors: Huang Bing S , Leenen Frans Hh
Published: July 2021

P46

Increased responsiveness to increases in CSF Na
+
by high salt intake may contribute to salt-sensitive hypertension in Dahl S rats. To test this hypothesis, Dahl S and R rats received a chronic icv infusion of artificial CSF (aCSF) or aCSF containing 0.8 M Na
+
(5 μl/hr) for 14 days, using osmotic minipumps. On day 14 in conscious rats, CSF was sampled and BP, HR and renal sympathetic nerve activity (RSNA) were recorded at rest and in response to air stress, icv α
2
-adrenoceptor agonist guanabenz (50 μg), icv ouabain (0.6 μg), and iv phenylephrine and nitropusside to estimate baroreflex function. Data are mean±SEM (n=8-13). *p<0.05 vs others.†p<0.05 vs aCSF. Similar chronic increases in CSF Na
+
caused more severe hypertension, larger responses to air stress and guanabenz, and more blunting of the arterial baroreflex gain in S vs R rats. These findings suggest that enhanced neuronal responsiveness to CSF Na
+
is one of the mechanisms contributing to salt-sensitive hypertension in Dahl S rats.
Role of the At
2
Receptor in the Cardioprotective Effect of At
1
Antagonist in Mice with Chronic Heart Failure Induced by Coronary Ligation
Authors: Xu Jiang , Carretero Oscar A , Liu Yun-He , Shesely Edward G , Yang Fang , Yang Xiao-Ping
Published: July 2021

P63

Angiotensin II (Ang II) acts mainly on two receptor subtypes: type 1 (AT
1
) and type 2 (AT
2
). Most of the known biological actions of Ang II are mediated
via
AT
1
receptors; however, the role of the AT
2
receptor is less well defined. We hypothesized that blockade of AT
1
receptors increases circulating Ang II levels, which in turn activates the AT
2
receptor and induces cardioprotection. In mice which lack AT
2
receptors, the effect of an AT
1
antagonist (AT
1
-ant) would be diminished or absent. AT
2
receptor knockout mice (-/-) and their wild-type littermates (+/+) were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. One month after MI, each strain of mice received either vehicle or AT
1
-ant (valsartan, 50 mg/kg/day in drinking water) for 3 months. Systolic blood pressure (SBP) was measured weekly and echocardiography performed once a month. Basal SBP and cardiac function did not differ between +/+ and -/-. Three months after MI, SBP and cardiac function changed similarly in both strains receiving vehicle. Valsartan significantly increased EF and decreased LVDd and mass and these effects were diminished in -/- (table). Our results suggest that under basal conditions, the AT
2
receptor may not play an important role in regulation of blood pressure and cardiac function; however, it does appear to be an important component in the cardioprotective effect of AT
1
-ant.
Catecholamines Abrogate the Anti-Mitogenic Effects of 2-Hydroxy Metabolite of Estradiol on Vascular Smooth Muscle Cells by Inhibiting Catechol-O-Methyltransferase (COMT) Activity and 2-Methoxyestradiol Formation
Authors: Zacharia Lefteris C , Jackson Edwin K , Gillespie Delbert G , Dubey Raghvendra K
Published: July 2021

P70

Methylation of 2-hydroxyestradiol(2OHE; endogenous estradiol metabolite) to 2-methoxyestradiol (2MeOE; angiogenesis inhibitor)by COMT plays a key role in mediating the anti-mitogenic effects of 2OHE on vascular smooth muscle cell (SMC)growth. Catecholamines such as norepinephrine (NE) are also substrates for COMT and increased levels of NE are associated with vasoocclusive disorders. We hypothesize that increased endogenous synthesis/levels of NE under pathophysiological conditions may abrogate the vasoprotective effects of 2OHE by competing for COMT and inhibiting 2MeOE formation. To test this hypothesis we investigated the anti-mitogenic effects of .001-10μM 2OHE on 2.5% FCS-induced SMC growth (cell number, DNA synthesis [thymidine incorporation], collagen synthesis [proline incorporatio])in rat and human aortic SMCs in the presence and absence of NE (0.1-40μM). NE concentration-dependently abrogated the inhibitory effects of 2OHE on SMC growth and in the presence of 10μM NE the inhibitory curve of 2OHE on SMC growth was shifted to the right(P<.05). In the presence of 10μM NE, the inhibitory effect of 1μM 2OHE on DNA synthesis was reduced from 70±3% to 24±2% (P<.05), and this effect of NE was mimicked by isoproterenol (ISO) and epinephrine (EPI). Additionally, NE (0.5-2.5mM) inhibited the metabolism of 10μM 2OHE to 2MeOE in a concentration-dependent manner and the effects of NE were mimicked by ISO, EPI, metanephrine, normetanephrine and 3,4-dihydroxymandelic acid. At 0.5 mM ISO, NE and EPI inhibited 2MeoE formation by 70±4%,20±2% and 40±2%, respectively. Our findings suggest that increases in local synthesis of catecholamines within the vasculature may abrogate the anti-vasoocclusive effects of estradiol and 2OHE by blocking 2MeOE formation. In conclusion, the interaction between catecholamines and 2OHE may play a key role in the biology of vascular SMC growth.
Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43
Authors: Slovut David Paul , Dorrance Anne N , Brosius Frank C , Watts Stephanie W , Webb R. Clinton
Published: July 2021

P71

Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10
-9
M-10
-5
M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC
50
[mol]/L control -6.89 vs reserpine -7.16, p<0.05); control -6.86 vs phenol -7.32, p<0.0001). Incubation in K
+
-free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10
-6
M, p<0.001). These findings are consistent with adrenergic denervation. Agonist-induced oscillatory activity was observed in 5/5 reserpine- and 4/7 phenol-treated vessels vs 0/12 controls (Fischer’s exact test p<0.05). The highly selective gap junction inhibitor, Gap27 (10 mM), nearly abolished NE-induced vascular smooth muscle oscillations. Reverse transcriptase polymerase chain reaction was carried out for connexin43 (Cx43) and c-Kit, a marker for non-neural interstitial cells that serve as pacemaker cells, using specific oligonucleotide primers. Following denervation, mRNA levels of Cx43 remained unchanged while levels of c-Kit increased markedly (0.16±0.03 vs 0.62±0.13 a.u., p<0.05). Western blot analysis revealed near doubling of Cx43 protein after denervation (80.0±13.7 vs 41.4±5.7 a.u., p<0.05). In conclusion, sympathetic denervation produced increased agonist-induced oscillatory contractile activity in vascular smooth muscle. The heightened vascular reactivity may result from upregulation of pacemaker cells and increased Cx43 expression.
Pre-Clinical Studies of Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor (HGF) Gene in Rat and Rabbit Hind Limb Ischemia Models
Authors: Taniyama Yoshiaki
Published: July 2021

P72

A novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy. Initially, we examined the transfection of “naked” human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imager. Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (C100% vs H 245%, P<0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hind limb models, as potential therapy for peripheral arterial disease.
Calcium-Mediated Cox-2 Expression and Pge
2
Production in the Rat Medullary Thick Ascending Limb(mTAL) Is Tnf
α
-Dependent
Authors: Wang Dairong , McGiff John C , Ferreri Nicholas R
Published: July 2021

P89

Severe hypercalcemia often is associated with polyuria in patients. We have shown that mTAL cells express cyclooxygenase-2(COX-2) when challenged with tumor necrosis factor-alpha(TNF
α
), an effect that was linked to the decrease in TNF
α
-mediated
86
Rb
+
uptake, an
in vitro
correlate of natriuresis. The mTAL expresses calcium sensing receptor(CaR), a G-protein coupled receptor that senses small changes in extracellular calcium concentration([Ca
2+
]
o
) and ultimately increases intracellular calcium concentration([Ca
2+
]
i
) and protein kinase C(PKC) activity. We assessed the effects of [Ca
2+
]
o
on COX-2 and TNF
α
protein expression as calcium increases TNF
α
synthesis in certain cell types, and PMA, a PKC activator, induces COX-2 gene expression in mTAL cells. Western blot analysis showed that COX-2 protein expression was increased two-fold after challenge for 9 hours with 1.7 mM CaCl
2
. ELISA analysis revealed that PGE
2
formation was increased from 11 ± 1.5 to 68 ± 21 pg/μg protein (p<0.05) by 1.7 mM CaCl
2
; no effect was observed when cells were exposed to 3.4 mM NaCl suggesting that the increase in PGE
2
synthesis was a function of increasing [Ca
2+
]
o
. TNF
α
synthesis increased from 1.3 ± 0.1 to 5.4 ± 0.4 pg/μg protein (p<0.05) after challenge with 2 mM CaCl
2
. CaR agonist poly-L-arginine (100nM) increased PGE
2
production from 15 ± 0.1 to 66.3 ± 4.4 pg/μg protein (p<0.0001), and increased TNF
α
production from 0.7 ± 0.1 to 3.7 ± 0.3 pg/μg protein after a 9 hr incubation period. Pretreatment of cells with 1 μg/ml neutralizing TNF
α
antibody reduced COX-2 protein expression and PGE
2
production by approximately 40%. These data indicate that Ca
2+
o
, as well as CaR selective agonists, increase mTAL COX-2 expression, PGE
2
formation, and TNF
α
production in primary cultures of mTAL cells. We conclude that the increases in COX-2 expression and PGE
2
production are, in part, TNF
α
-dependent, suggesting that Ca
2+
o
may be a modulator of extracellular fluid volume regulation via a cytokine-mediated autocrine mechanism.
Increased Expression of a Constrictor Mechanism Mediated by 20-Hydroxyeicosatetraenoic Acid in Renal Interlobular Arteries of Rats Fed a K Deficient Diet.
Authors: Zhang Fan , Wang Mong-Heng , Kaide Jun-Ichi , Schwartzman Michal Laniado , Nasjletti Alberto
Published: July 2021

P91

Renal preglomerular vessels metabolize arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that amplifies vasoconstrictor responsiveness. This study examines (a) the effect of variations in dietary K intake on renal 20-HETE synthesis, and (b) the significance of 20-HETE synthesis in relation to the regulation of renal interlobular artery (ILA) internal diameter (ID) in rats fed a diet with a low (0.004 mmol/g) or high (1.351 mmol/g) K content (LK and HK rats) for 10-12 days. Metabolism of
14
AA to 20-HETE by renal microsomes from LK rats exceeded values by renal microsomes from HK rats (800±140 versus 263±29pmol/mg/min); P<0.05). Complementary studies were conducted on ILA mounted on a pressure-myograph and superfused with Krebs buffer. In ILA from LK rats, the ID after equilibration at an intraluminal pressure (IP) of 100 mmHg was 58±10 μm and increased (P<0.05) to 81±8μm after inclusion of the 20-HETE synthesis inhibitor DDMS (30 μM) into the buffer. In ILA from HK rats, the ID at 100mmHg was 77±3 μm and increased (P<0.05) to 87±4μm after DDMS treatment. The DDMS-induced increase of ID in ILA of LK rats (23±3 μm) exceeded (P<0.05) that in ILA of HK rats (10±3 μm). We also evaluated IP-ID relationships. Stepwise increases in IP within the range 40-100 mmHg elicited a myogenic response that decreased ID (expressed as % of passive ID). IP-induced decreases of ID in LK rats surpassed (P<0.05) those in HK rats. However, after treatment with DDMS myogenic constrictor responses were comparable in ILA from LK and HK rats. These data suggest that renal 20-HETE synthesis increases as dietary K decreases and that a constrictor mechanism involving 20-HETE is more prominently expressed in ILA of LK than of HK rats.
Beneficial or Detrimental Effects on Hemodynamics Depends on Dose of Dietary Potassium in Rapp Dahl Salt-Sensitive (DS) Rats.
Authors: Manger William M , Simchon Shlomoh , Jan Kung-Ming , Haddy Francis , Stier Charles T , Loscalzo Joseph , Jan Rex
Published: July 2021

P103

Dietary K supplementation was reported to lower blood pressure and prevent strokes in humans and to prevent strokes in hypertensive DS rats. We report a biphasic effect as a function of KCl dose in DS rats that were fed 1% NaCl with increasing dietary KCl, namely, 0.7, 2.6, 4, and 8%. After 8 months on 1% NaCl supplemented with 0.7% KCl, mean arterial pressure (MAP), plasma volume (PV), cardiac output (CO), total peripheral, renal and cerebral vascular resistances (TPR, RVR, CVR) increased compared to salt-resistant DR rats; on 2.6% KCl all these parameters decreased compared with DS on 0.7% KCl diet. When KCl was increased to 4 and 8%, MAP, PV, CO and RVR progressively increased in DS and DR rats, without changing TPR; these changes were accompanied by parallel increases in plasma aldosterone. Only DS rats on the ”optimal“ 2.6% KCl supplement maintained hemodynamics most similar to control DR rats and thus prevented Na retention, hypertension, increases in RVR and CVR. These beneficial hemodynamic effects may explain stroke prevention.
Role of ET
B
Receptors in the Renal Response to Big Endothelin-1: Contrasting Pharmacologic ET
B
Receptor Blockade with Genetic ET
B
Deficiency
Authors: Pollock David M , Portik-Dobos Vera
Published: July 2021

P98

Renal clearance studies were conducted to determine the role of ET
B
receptors in the renal response to the endothelin-1 precursor, big endothelin-1 (Big ET). Two series of experiments were conducted on Inactin anesthetized rats to contrast acute pharmacologic blockade of ET
B
receptors versus genetic ET
B
receptor deficiency. In the first series, separate groups of normal SD rats were given the ET
B
-selective antagonist, A-192621, or vehicle (0.9% NaCl) prior to infusion of Big ET (20 pmol/kg/min) for a 60 min period (n=9 in each group). ET
B
receptor blockade alone significantly increased baseline mean arterial pressure (MAP; 102±4 vs. 141±6 mmHg, P<0.05) and urine flow rate (UV; 0.5±0.1 vs. 1.3±0.2, P<0.05) without any effect on GFR or renal plasma flow (RPF). Big ET significantly increased MAP in both groups but to a higher level in rats given antagonist (120±6 vs. 169±6 mmHg, P<0.05). Big ET increased UV in control rats but decreased in rats given antagonist. GFR and RPF were decreased in rats given Big ET, an effect which was exaggerated by ET
B
blockade. An additional series of experiments examined the response to Big ET in rats lacking functional renal ET
B
receptors. Spotting lethal (sl) rats have a naturally occurring ET
B
deficiency but can be ”rescued“ from fatal intestinal aganglionosis by directed transgenic expression of ET
B
receptors. Rats heterozygous (sl/+) or homozygous (sl/sl) for ET
B
receptor deficiency, were given Big ET as in the first series (n=5 in each group). Surprisingly, baseline MAP was significantly higher in sl/+ compared to sl/sl rats (147±3 vs. 111±11 mmHg, P<0.05) although other variables were similar. Big ET had no significant effect on MAP in either group although there was a tendency for MAP to increase in sl/+ rats (7±2%, P=0.052). UV was significantly decreased in both groups although the change was much larger in sl/sl rats. GFR and RPF were significantly decreased in sl/sl, but not sl/+ rats. Both series of experiments indicate that the ET
B
receptor plays an important role in limiting the renal hemodynamic response to Big ET. Furthermore, the diuretic actions of Big ET require a functional ET
B
receptor.
Short-Term NOS II Inhibition Leads to Long-Term Salt-Sensitivity in Dahl Salt-Resistant Rats
Authors: Rudd M. Audrey , Toolan George , Trolliet Maria R , Cloutier Timothy , Maitland Karlene , Loscalzo Joseph
Published: July 2021

P111

We have previously shown that inhibition of inducible nitric oxide synthase (NOS II) evokes a salt-sensitive increase in blood pressure in Dahl salt-resistant rats when given a high salt (8% NaCl) diet for 7 days. To determine whether or not the effect of NOS II inhibition is short-lived, we continued the high salt diet for an additional 3 weeks following the discontinuation of the NOS II inhibitor. DR rats were given one of two NOS II selective inhibitors, AMT (300 nmoles/hr) and 1400W (35 nmoles/hr) for 2 weeks. A high salt diet was initiated after the first week of NOS II inhibition and continued for an additional 3 weeks for a total of 4 weeks of high salt treatment. Control DR rats received high salt alone for 4 weeks. Systolic blood pressure was taken at baseline and once weekly for the treatment period. Blood pressure significantly increased in DR rats after 1 week of high salt following NOS II inhibition. The blood pressure remained elevated throughout the 4-week period of high salt treatment despite the discontinuation of NOS II inhibitors 1 week following the initiation of the high salt diet. There was no significant change in blood pressure in DR rats on high salt diet alone. These data suggest that salt-sensitive hypertension can be evoked by transient NOS II inhibitor exposure. We conclude that tranisent NOS II inhibition may initiate events or processes that high salt maintains leading to sustained elevation in systolic blood pressure.