bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Lasting Pressor Effect of Liposome-Encapsulated Angiotensin-(1-7) Following Unilateral Microinjection into the Rat Rostral Ventrolateral Medulla
Authors: Silva-Barcellos Neila M , Santos Robson A S , Caligiorne Sordaine , Lemos Marisa , Frezard Frederic
Published: March 2022

P54

Several studies with vasoactive peptides in the rostral ventrolateral medulla (RVLM) have focused on their acute cardiovascular effects. However, information is still lacking on their long term effects in this key region for the central control of blood pressure. Sterically stabilized liposomes (SSL) represent an important tool for the slow release of bioative agents and the prolongation of their effect
in vivo
. In this work, we administered Angiotensin(Ang)-(1-7)-containing SSL in the RVLM and evaluated its effects on blood pressure (BP) and heart rate (HR). Ang-(1-7) was encapsulated in SSL made from disteroylphosphatidylcholine, cholesterol and polyethyleneglycol-phosphatidylethanolamine (PEG-2000PE) at a molar ratio of 5:4:0.3. Peptide-containing liposomes were separated from unencapsulated peptide by dialysis and then sterilized by filtration. The final preparation showed a mean vesicle diameter of 0,2 μm and a weight ratio of encapsulated peptide to lipid of 0.03 (w/w). Ang-(1-7)-containing liposomes (36 ng peptide/200 nl) and empty lipossomes (control) were unilaterally microinjected in the RVLM of Wistar rats. The effects on BP and HR in freely-moving rats were evaluated by telemetry. The group microinjected with SSL-Ang-(1-7) exhibited a significant increase in mean arterial blood pressure (MAP) that was maintained for 4 days (MAP = 112 ± 2.3 mmHg vs 97 ± 2.0 mmHg, p<0.05). On the other hand, the microinjection of empty liposomes did not produced any significant alteration of BP. Strikingly, the normal circadian variations of the BP and HR were markedly attenuated in SSL-Ang-(1-7) group only. In conclusion, the site specific (RVLM) microinjection of SSL-Ang-(1-7) produced a long-lasting pressor effect. Furthermore, these data unmasked a new physiological role for Ang-(1-7) at the level of RVLM: modulation of circadian rhythm of BP.
Effects of Ac-Sdkp, a Natural Inhibitor of Hematopoietic Stem Cell Proliferation, on Cardiac Collagen Deposition in Rats with Myocardial Infarction
Authors: Yang Fang , Yang Xiao-Ping , Liu Yun-He , Xu Jiang , Rhaleb Nour-Eddine , Peng Hong-Mei , Carretero Oscar A
Published: March 2022

P56

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a potent natural inhibitor of hematopoietic stem cell proliferation. Recently, it has been shown to inhibit collagen production by cardiac fibroblasts
in vitro
and collagen content in the left ventricle (LV) of 2K1C hypertensive rats
in vivo
. We hypothesized that Ac-SDKP may decrease myocardial collagen deposition in rats with myocardial infarction (MI), thereby reducing interstitial fibrosis and improving cardiac function. Lewis inbred rats were subjected to MI by ligating the left anterior descending coronary artery. Either vehicle or Ac-SDKP (400 mg/kg/day, i.p. by osmotic mini-pump) was given 2 months after MI and continued for another 2 months. Systolic blood pressure (SBP) was measured once a month by tail cuff. LV diastolic dimension (LVDd), mass, ejection fraction (EF) and cardiac output (CO) were measured monthly by echocardiography. At the end of the experiment, mean blood pressure (MBP), LV end-diastolic pressure (LVEDP) and dP/dt were determined. Collagen content in the non-infarcted area of the LV was measured by hydroxyproline assay. We found that Ac-SDKP had no effect on most hemodynamic and cardiac functional parameters. However, it significantly decreased collagen content from 26.1 ±2.2 (vehicle) to 14.7 ±4.8 mg/mg dry weight (Ac-SDKP); P < 0.001, associated with an increase in negative dP/dt from 5451.8 ±1240.9 (vehicle) to 7030.3 ±763.2 mm Hg/sec (Ac-SDKP); P < 0.01. These results suggest that Ac-SDKP could be a potent tool to slow down the process of myocardial interstitial fibrosis and improve post-MI diastolic function.
NF1 Negatively Regulates Gadd153 Expression in Vascular Smooth Muscle Cells.
Authors: Nakamura Michitsugu
Published: March 2022

P67

OBJECTIVES: Vascular remodeling is determined by the balance of proliferation and apoptosis in vascular smooth muscle cells (VSMC). Endoplasmic reticulum (ER) stress has been reported as an inducible factor of apoptosis. We have recently reported that growth arrest and DNA damage inducible gene (Gadd)153, which is known to be upregulated by ER stress, plays a pivotal role in the regulation of apoptosis in VSMC. The aim of this study is to characterize a promoter region of the Gadd153 gene for further elucidation of underlying mechanisms of VSMC apoptosis mediated by Gadd153. METHODS: Promoter region was isolated by screening of a rat genomic library. Transcription start sites were determined by primer extension analysis. Progressive 5′-deletions were prepared and used for the construction of luciferase-fusion vectors. For all experiments, VSMC were maintaining in the presence of 10% FCS to make them a proliferative state. Promoter activity was measured by luciferase assay, and DNA-binding studies were performed by electromobility shift and supershift assays. RESULTS: A 1500-bp fragment containing a Gadd153 promoter region was obtained, and its sequence alignment was determined. Primer extension analysis revealed that there were two major transcription start sites. Further, one TATA-like box was found within 30 bp from the transcription start sites. Although the 5′-deletion that was deleted the sequence spanning -1500 through -400 showed the highest promoter activity in VSMC, the presence of sequence spanning -447 and -400 reduced promoter activity drastically to the almost equal level of promoter less control. This domain between -447 and -400 contained a well-known consensus element, NF1 binding site, located at positions between -447 and -417. DNA-binding studies indicated that nuclear extracts from VSMC specifically produced a retarded band that was supershifted by addition of antibodies against NF1. CONCLUSION: In proliferative VSMC, basal transcriptional activity of the Gadd153 gene was regulated mainly by a nuclear factor NF1 via a negative regulatory element located at positions between -447 and -417, suggesting that NF1 may act as an anti-apoptotic factor of VSMC.
Tumor Necrosis Factor-Alpha Dependent Stimulation of Cyclooxygenase Involves Nitric Oxide
Authors: Haider Asifa , Young Wilson , Ferreri Nicholas Raymond
Published: March 2022

P68

The aim of this study was to establish the interaction of nitric oxide (NO) and cyclooxygenase (COX) isoforms in vascular smooth muscle cells (VSMC) of rat aorta. We hypothesize that increased NO levels derived from inducible nitric oxide synthase (iNOS) after challenge with tumor necrosis factor-alpha (TNF) stimulates COX-2. TNF [1ng/ml] induced a transient increase in VSMC COX-2 and iNOS mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 and iNOS mRNA accumulation were similar after challenge with TNF; mRNA peaked at 2 hr (COX-2: ∼8 fold and iNOS: ∼2 fold) and decreased to control levels by approximately 6 hr. COX-2 and iNOS protein expression also increased after TNF challenge (COX-2: ∼8-fold and iNOS: ∼1.5fold). Stimulation with TNF for 24 hrs increased nitrite levels by 4-fold (control: 5.26±0.37 vs. TNF: 18.33±0.66 pmol/μg protein; p<0.001). Aminogunidine (AG; [1mM]), a specific iNOS inhibitor, abolished this stimulation. TNF-mediated PGI
2
production increased 4.5-fold (control: 120±14.47 vs. TNF: 541±55.05 pg/μg protein; p<0.001) and 2-fold for TXA
2
(control 30.67±4.17 vs. TNF: 59+4.0 pg/μg protein; p<0.01). AG pretreatment attenuated TNF mediated increase in PGI
2
by 45% (TNF: 541±55.05 vs. TNF+AG: 296±29 pg/μg protein; p<0.001) and TXA
2
by 34% (TNF: 59±4.0 vs. TNF+AG: 39±3 pg/μg protein; p<0.01). Sodium Nitroprusside (SNP), an NO donor increased COX-2 protein expression by ∼3 fold after 1 hr of treatment. PGI
2
levels also increased after incubation with SNP (control: 136±4.95 vs. SNP: 188±10.39 pg/μg protein; p<0.01); TXA
2
levels were unaffected. The data indicate, that TNF-mediated NO production in VSMC is iNOS dependent. Moreover, increased NO levels after exposure to TNF stimulates prostanoid production, possibly via a COX-2 dependent mechanism. We conclude that stimulation of the COX pathway by TNF may involve iNOS-mediated increases in NO levels. This interaction may be relevant to the regulation of hyperplasia/hypertrophy/apoptosis in diseases such as arteriosclerorsis and hypertension, as recent evidence has shown that TNF-mediated increases in VSMC proliferation are COX-2 dependent.
Antisense to Both 11β-Hsd1 and 2 Increase Glucocorticoid (GC) Activity in Vascular Tissue
Authors: Souness Graham W , Brem Andrew S , Morris David J
Published: March 2022

P69

GC’s amplify the pressor effects of catecholamines and Ang II in VSM. Thus, the level of vasoconstriction in vascular tissue can be influenced by local GC concentrations. GC’s are metabolized to their inactive 11-dehydro derivatives by the enzyme 11β-HSD which exists in at least two isoforms, 11β-HSD1 and 11β-HSD2. 11β-HSD2 is unidirectional, with only dehydrogenase activity (inactivating GC’s) while 11β-HSD1 is bi-directional, also possessing reductase activity and thus the ability to reactivate 11-dehydroGC’s back to the active parent steroid. The aim of the present study was to investigate the role of each isoform in the regulation of GC metabolism in vascular tissue. Aortic rings (2-3mm), from male SD rats, were incubated for 24 hrs in DMEM media with either corticosterone (B) or 11-dehydrocorticosterone (11-dehydroB)(10
-8
-10
-7
M) ± specific Antisense oligos (20 μg/ring) targeted against rat 11β-HSD1 or 11β-HSD2. The contractile responses to graded doses of phenylephrine (PE; 10
-9
- 10
-6
M)in individual rings were then measured. In aortic rings co-incubated with B and 11β-HSD2 Antisense, the contractile responses to PE were amplified 30-50% versus rings incubated with B + nonsense oligonicleotide (n = 21, p < 0.01). The co-incubation of aortic rings with B + 11β-HSD1 Antisense resulted in a 50% increase in the contractile responses to PE (n = 8, p<0.001). In parallel experiments, 11β-HSD2 Antisense caused a 25% reduction(n =16 aortic rings) and 11β-HSD1 Antisense a 12% reduction (n = 8 aortic rings)in the metabolism of B to 11-dehydroB. These results indicate that 11β-HSD1 and 11β-HSD2 are both important in the metabolism of GC’s in vascular tissue and hence in the regulation of vascular tone. Additionally, rat aortic rings incubated with 11-dehydroB have an increased contractile response to PE versus controls, an effect attenuated by 11β-HSD1 Antisense. This suggests 11-dehydroB is re-activated back to B by 11β-HSD1-reductase making this enzyme a target for possible antihypertensive therapies.
Uupregulation of High Affinity Cyclic AMP Phosphodiesterase (PDE) Genes Following BALloon Catheter Deendothelialization (Bal) in Rat Aorta
Authors: Smith Carolyn J , He Jing , Ding Jia-Zhen , Ricketts Sidonnie , Huston Elaine , Houslay Miles , Moggio Richard A , Stemerman Michael B
Published: March 2022

P75

Inhibitors of PDE 3 and/or PDE4 block smooth muscle cell (SMC) proliferation/migration in vitro and reduce restenosis. To evaluate chronic regulation of SMC PDEs, rats were subjected to BAL. At several times after BAL (30 min to 10days/10D), medial SMC were isolated from the thoracic aortae. Tissues were fractionated for total RNA (analyzed by Northern blots/ RNAase protection) or cytosolic protein (Western blots or low Km [100 nM] cAMP PDE activity). BAL-dependent increases in PDE4B mRNA were biphasic: 2.7-fold at 1hr, which declined by 24hr below control values, followed by 2-3-fold increases by 4-7D (p<0.05 vs controls, n=3-6). PDE4B and c-myc mRNAs were selectively superinduced in cycloheximide-treated rats. Despite modest changes in PDE3 mRNAs, both 80 and 120 kDa PDE3A proteins were detected and only the 80kDa increased 7D post-BAL (10-fold increase, p<0.05, n=6). PDE4B2 (80kDa) persistently increased 2.5-3-fold (p<0.05, n=4-6) from 24 hr to 10D post-BAL, while 2-fold increases in PDE4D3 (90kDa) and PDE4B1 (104kDa) were evident at 24hr or 7D, respectively. PDE activity increased 50-60% (p<0.05; n=4) at 24 hr and 7D after BAL. Thus PDE4B resembles an immediate-early gene. Selective induction of the PDE4 family is associated with 2 waves of SMC proliferation in vivo, the latter of which is also accompanied by induction of PDE3A/80kDa. Upregulation of specific SMC PDEs following angioplasty represents an important response to injury that may be a useful therapeutic target in vascular disease.
P38 MAP Kinase Regulates Vascular Smooth Muscle Cell (VSMC) Collagen Synthesis by Ang II in Shr But not in Wky.
Authors: Touyz Rhian M , He Gang , Wu Xiao-Hua , Mabrouk Mohammed El , Schiffrin Ernesto L
Published: March 2022

P81

Vascular remodeling in hypertension is associated with cell growth and increased deposition of extracellular matrix components, particularly collagen. Molecular mechanisms underlying these processes are unclear, but MAP kinases, particularly ERK1/2 and p38, may be important. We studied the role of ERK1/2 and p38 in vascular growth effects mediated by Ang II in SHR. Cultured mesenteric VSMC from WKY and SHR were used. Phosphorylation of ERK1/2 and p38 was assessed by Western blot using phospho-specific antibodies.
c-fos
mRNA expression was determined by RT-PCR. Ang II-stimulated DNA, protein and proline synthesis were assessed as indices of VSMC proliferation, hypertrophy and collagen production and were determined by measuring incorporation of
3
H-thymidine,
3
H-leucine and
3
H-proline respectively. Ang II dose-and time-dependently increased ERK1/2 and p38 phosphorylation. Responses were increased (p<0.01) 3-4 fold in SHR. PD98059, selective MEK1/2 inhibitor, and SB202190, selective p38 inhibitor, abolished Ang II-induced ERK1/2 and p38 activation respectively. Ang II increased (p<0.05)
c-fos
mRNA expression in SHR (173 ±12 % of control). These actions were inhibited by PD98059, but not by SB202190. Ang II stimulated
3
H-thymidine,
3
H-leucine and
3
H-proline incorporation. Responses were enhanced (p<0.02) 2-3 fold in SHR. PD98059 attenuated Ang II-induced growth effects and normalized responses in SHR. SB212190 did not alter Ang II-elicited DNA synthesis, but reduced (P<0.05) collagen production in SHR. Effects were inhibited by irbesartan, AT
1
receptor antagonist, but not by PD123319, AT
2
receptor blocker. Thus 1)Ang II-mediated activation of vascular ERK1/2 and p38 is increased in SHR, 2) augmented
c-fos
and growth responses are generated primarily via ERK1/2, 3) p38 influences Ang II-induced collagen production in SHR but not in WKY. In conclusion, these results indicate differential roles of ERK1/2 and p38 in AT
1
-stimulated VSMC growth and collagen production, which may contribute to vascular remodeling in genetic hypertension. (Supported by a research grant from Bristol-Myers Squibb/Sanofi)
Mitogenic and Anti-Apoptotic Actions of Hepatocyte Growth Factor Through Erk, Stat3 and Akt in Human Aortic Endothelial Cells
Authors: Nakagami Hironori , Morishita Ryuichi , Yamamoto Kei , Taniyama Yoshiaki , Aoki Motokuni , Kaneda Yasufumi , Horiuchi Masatsugu , Ogihara Toshio
Published: March 2022

P83

Background: Hepatocyte growth factor (HGF), an angiogenic growth factor, may play a pivotal role in the regulation of endothelial cells (EC), since HGF demonstrated the mitogenic and anti-apoptotic actions in endothelial cells. However, it is still unclear how HGF demonstrated those actions. Therefore, in this study, we have focused on the signal transduction systems. Methods: Human aortic EC death was induced by serum-deprived condition. Cell growth was assessed by WST assay, and thymidine incorporation and cell death was measured by LDH release and caspase 3 activity. Phosphorylation of ERK, STAT and Akt was measured by western blotting as assessed by the specific phospho-specific antibody. Results: Treatment of EC with recombinant HGF (rHGF) resulted in a significant increase in cell growth (WST: control; 0.267±0.02, rHGF 100ng/ml; 0.385±0.003, p≤0.01) and also attenuated the cell death (LDH release: control; 0.94±0.04, rHGF 100ng/ml; 0.43±0.024, p≤0.01). ERK and STAT3 (Ser727) was phosphorylated by rHGF (100 ng/ml) as assessed by the specific phospho-specific antibody, while PD98059, the inhibitor of MEK, attenuated endothelial cell growth (HGF; 0.385±0.003, HGF+PD 30uM; 0.203±0.003, p≤0.01) and the phosphorylation of STAT3 (Ser727). On the other hand, STAT3 (Tyr705), which was usually stimulated by JAK, was not phosphorylated by rHGF. In addition, Akt on the down stream of PI3 kinase was also phosphorylated by rHGF, while the specific inhibitor of PI3kinase, wortomanin and LY294002, markedly attenuated endothelial cell death assessed by LDH release and caspase3 activity. Conclusions: Overall, this study demonstrated that HGF stimulated cell growth through ERK cascade including STAT3(Ser727) activation, not JAK-STAT3(Tyr705) pathway, and inhibited endothelial cell death through PI3 kinase-Akt pathway. HGF may stimulate angiogenesis through both mitogenic and anti-cell death function in endothelial cells.
Angiotensin II Enhances Tubuloglomerular Feedback
Via
Activation of Luminal At
1
Receptors on the Macula Densa
Authors: Garvin Jeffrey L , Carretero Oscar A
Published: March 2022

P87

Angiotensin II (Ang II) enhances tubuloglomerular feedback (TGF) through its actions on the afferent arteriole (Af-Art). Recent studies revealed AT
1
-receptors on macula densa cells, raising the possibility that Ang II could enhance TGF by affecting macula densa cell function. We hypothesized that Ang II enhances TGF
via
activation of AT
1
receptors on the luminal membrane of the macula densa. Rabbit Af-Arts and the attached macula densa were simultaneously microperfused in vitro. Af-Art diameter was measured while the macula densa was perfused with low NaCl (Na
+
: 5 mM; Cl
-
: 3 mM) and then with high NaCl (Na
+
: 79 mM; Cl
-
: 77 mM). In the absence of Ang II, Af-Art diameter decreased by 2.4 ± 0.5 μm (from 17.3 ± 1.0 to 14.9 ± 1.2 μm) when the NaCl concentration at the macula densa was increased. After adding Ang II (0.1 nM) to the lumen of the macula densa, the diameter decreased by 3.8 ± 0.7 μm (
p
<0.02 compared to control; n=8). To make sure that this effect was not due to Ang II entering the bath and affecting the Af-Art, we repeated this experiment with losartan in the bath. In the absence of Ang II with losartan (1 μM) in the bath, diameter decreased by 2.7 ± 0.5 μm (from 19.3 ± 1.2 to 16.6 ± 0.8 μm). After Ang II was added to the macula densa perfusate, the TGF response was 4.0 ± 0.7 μm (p<0.01; n=8). This concentration of losartan completely blocked the direct effects of Ang II on the Af-Art. To test whether Ang II enhances TGF
via
activation of AT
1
receptors on the luminal membrane of the macula densa, we compared the TGF response in the presence and absence of Ang II plus losartan in the macula densa perfusate. With losartan in the macula densa perfusate, Af-Art diameter decreased by 2.5 ± 0.5 μm (from 16.7 ± 1.8 to 14.2 ± 2.1 μm). When Ang II was added to the macula densa perfusate with losartan, the TGF response was 2.7 ± 1.1 μm (n.s.). We concluded that Ang II enhances TGF
via
activation of AT
1
receptors on the luminal membrane of the macula densa.
Contribution of EGF-Receptor Tyrosine Kinase to Angiotensin II-Induced Renal Arteriolar Constriction
Authors: Carmines Pamela K
Published: March 2022

P88

Tyrosine kinase activity has been reported to modulate voltage-gated Ca
2+
channels (VGCCs), and has been implicated in both the Ca
2+
mobilization and Ca
2+
influx responses to angiotensin II (AngII). Because renal afferent arteriolar contractile responses to AngII rely on opening of VGCCs while efferent arteriolar AngII responses arise independent of VGCCs, we designed experiments to determine if AngII contracts these arteriolar segments via a mechanism involving tyrosine kinase activation and to examine the role of VGCCs in this phenomenon. Rats were subjected to acute enalaprilat treatment to decrease endogenous AngII formation prior to harvesting tissue for experiments using the
in vitro
blood-perfused juxtamedullary nephron technique. Papillectomy was employed to avoid the indirect afferent arteriolar effect of AngII that arises via increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nM AngII were monitored using videomicroscopic methods before and during inhibition of EGF-receptor tyrosine kinase activity (100 μM tyrphostin A23). Afferent arteriolar baseline diameter averaged 23.5 ± 1.2 μm (
n
= 12) and was unaffected by tyrphostin A23. AngII (10 nM) decreased afferent diameter by 13.7 ± 1.9 μm before and 9.6 ± 1.4 μm during tyrphostin A23 treatment (
n
= 7;
P
< 0.05), indicating 28 ± 8% inhibition of AngII-induced constriction. However, tyrphostin A23 did not suppress afferent arteriolar contractile responses to membrane depolarization (20-55 mM bath K
+
;
n
= 5). Neither baseline diameter nor AngII responsiveness was altered by exposure of afferent arterioles to tyrphostin A1 (inactive analog). Efferent arteriolar baseline diameter averaged 23.6 ± 1.1 μm (
n
= 6) and was not influenced by tyrphostin A23, but this agent evoked a 52 ± 9% inhibition of the efferent arteriolar diameter response to 10 nM AngII. These observations indicate that 1) EGF-receptor tyrosine kinase activation contributes to AngII-induced contraction of both afferent and efferent arterioles, and 2) a direct influence of the EGF-receptor tyrosine kinase on VGCCs is not likely involved in the afferent arteriolar response to AngII.
Angiotensin II Induces Carbon Monoxide Production in Isolated Rat Kidney
Authors: Li Ping , Jiang Houli , Yu ChangHua , Quan Shou , Abraham Nader G , Balazy Michael , Nasjletti Alberto
Published: March 2022

P106

Chronic angiotensin II (Ang II) infusion induces vascular expression of heme oxygenase (HO)-1. HO-dependent synthesis of carbon monoxide (CO) subserves vasodilatory mechanisms. We examined whether Ang II stimulates CO production in isolated rat kidneys perfused at 7 ml/min with oxygenated Krebs buffer. We monitored perfusion pressure (PP) continuously and measured the concentration of CO in the renal venous effluent by gas chromatography/mass spectroscopy. At the conclusion of perfusion for 120 min after the onset of treatment with Ang II or vehicle, the kidneys were processed for measurement of HO activity and isoform expression. In control experiment without experimental interventions, PP and concentration of CO in the venous effluent remained stable throughout the perfusion, ranging between 78 ± 7.7 and 83 ± 5.5, mmHg and 12.3 ± 5.7 and 15.1 ± 6.6 nM, respectively. Inclusion of Ang II (1μM) into the perfusion increased PP by 31.5 ± 14, 43.5 ± 14.5, 47 ± 13, 53 ± 11.2, after 30, 60,90,120 min, respectively. Ang II also increased (p<0.05) the concentration of CO in the venous effluent from 27.1 ± 11.9, to 45.6 ± 11.7, 62.6 ± 16.7, 94.8 ± 20.7, and 101.9 ± 13.1 nM after 30, 60, 90,120 min, respectively. Importantly, the ability of Ang II to increase PP and CO concentration in the effluent were greatly attenuated in the kidneys perfused with AT
1
receptor antagonist, losartan (1 μM) or protein kinase C (PKC) inhibitor, staurosporine (100nM). HO activity in control kidneys (111.03 ± 2.25, pmol/mg/4h) was exceeded by HO activity in kidneys perfused with Ang II (162.40 ± 6.95, p<0.05). These data suggest that Ang II stimulates renal production of CO via a HO dependent pathway that is subject to stimulatory regulation by PKC. The known vasoregulatory actions of CO suggest that it may serve as a counter-regulatory influence to the constrictor actions of Ang II.
Vascular Reactivity to Phenylephrine Is Reciprocally Regulated by Carbon Monoxide and 20-Hydroxyeicosatetraenoic Acid of Vascular Origin
Authors: Kaide Jun-Ichi , Kemp Rowena , Zhang Fan , Jiang Houli , Balazy Michael , Carroll Mairead A , Nasjletti Alberto
Published: March 2022

P107

Carbon monoxide (CO) inhibits CYP-450 enzymes that synthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that enhances vascular reactivity to phenylephrine (PE). This study examines the hypothesis that an interaction between CO and 20-HETE of vascular origin regulates the reactivity of rat renal interlobar arteries (ILA) to PE. ILA incubated (1h) in Krebs buffer released CO into the headspace (95±16 pmol/mg) and manufactured 20-HETE (18±2 ng/mg). Chromium mesoporphyrin (CrMP, 30 μM), an inhibitor of heme oxygenase-dependent CO synthesis, reduced (p<0.05) CO release (29±5 pmol/mg) but not 20-HETE production (15±4 ng/mg). In studies conducted on ILA mounted on a wire-myograph, CrMP caused a leftward shift in the PE concentration-response curve, decreasing the EC
50
(0.27±0.03 to 0.12±0.05 μM, p<0.05) without affecting the maximal response. However, CrMP did not affect any aspect of the PE concentration-response relationship in ILA treated with dibromo-dodecenyl-methylsulfimide (DDMS, 30 μM), a 20-HETE synthesis inhibitor. Like CrMP, exogenous 20-HETE (10 μM) caused a leftward displacement of the PE concentration-response curve, decreasing (p<0.05) the EC
50
from 0.29±0.01 to 0.07±0.01 μM. Superimposed treatment with exogenous CO (1 μM) shifted back to the right the PE concentration-response curve, increasing the EC
50
to 0.31±0.03 μM (p<0.05). Exogenous CO had a similar effect in ILA treated with exogenous 20-HETE and DDMS in combination. These data suggest that CO of vascular origin inhibits vascular reactivity to PE via a mechanism independent of 20-HETE synthesis. However, vascular 20-HETE is a stimulatory regulator of vascular reactivity to PE and thus opposes at a functional level the inhibitory action of CO.
Birth Weight as a Determinant of Ambulatory Blood Pressure Variability in Children and Adolescents
Authors: Lurbe Empar F , Torro Isabel D , Cremades Beatriz R , Alvarez Vicente A , Redon Josep M
Published: March 2022

P114

To study the impact of birth weight (BW), as a crude measure of fetal growth, in the variability of ambulatory BP in children. Six hundred and thirty subjects (369 girls), with a BW ranging 2.3 to 4.8 kg, were included. Ambulatory BP was assessed at the age of 4 to 18 yr during a 24-hour period (Spacelabs 90207). In each subject average and standard deviation of SBP, DBP, HR for 24hour, daytime and nighttime were calculated. The 24hour SBP (mmHg) and variability (mmHg) values for each tertile of BW were 112.7±9.8/10.9±2.7 (BW <3.1 kg), 109.1±8.8/10.4±2.0 (BW 3.1-3.6 kg), 111.8±9.5/10.2±2.0 (BW >3.6 kg). In a multiple regression model, current weight and BW were independent determinants of 24hour SBP variability. At the same 24hour SBP, children with the lowest BW had the highest SBP variability when the parameters were adjusted by age, sex, current height and weight (figure). In conclusion children who had lower BW, not only tended to have the highest BP values but also the highest variability. The increased variability might predispose to hypertension later in life.
Nacl Loading Impairs Endothelium-Dependent Vasodilation in Renal Artery, But not in Brachial Artery, in Salt-Sensitive Patients with Essential Hypertension
Authors: Higashi Yukihito , Sasaki Shota , Nakagawa Keigo , Matsuura Hideo , Kajiyama Goro , Oshima Tetsuya
Published: March 2022

P118

The purpose of this study is to determine whether there was a difference in endothelial function response to NaCl loading between vascular beds in essential hypertensive patients. We evaluated the effects of the intravenous administration of L- and D-arginine (500 mg/kg over 30 min.) on renal hemodynamics in 22 patients with essential hypertension (13 salt-resistant (SR) and 9 salt-sensitive (SS)) and the effects of intraarterial infusion of acetylcholine and isosorbide dinitrate (ISDN) on forearm hemodynamics in 18 patients (10 SR and 8 SS) on each salt diet period (low-salt; NaCl 50 mmol/day, high-salt; NaCl 340 mmol/day, for 1 week, respectively). L-arginine-induced renovascular relaxation was less in SS than in SR during a high salt diet (renal blood flow: 5.6±1.9 vs. 12.4±2.8%; renal vascular resistance: -7.0±2.1 vs. -13.4±2.5%, p<0.05, respectively), but not during a low salt diet. The increase in urine nitraite/nitrate (NOx) excretion evoked by L-arginine was also less in SS than SR during a high salt diet (86±18 % vs. 126±23 %, p<0.05). D-arginine infusion did not change the urine NOx excretion. The forearm blood flow responses to acetylcholine and ISDN were similar in SS and SR during the both salt diets. These findings suggest that renal, but not forearm, endothelial function may be modified by salt loading in SS and this modification may participate in the salt-sensitivity in patients with essential hypertension.
Iron as a Novel Risk Factor for Endothelial Dysfunction in Humans
Authors: Fukami Kei , Matsuoka Hidehiro , Iida Shuji , Satoh Akira , Okuda Seiya , Imaizumi Tsutomu
Published: March 2022

P130

Iron is a transition metal which generates reactive oxygen species (ROS). ROS-induced oxidative stress impairs endothelial function and plays a central role in the pathogenesis of atherosclerosis. To investigate whether body iron store is associated with endothelial dysfunction via oxidative stress, the effects of 1) parenteral iron loading and 2) iron chelation on endothelial function were measured. 1) In healthy volunteers, we administrated therapeutic dose of iron dextran (0.7 mg/kg body weight, i.v.) or vehicle, and examined flow-mediated vasodilation (FMD) of the brachial artery by high resolution ultrasonography, as a non-invasive measure of endothelial function. We also measured plasma levels of malondialdehyde (MDA), as an index of oxidative stress. Iron loading increased plasma levels of iron (from 89±13 to 232±12 mg/dl, p<.0001) and decreased FMD (from 9.2±1.4 to 5.3±1.1 %, p<.0003). Plasma levels of MDA were significantly increased after iron loading (from 2.4±0.2 to 3.1±0.2 nmol/ml, p<.0005) and were positively correlated with plasma iron concentrations (r=.64, p<.005). 2) In smokers without apparent cardiovascular disease, we administrated an iron chelator, deferoxamine (8.3 mg/kg body weight, i.v.) or vehicle, and measured FMD and MDA. Iron chelation by deferoxamine eliminated plasma iron (from 105±16 to 88±11 mg/dl, p<.02) and restored FMD in smokers (from 4.6±0.4 to 7.7±0.7 %, p<.0008), whereas plasma MDA was significantly decreased after deferoxamine (from 2.5±0.2 to 2.2±0.2 nmol/ml, p<.01). In each experiment, vehicle had no effects on vascular function or MDA and endothelium-independent vasodilation by nitroglycerin was not changed. Thus, iron loading exacerbated and iron chelation restored endothelial function paralleled with lipid peroxidation. Our series of experiments suggest that increased body iron stores may be associated with cardiovascular events via ROS-related endothelial dysfunction.
Maternal Influence on the Inheritance of Hypertension
Authors: DeStefano Anita L , Gavras Haralambos , Heard Nancy , Bursztyn Michael , Manolis Athanasios , Farrer Lindsay A , Baldwin Clinton T , Gavras Irene , Schwartz Faina
Published: March 2022

P138

Genetic determinants of essential hypertension remain poorly understood. To date, the focus of molecular genetic research has been primarily on the nuclear genes, whereas the role of the mitochondrial genome has not been ascertained. Although mitochondrial genome is just 16.5 kilobase pairs in length and comprises less than 1% of the total nucleic acid content in mammalian cells, mitochondria occupy pivotal position in human cellular bioenergetics, and mitochondrial defects have been implicated in a wide spectrum of human genetic disorders. In contrast to nuclear inheritance, mitochondrial genome is inherited exclusively through the mother. To assess the relative maternal and paternal contributions to the familial aggregation of hypertension, we examined family history data obtained from 310 hypertensive probands (65 African American, 149 US Caucasian, 96 Greek Caucasian) ascertained without respect to parental hypertension status. Proportions of affected mothers and fathers were compared using a χ
2
test. Cumulative lifetime risk for hypertension in mothers and fathers was estimated using a maximum likelihood extension of Kaplan Meier survival analysis. We found that the proportion of hypertensive mothers was significantly greater than the proportion of hypertensive fathers in all three ethnic groups. The proportions of hypertensive mothers were 83.9, 63.2 and 83.3% for African Americans, US Caucasians and Greek Caucasians, respectively. In contrast, the observed proportions for fathers were, respectively, 53.2, 47.3 and 48.8%. In conclusion, we observe a consistent maternal component in the inheritance of hypertension in three ethnic groups. Although environmental and epigenetic influences can not be excluded, our findings provide a new dimension in the search for the genetic components of hypertension and suggest that hypertension may prove to be not only polygenic but also “polygenomic” disorder. Furthermore, our data indicate that when assessing a patient’s risk for hypertension, particular attention should be paid to the maternal portion of the family history.
Characterization and cADP-Ribose Activation of Reconstituted Ryanodine Receptors from Coronary Arterial Smooth Muscle
Authors: Li Pin-Lan , Tang Wangxian , Zou Ai-Ping , Campbell William B
Published: March 2022

P144

Recent studies indicate that cyclic ADP-ribose (cADPR) serves as a second messenger to mobilize Ca
2+
from sarcoplasmic reticulum (SR) independent of IP
3
. However, the mechanism by which cADPR activates Ca
2+
mobilization is poorly understood. In the present study, ryanodine receptors from SR of coronary arterial smooth muscle cells (CASMCs) were reconstituted into planar lipid bilayer. A unitary Ca
2+
current through this receptor/channel complex was detected at membrane potentials from -40 to +40 mV. Mean slope conductance of this current was 245 ± 4.5 pS in a symmetric cesium methanesulfonate solution. Ryanodine at 0.1-10 μM produced a typical subconductance with increased channel open probability (NP
O
), and 50 μM ryanodine, however, decreased the NP
O
of the channels. These ryanodine effects represent a unique feature of this SR Ca
2+
release channel confirmed in other tissues. Addition of Ca
2+
into bath solution (0.01-10 μM) significantly increased the NP
O
of these SR Ca
2+
channels. cADPR significantly increased the channel NP
0
in a concentration-dependent manner. A 9.7-fold increase in the NP
O
was observed at a concentration of cADPR of 1 μM. This cADPR-induced activation of the SR Ca
2+
channels was completely abolished by 50 μM ryanodine. IP
3
had no effect on the activity of these reconstituted SR Ca
2+
channels even at the highest concentration studied (10 μM). It is concluded that ryanodine receptor/Ca
2+
channels are present in CASMCs and can be reconstituted into planar lipid bilayer. cADPR stimulates Ca
2+
release through the activation of ryanodine receptors on the SR of CASMCs.
Kinase Regulation of Angiotensin II-Mediated Activation of Phospholipase A
2
and Growth in Vascular Smooth Muscle Cells
Authors: Silfani Tonous N , Freeman Ernest J
Published: March 2022

P149

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via multiple signaling mechanisms, including activation of tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, we have reported that cytosolic phospholipase A
2
(cPLA
2
)-dependent release of arachidonic acid (AA) is linked to Ang II-induced VSMC growth independent of tyrosine kinase. Ang II-mediated activation of cPLA
2
involves translocation to the nucleus, as well as serine phosphorylation. Furthermore, VSMC growth stimulated by Ang II has been shown to involve phosphoinositol 3-kinase (PI3K). Thus, Ang II-mediated cPLA
2
activation may require a MAPK-dependent phosphorylation, while additional kinases may couple Ang II to the activation of MAPK. Therefore, we used cultured rat VSMC to examine the role of PI3K and MAPK in the phosphorylation of cPLA
2
, release of AA and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased
3
H-thymidine incorporation and cell number by 82% and 75%, respectively when compared to controls (5976 ± 861 cpm/well; 1.9 ± 0.25 x 10
5
cells/mL, respectively). Ang II increased
3
H-AA release by 145% when compared to controls (411 ± 81 cpm/well). The PI3K inhibitor LY294002 (10 μM) blocked the Ang II-induced thymidine incorporation completely and the release of AA by 66%. Similarly, the MAPK-kinase (MEK) inhibitor PD98059 (10 μM) abolished Ang II-mediated growth and attenuated AA release by 35%. Exogenous AA restored VSMC growth in the presence of PD98059 but not in cells treated with LY294002. Western blot analysis revealed that inhibition of PI3K or MEK reduced the Ang II-mediated increase in phosphorylation of MAPK, as well as the amount of phosphoserine-cPLA
2
associated with the nucleus. Thus, it appears that cPLA
2
-dependent release of AA is required for Ang II-induced growth of VSMC and may involve the PI3K-dependent phosphorylation of MAPK and subsequent phosphorylation-dependent activation of cPLA
2
.
Crystal Structure of the Dimerized Hormone Binding Domain of the Atrial Natriuretic Factor Receptor
Authors: Misono Kunio S , Zhang Xiaolun
Published: March 2022

P150

Atrial natriuretic factor (ANF) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. Such actions of ANF are mediated by the transmembrane ANF receptor coupled to guanylate cyclase. The mechanism of signal transduction by this receptor remains as yet unknown. Toward elucidating the signaling mechanism, we have expressed the extracellular hormone biding domain of the ANF receptor (ANFR-ECD) and have investigated its structure and function. We have found that ANFR-ECD binds ANF at a 1:1 stoichiometry and this binding induces formation of 2:2 complexes, and have identified the bind-site residues by stepwise-affinity labeling combined with site-directed mutagenesis. Here we report the crystal structure of the dimerized hormone binding domain of the receptor at 2.0-angstrom resolution. The monomer molecule contains two subdomains, each consisting of a central β-sheet flanked by α-helices, that are interconnected by three flexible peptide loops. Dimerization occurs through the membrane-proximal subdomain via juxtaposed 2x2 parallel helices, bringing the 2 protruding C-terminal hinge structures in close proximity. The binding-site residues identified by affinity labeling and mutagenesis localize the ANP binding site to the side of each monomer extending to near the dimer interface. A conserved chloride binding site is found in the membrane-distal domain and hormone binding is found to be chloride dependent. These studies suggest mechanisms for hormone activation and possible allosteric regulation of the ANP receptor mediated by chloride.
Flow Cytometric Analysis of At
1a
Receptor Protein Expression in Renal Cortical Endosomes in Angiotensin Ii-Induced Hypertensive Rats
Authors: Zhuo Jia L , Imig John D , Benes Edmund , Raibstein Sheyla R , Hammond Timothy G , Navar L. Gabriel
Published: March 2022

P152

The positive feedback regulation of angiotensinogen and AT
1
receptor expression in renal cortical cells by angiotensin II (Ang II) may play an important role in augmenting intrarenal Ang II formation/accumulation or in enhancing tubular reabsorptive responses in Ang II-dependent hypertension. The aim of the present study was to determine the AT
1A
receptor expression in rat renal cortical endosomes and intermicrovillar clefts during Ang II-induced hypertension using flow cytometry. Adult male Sprague-Dawley rats were treated with either vehicle (n=8) or chronic Ang II infusion via an osmotic minipump (n=8; 80 ng/min, s.c.) for 13 days. Rats receiving chronic Ang II infusion developed hypertension progressively over 12 days (Control: 120 ± 9 mmHg; Ang II: 191 ± 17 mmHg; p<0.05). To quantitate the expression of the AT
1A
receptor in isolated/purified renal cortical endosomes and intermicrovillar clefts, AT
1A
receptor antibody binding curves were performed using a rabbit polyclonal antibody to the cytosolic tail of the AT
1A
receptor. AT
1A
receptor binding was significantly increased by 40%-60% in both renal endosomes (Control: 115.2 ± 5.4 fluorescence units; Ang II: 160.9 ± 17.6 fluorescence units, p<0.05) and intermicrovillar clefts (Control: 26.4 ± 4.8 fluorescence units; Ang II: 44.7 ± 8.6 fluorescence units, p<0.05) in the Ang II-induced hypertensive rats. No significant difference was observed in basolateral membranes between the groups. These results indicate that increased expression of AT
1A
receptors in renal cortical endosomes and intermicrovillar clefts may promote trafficking of Ang II into the intracellular endosomal compartment and enhance tubular reabsorption during Ang II-induced hypertension.
Angiotensin II Signals to Phospholipase D in the Spontaneously Hypertensive and Wistar-Kyoto Rats by Pharmacologically Distinct Mechanisms
Authors: Andresen Bradley T , Jackson Edwin K , Romero Guillermo G
Published: March 2022

P158

Angiotensin II (Ang II) differentially activates phospholipase D (PLD) in vascular smooth muscle from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. We show that the Ang II concentration-response curve with regard to Ang II-induced PLD activity in preglomerular arteriolar smooth muscle cells (RASMCs) is shifted 10-fold to the left (SHR EC
50
= 6.0 x 10
-9
M and WKY EC
50
= 7.2 x 10
-8
M) indicating that PLD activation is hypersensitive to Ang II in SHR RASMCs. We hypothesize that this increase in PLD activity is due to a different signaling cascade in the SHR leading to increased activation of PLD. To examine this hypothesis, we systematically blocked, with pharmacological inhibitors and dominant negatives, all known pathways leading to PLD activation from a G-protein linked receptor (see figure). We observed that PLD activity in both the SHR and WKY RASMCs was inhibited by expression of a Rho A dominant negative, but only PLD activity in the SHR was sensitive to the ARF specific inhibitor Brefeldin A (BFA). Importantly, PLD activation in the SHR and WKY was not sensitive to expression of either an ARF 1 or ARF 6 dominant negative protein. This indicates that there is a different and novel BFA target in the SHR.
Pyk2 Is a Redox-Sensitive Tyrosine Kinase in Vascular Smooth Muscle Cells
Authors: Frank Gerald D , Motley Evangeline D , Inagami Tadashi , Eguchi Satoru
Published: March 2022

P162

PYK2 is a focal adhesion kinase-related protein tyrosine kinase (PTK) and is implicated in downstream mitogen-activated protein kinase activation. In cultured vascular smooth muscle cells (VSMCs), Ca
2+
and/or protein kinase C- (PKC) dependent PYK2 activation by angiotensin II and platelet-derived growth factor has been reported. Because reactive oxygen species (ROS) have been shown to mediate mitogenic signals by these agonists in VSMCs, we hypothesized that PYK2 represents a redox-sensitive PTK used by vascular mitogens. Activation of PYK2 was assessed by three distinct methods: (1) immunoblotting with phospho-specific antibody against autophosphorylated PYK2 at Tyr
402
; (2) immunoprecipitation of PYK2 by anti-PYK2 antibody and immunoblotting with anti-phosphotyrosine antibody; (3) measurement of PYK2’s kinase activity towards the tyrosine kinase specific substrate poly-[Glu
80
Tyr
20
] after immunoprecipitating with anti-PYK2 antibody. In cultured rat VSMCs, H
2
O
2
concentration- and time- dependently induced PYK2 phosphorylation at its autophosphorylation site Tyr
402
. Maximal phosphorylation occurred using 10-20 μM H
2
O
2
at 5-10 min and the phosphorylated protein co-migrated with a major tyrosine-phosphorylated 120 kDa protein induced by H
2
O
2
. Diamide (1 mM), a thiol-oxidizing agent, gave similar results to that of H
2
O
2
. H
2
O
2
also increased tyrosine phosphorylation of PYK2 immunoprecipitated with anti PYK2 antibody. Moreover, treatment of VSMCs with 10 μM H
2
O
2
for 5 min increased PTK activity immunoprecipitated with anti PYK2 antibody. However, 10 μM H
2
O
2
had no effect on intracellular Ca
2+
concentration detected by the fluorescent dye, fura-2 and GF109203X, a PKC inhibitor, failed to inhibit H
2
O
2
-induced PYK2 phosphorylation indicating that ROS-dependent PYK2 activation does not require Ca
2+
or PKC. These data demonstrated for the first time that PYK2 represents a major redox-sensitive PTK in the vessel wall, suggesting its pivotal role in vascular remodeling.
Raising Plasma Fatty Acids Reduces Baroreflex Sensitivity
Authors: Gadegbeku Crystal A , Dhandayuthapani Arachelvi , Sadler Zara E , Egan Brent M
Published: March 2022

P176

We have shown that lipid abnormalities present in obese hypertensives could contribute to their enhanced α
1
-adrenoceptor sensitivity. In this study, we examined if changing lipids with an intralipid/heparin infusion could contribute to impaired baroreflex sensitivity (BRS). To test this hypothesis, we compared the dose of the α
1
-adrenoceptor agonist, phenylephrine, to raise MAP 20 mmHg (PD
20
) before and after an infusion of intralipid/heparin. Also, we determined BRS by the ratio of the change in heart rate recorded in msec over the change in systolic BP in mmHg. We studied six obese hypertensive subjects with a mean age of 40.6 ± 2 and % body fat by DEXA of 30 ± 4%. Raising plasma NEFA levels lowered the PD
20
from a baseline of 1.21 to 0.8 μg/m
2
/min (p=0.05). With raising NEFAs, BRS was decreased from 6.3 ± 0.55 to 3.6 ± 0.42 msec/mmHg (p=0.02)(Δ = baseline measurements, ♦ = intralipid/heparin measurments). These data suggest that acute increases in plasma NEFAs enhance α
1
-adrenoceptor vascular reactivity and reduce BRS which may contribute to the maintenance of elevated BP in patients with obesity hypertension.
Increased At1 Receptors and Giα Expression Contribute to Greater Angiotensin Ii-Mediated Stimulation of Na,H-Exchanger in Proximal Tubules of Hypertensive Obese Rats
Authors: Hussain Tahir , Becker Misti L
Published: March 2022

P184

Angiotensin II (ang II) is a powerful stimulator of sodium and water reabsorption in the proximal tubules (PTs) through the activation of AT1 receptors and subsequent stimulation of sodium transporters, Na,H-exchanger (NHE), Na,K-ATPase and Na/HCO
3
co-transporter. AT1 receptors are linked to adenylyl cyclase via Gi proteins. Ang II is implicated in the development of obesity related hypertension, however the mechanism is unknown. Present study was designed to investigate the effects of ang II on NHE activity, cAMP accumulation in PTs and to measure AT1 receptors and the expression of Giαin brush border membranes (BBM) of obese and lean Zucker rats. Obese rats exhibited higher blood pressure (165±2/114±3 mmHg) when compared with lean rats (133±5/99±2 mmHg), a modest increase (12%) in blood glucose and a 6-fold increase in plasma insulin. Plasma renin activity was similar in lean and obese rats. Ang II (0.01-10 pM) stimulated NHE activity in PTs of lean and obese rats, however the stimulatory response was markedly greater in obese rats. Ang II (0.1-100 pM) inhibited cAMP accumulation in PTs of lean and obese rats, but the inhibitory effect was greater in obese rats. There was no difference in basal and forskolin-mediated stimulation of cAMP between lean and obese rats. [
125
I]sar-ang II binding revealed 100% increase in AT1 receptor binding sites without a change in dissociation constant (Kd) to the ligand in BBM of obese compared to lean rats. Western blot analysis revealed 2-3-fold increase in Giα1&3 in BBM of obese compared to lean rats. We conclude that increased AT1 receptor binding sites and expression of Giαmay be responsible for greater ang II-mediated inhibition of cAMP and subsequent increased stimulation of NHE in PTs of obese rats. This phenomenon of increased stimulation of NHE by ang II may contribute to increased tubular sodium retention and development of hypertension in obese Zucker rats.