bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional adrenoreceptor distribution in arteriolar networks of mouse gluteus maximus muscle
Authors: Moore Alex W. , Jackson William F. , Segal Steven S.
Published: June 2021

Adrenoreceptor (AR) activation governs the magnitude and distribution of exercise hyperemia in accord with heterogeneity of AR subtype distribution. Functional studies in rat cremaster muscle (CM) indicate α1 AR predominate in proximal (1A) arterioles and α2 AR in distal (3A) arterioles yet little is known of arteriolar AR subtype distribution in locomotor muscles, particularly for the mouse. We tested the hypothesis that functional AR subtype exhibits regional heterogeneity in arteriolar networks of gluteus maximus muscle (GM). In anesthetized male C57BL/6J mice (3 mo), concentration‐response curves (1 nM – 1 μM, 0.5 log increments) were performed in superfused GM (35 °C, pH 7.4; n 5/group). Norepinephrine (NE, nonselective α AR agonist) constricted 1A, 2A and 3A with similar potency and efficacy. In contrast to rat CM, phenylephrine (PE; α1 AR agonist) evoked greater (p<0.05) constriction in 3A (inhibited by prazosin, 10 nM; α1 AR antagonist) while UK14304 (UK; α2 AR agonist) evoked greater (p<0.05) constriction in 1A (inhibited by rauwolscine, 0.1 μM; α2 AR antagonist). Isoproterenol (β AR agonist) dilated 1A, 2A, and 3A with similar potency and efficacy; each was inhibited by propanolol (β AR antagonist, 0.1 μM) which otherwise had no effect on responses to NE, PE, or UK. We conclude that functional AR distribution in arteriolar networks of mouse GM is heterogeneous and differs from previous reports. (HL086483)
Flow‐Mediated, Redox‐Dependent AT1R Regulation Determines Collateral Growth Capacity
Authors: Unthank Joseph L. , Miller Steven J. , Labarrere Carlos A.
Published: June 2021

We previously observed flow‐related suppression of AT1R mRNA in collaterals of the SHR (Miller, 2008) which is characterized by oxidative stress, metabolic abnormalities and impaired collateral growth (CG). This study was undertaken to determine the role of flow‐related AT1R regulation in both the promotion of CG in normotensive WKY and CG impairment in SHR.

RT‐PCR demonstrated upregulation (3.0±0.7X) in WKY but suppression in SHR (−2.4±0.5X). Immunostaining for AT1R demonstrated significant upregulation throughout the arterial wall in WKY but not SHR collaterals (see table). Losartan prevented CG and the associated endothelial proliferation in WKY. Captopril but not losartan restored CG in SHR and prevented AT1R down‐regulation. Captopril also restored normal p22phox expression in SHR. We propose that AT1R upregulation and stretch‐dependent AT1R activation is induced by elevated shear stress in WKY but prevented by oxidative stress in SHR.

Number of cells in each wall layer with significant immunoreactivity, as percent of total cells. A remarkable increase is observed in all wall layers of the WKY collateral.

% Cells with AT1R Immunoreactivity

Intima
Media
Adventitia

WKY‐Control
8±2.0
10±2.1
10±1.2

WKY‐Collateral
45±5.6*
74±8.2*
43±8.2*

SHR‐Control
6±0.6
9±1.1
11±2.1

SHR‐Collateral
3±0.4
9±1.8
3±2.6*

N=3

p≤0.05.

Supported by HL42898.
The ZDSD, a New Rat Model of Metabolic Syndrome: Altered Arterial Muscle Mechanics
Authors: Packer C Subah , Boice Alexa M , Meulen Leeanna M , Coy Kathy , Peterson Richard G
Published: June 2021

The goal was to determine if altered arterial muscle contractility or compliance plays a role in cardiovascular disease reported for ZDSD. ZDSD and control SD systemic BP were not different from 18 to 24 weeks of age. At the end of the study period, ZDSD were found to have larger hearts but lower heart/body weight ratios indicating that increase in heart size did not keep pace with body mass increase. ZDSD had elevated right/left ventricular weight ratios (RV/LV+S) indicating right heart hypertrophy and pulmonary hypertension. ZDSD and SD caudal arterial muscle resting length‐tension (L‐T) curves were similar indicating no change in tissue compliance. The ZDSD active L‐T curve was elevated above the SD active L‐T curve indicating that ZDSD arterial muscle is more contractile. Hypercontractile arterial muscle could be causally related to systemic hypertension. Systemic arterial muscle hypercontractility, enlarged hearts, low heart/body weight, pulmonary hypertension, trend towards systemic hypotension are consistent with an initial phase of diabetes‐associated systemic hypertension followed by cardiac insufficiency and congestive heart failure in ZDSD.

Supported by PreClinOmics. A Boice and L Meulen were 2009 Indy Project SEED Scholars supported by American Chemical Society (ACS), Lilly, DowAgroSciences, IUPUI Indiana Clinical and Translational Sciences Institute (CTSI)
Niacin reduces renal damage and insulin insensitivity in Charles River Obese SHR (SHROB/cp): a model of the metabolic syndrome
Authors: Kang Youn‐Jung , Manero Tabitha , Levine Seymour , Owens Dwight , Chander Praveen , Stier Charles
Published: June 2021

Niacin is a hypolipemic drug which lowers triglyceride levels. We examined the effect of niacin (5% in chow) from 8 to 15 weeks of age on renal damage in SHROB/cp from Charles River Laboratories. SHROB/cp (n=4) exhibited increased body weight, urinary protein excretion (UPE), focal segmental glomerulosclerosis (FSGS) and plasma insulin as compared with lean control rats SHROB/kol (n=5, Table). Plasma triglyceride (605 ± 101 v 140 ± 12 mg %) and blood glucose (147 ± 21 v 114 ± 4 mg %) levels were also greater in SHROB/cp than SHROB/kol, respectively. Niacin (n=5) markedly reduced UPE, FSGS and plasma insulin while blood glucose was unaffected. Niacin strikingly reduced renal inflammation and tubular scarring. SBP did not differ between SHROB/kol and SHROB/cp and was unaffected by niacin treatment of SHROB/cp. Niacin reduced hepatic steatosis and plasma 8‐isoprostane levels in SHROB/cp.

SHROB/kol
SHROB/cp
SHROB/cp &Niacin

Body Weight (g)
349±15
462±15 **
418±10 **†

UPE (mg/day)
19±3
225±45 **
48±12 ††

SBP (mmHg)
211±4
199±5
192±4 *

FSGS (%)
0.5±0.2
16.6±5.9 **
1.4±0.4 ††

Plasma Insulin (ng/ml)
5.9±2.1
76.1±9.1 **
26.5±7 *††

Plasma Isoprostane (pg/ml)
8.4±0.6
27±10 **
10.4±0.8 ††

P≤.05

P≤.01 v SHROB/kol;

P≤.05

P≤.01 v SHROB/cp

In conclusion, niacin may be of particular benefit in combating renal damage in the metabolic syndrome and diminished oxidative stress may be an important mechanism for this effect.
Telmisartan improves cognitive function through the increase in neurotrophic factor and inhibition of apoptosis in hippocampus of rats with metabolic syndrome
Authors: Kishi Takuya , Hirooka Yoshitaka , Sunagawa Kenji
Published: June 2021

Background

In metabolic syndrome (MetS), impaired cognitive function has been indicated. We investigated whether telmisartan, an AT1 receptor blocker, improves cognitive function of MetS.

Methods and Results

Male Sprague‐Dawley rats were fed on a high‐fat diet and segregated into obesity‐prone (OP) showed a MetS profile with hypertension and obesity‐resistant (OR) after 13 weeks. OP was treated either with telmisartan (TLM‐OP) or hydralazine (HYD‐OP) for 12 weeks. Morris water maze test, a cognitive function test determined by latency (the shorter the better) to reach platform, was significantly longer in OP than in OR (42±3 sec vs. 29±2 sec, n=4, P<0.01), and significantly shorter in TLM‐OP than in HYD‐OP despite of similar blood pressure. Brain‐derived neurotrophic factor (BDNF) in hippocampus was significantly lower in OP than in OR, and significantly higher in TLM‐OP than in HYD‐OP. Apoptosis in hippocampus was significantly higher in OP than in OR, and significantly lower in TLM‐OP than in HYD‐OP.

Conclusion

Telmisartan improves cognitive function through the increase in BDNF and inhibition of apoptosis in hippocampus in rats with MetS independent of blood pressure lowering effect.
Cerebrovascular reactivity after experimental subarachnoid hemorrhage (SAH) in insulin resistant (IR) rats
Authors: Institoris Adam , Domoki Ferenc , Bari Ferenc , Busija David W.
Published: June 2021

IR impairs cerebrovascular reactions to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after SAH have not been described in IR. Hemolyzed blood (300μl) or saline was infused (10μl/min) into the cisterna magna of 11–13 week‐old ZO (n=15) and lean (ZL) rats (n=15). One day later, dilator responses of the basilar artery (BA) and its side branch (SB) to acetylcholine (ACh)(10−6M), cromakalim (10−7M, 10−6M) and sodium nitroprusside (SNP)(10−7M) were recorded with intravital videomicroscopy. Saline injected ZO animals showed reduced dilation to ACh (BA=7±4% vs. 21±5%; SB=20±5% vs. 37±8%) compared to ZL rats. Blood injection blunted the response to ACh in both the ZO (BA=4±3%; SB=11±3%) and ZL rats (BA=7±2%; SB=16±4%). Cromakalim (10−6M)‐induced dilation was significantly reduced both in the blood injected ZO animals compared to the saline control (BA=11±3% vs. 27±5%; SB=23±7% vs. 43±11%), and in the blood injected ZL rats vs. its saline control (BA=24±4% vs. 29±3%; SB=39±3% vs. 58±9%). No difference in SNP reactivity was observed. In summary, cerebrovascular reactivity to both endothelium‐ and smooth muscle‐dependent stimuli is severely compromised by SAH in IR animals.
Inhibition of PPARγ during pregnancy causes inward remodeling of brain parenchymal arterioles
Authors: Cipolla Marilyn J. , Chan Siu‐Lung , Chapman Abbie C. , Godfrey Julie A.
Published: June 2021

We previously showed that pregnancy causes outward remodeling of brain parenchymal arterioles (PA) and decreased small vessel resistance in the maternal brain, an effect that could contribute to hemorrhage and edema during conditions such as eclampsia. In the present study, we hypothesized that the mechanism by which pregnancy causes outward remodeling is through activation of PPARγ, a ligand activated transcription factor expressed in vascular cells and shown to be highly activated during pregnancy. Female SD rats were either nonpregnant (NP;n=7), late‐pregnant (LP;d20;n=7) or LP and treated with the PPARγ inhibitor GW9662 (10mg/kg/day) in food for the last 10 days of gestation (LP+GW;n=8). Brain PA were dissected, mounted in an arteriograph chamber and treated with papaverine (0.1mM) and diltiazem (10−5M) for measurements of passive diameter and wall thickness at pressures from 5–200 mmHg. Pregnancy caused significant outward hypotrophic remodeling of PA compared to NP, an effect that was reversed by inhibition of PPARγ. Lumen diameter and wall thickness of PA from NP, LP and LP+GW at 40 mmHg was (μm): 78±3 and 8.6±0.5, 92±4 and 6.6±0.4 (p<0.05 vs. NP and LP+GW), and 80±5 and 8.8±0.5 (ns vs. NP;p<0.05 vs. LP). There were no differences in outer diameter between groups. These results suggest that the underlying mechanism by which pregnancy causes outward hypotrophic remodeling of PA is due to PPARγ activation.
Akt/Bcl‐2 signaling is critical for anti‐apoptotic effects of carbon monoxide (CO) against TNF‐alpha‐mediated inflammatory damage in cerebral vascular endothelial cells.
Authors: Basuroy Shyamali , Bhattacharya Sujoy , Leffler Charles W. , Parfenova Helena
Published: June 2021

The pro‐inflammatory cytokine TNF‐alpha causes oxidative stress and apoptosis in cerebral vascular endothelium. CO, an endogenous gaseous mediator, provides cytoprotection from inflammatory endothelial damage. Phosphorylation signaling via Protein Kinase B/Akt and the anti‐apoptotic protein Bcl‐2 is involved in various cell survival mechanisms. We addressed the hypothesis that Akt/Bcl‐2 signaling mediates anti‐apoptotic effects of CO and promotes endothelial cell survival during brain inflammatory disease. In primary cultures of cerebral microvascular endothelial cells from newborn pigs, TNF‐alpha (15 ng/ml) caused transient phosphorylation of Akt at Ser473 (5–10 min) and increased Bcl‐2 protein (3h). Triciribine, an Akt phosphorylation inhibitor, or Akt siRNA knockdown potentiated TNF‐evoked DNA fragmentation and blocked the anti‐apoptotic capacity of a CO‐releasing molecule (CORM‐A1), indicating Akt is essential for endothelial survival and is required for COmediated cytoprotection during inflammation. Triciribine reduced CO‐evoked increase in Bcl‐2 protein suggesting Akt is an upstream regulator of Bcl‐2. Bcl‐2 inhibitors (gossipol, Bcl2‐I, and HA14‐1) blocked the anti‐apoptotic capacity of CORM‐A1. These data indicate Akt/Bcl‐2 signaling is critical for CO‐mediated cytoprotection against TNF‐alpha‐mediated inflammatory damage in cerebral vascular endothelium.

Grant support: NIH/NINDS and NIH/NHLBI. S. Basuroy is supported by the Beginning‐Grant‐in‐Aid from AHA.
Cyclosporine upregulates KLF4 in vascular smooth muscle cells and drives phenotypic modulation in vivo
Authors: Sinden Daniel Stephen , Garvey Sean M , Rose Dominique L , Bortz Pamela D Schoppee , Lee Monica Y , Wamhoff Brian R
Published: June 2021

Cyclosporine‐A (CSA, a calcineurin inhibitor) is a fungus‐derived immunosuppressant that blocks proliferation of lymphocytes. CSA has also been shown to block both vascular smooth muscle cell (SMC) proliferation in culture and vessel neointimal formation following injury in vivo. The molecular and pathological effects of CSA on SMCs are not fully understood. We show here that CSA increased SMC expression of Krüppel like factor 4 (KLF4) mRNA and protein. KLF4 is a transcription factor that regulates SMC phenotype. KLF4 inhibits proliferation, promotes migration, and suppresses SMC differentiation marker genes. We show that CSA suppressed SMC differentiation markers SM α‐actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD), but increased cyclin‐dependent kinase inhibitor‐1A (CDKN1A) & matrix metalloproteinase‐3 (MMP3). CSA treatment of rat carotid arteries in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, & SM myosin heavy chain (MYH11) mRNA. Conversely, tumor suppressor genes KLF4, p53, & CDKN1A were increased, as well as MMP3, MMP9, & collagen‐VIII (COL8). CSA‐treated arteries showed breakdown of the internal elastic lamina and pathological reorientation of SMCs. KLF4 immunostaining in SMCs and endothelial cells was increased. In conclusion, CSA increases KLF4 expression and promotes SMC phenotypic modulation in vivo. Sources of support: APS UGSRF, APS post‐doc, NIH HL81682, AHA SDG.
In P14 rat middle cerebral arteries relations between myogenic tone and cytosolic Ca++ are enhanced by myofilament Ca++ sensitization and increased Ca++ influx, but attenuated by high proportions of non‐contractile smooth muscle cells.
Authors: Charles Shelton M , Zhang Lubo , Cipolla Marilyn J , Buchholz John N , Pearce William J
Published: June 2021

To test the hypothesis that relations between myogenic tone and cytosolic Ca++ are enhanced by myofilament Ca++ sensitization but attenuated by high proportions of non‐contractile smooth muscle cells, in immature compared to mature rat cerebral arteries, three protocols were performed in middle cerebral arteries (MCA) from P14 and adult rats. Myofilament Ca2+ sensitivity in β‐escin permeabilized arteries increased with pressure in P14, but not adult, MCA. Cyclopiazonic acid (a release inhibitor) increased diameter and reduced Ca++ in adult MCA, but increased diameter with no change in Ca++ in the P14 MCA. La3+ (an influx inhibitor) increased diameter and decreased Ca++ in adult MCA, but in P14 MCA diameter increased without any significant decline in Ca++. With La3+ and CPA, diameter was passive in adult and P14 MCA, but Ca++ declined only in adult MCA. Colocalization between α‐actin and SM2 myosin was >2‐fold greater in adult than in pup MCA.. These data suggest that compared to adult MCA, pup MCA rely more on myofilament Ca++ sensitization and Ca++ influx to maintain myogenic reactivity. The inability of La3+ to reduce cytosolic Ca++ in the pup MCA appears due to La3+‐insensitive non‐contractile smooth muscle cells that contribute to the spatially averaged measurements of Ca++ but not contraction. Supported by USPHS grant HL54120, HD31266, and HL64867.
Phenotypic differences between neonatal and adult ovine carotid arteries predict reactivity to chronic hypoxia and VEGF
Authors: Adeoye Olayemi O. , Butler Stacy M. , Abrassart Jenna M. , Mock Elizabeth A. , Williams James M. , Pearce William J.
Published: June 2021

Double label confocal studies of smooth muscle α‐actin and SM2 myosin indicate significantly greater colocalization between these markers in adult than neonatal arteries revealing that the fraction of non‐contractile smooth muscle cells is greater in immature than mature arteries. To explore the hypothesis that age‐related differences in phenotype determine responses to chronic hypoxia, and VEGF, which mediates many vascular effects of hypoxia, common carotid arteries were harvested from term fetal and adult non‐pregnant sheep maintained at sea level (FN, AN) or for 110 days at 3820m (FH, AH). Stress‐strain relations were determined in all arteries before organ culture, after 48h of serum starvation (S), or after 24h starvation + 24h treatment with 3 ng/ml VEGF (V). Aside from multiple effects of hypoxia that were similar in both age groups, S and V produced opposing effects in AN (S‐induced increases in diameter were reversed by V) but not AH arteries. Opposing effects of S and V were also observed in FH (S‐induced decreases in stiffness, diameter, maximum myogenic and depolarization‐induced tone were all reversed by VEGF) but not in FN. These results demonstrate that age‐dependent differences in smooth muscle phenotype predict reactivity to both VEGF and chronic hypoxia, and that VEGF modulates multiple functional characteristics of arterial smooth muscle. Supported by USPHS Grants HL54120, HD31266, & HL64867.
Gender specific regulation of hepatic aquaglyceroporin 9 and glycerol kinase expression
Authors: Lebeck Janne , Lund Sten A. , Praetorius Jeppe
Published: June 2021

Increased hepatic glucose production plays a pivotal role in the pathophysiology of diabetes. AQP9 has been suggested to facilitate the transport of glycerol into hepatocytes where glycerol kinase (GK) catalyses the initial step for its conversion into either glucose or glycerolipids. Hence, the regulation of AQP9 and thereby glycerol uptake could be a potential modulator of hepatic glucose production. This study aims to investigate the role of gender in regulation of hepatic AQP9 and GK protein. Immunoblotting showed that starvation of male rats resulted in increased hepatic AQP9 and GK abundance, whereas, starved female rats displayed no increase in AQP9 or GK. In contrast, plasma glycerol levels were doubled in female rats in response to starvation, whereas no statistically significant changes were found in male rats. Interestingly, immunoblotting revealed that castration of male rats resulted in a 1.5 fold increase in hepatic AQP9 expression, but a decrease in GK abundance. This was accompanied by a marked reduction in plasma glycerol together with a small but significant increase in plasma glucose when compared with controls. We conclude that sexual dimorphism exists in hepatic glycerol handling in response to starvation and that castration of male rats has a diverse effect upon liver abundance of AQP9 and GK.

Supported by the Danish National Research Foundation
Synaptic transmission in the inferior mesenteric ganglion of hypertensive and normotensive rats
Authors: Houchin Timothy , Kreulen David
Published: June 2021

The inferior mesenteric ganglion (IMG) is a prevertebral sympathetic ganglion that innervates the lower abdominal and pelvic viscera. While extensive studies have been performed on the IMG of several species, there is very little information available concerning the rat IMG. Because of the importance of the mesenteric circulation in the regulation of systemic blood pressure and in the genesis of salt‐sensitive hypertension, we compared the properties and response profiles to pre‐ganglionic input of IMG neurons in normotensive (NT) and hypertensive (HT) animals using intracellular recording in vitro. All rats were uninephrectomized 4 weeks prior to experiments. HT rats received 200mg/kg of deoxycorticosterone s.q. and 1%NaCl/0.2%KCl in drinking water. NT animals (mean arterial pressure < 130mmHg) received tap water. Resting membrane potential, rheobasic current, action potential amplitude, input resistance, and membrane time constant show no significant differences between NT and HT animals. In contrast to the findings in the IMG of other species, nearly all neurons (94% in HT, 91% in NT rats) showed a phasic response to direct current injection. This is the first study of the IMG of the hypertensive rat. While there is a predominance of phasic neurons in this ganglion, changes in sympathetic outflow that occur with hypertension are not due to alterations in the basic properties of the ganglionic neurons. Supported by NIH grant HL70687
Involvement of activation of Janus kinase (JAK)2 in the mechanisms utilized by Norepinephrine (NE) to cause hypertension.
Authors: Al‐Azawi Hind , Proctor Daniel , Elhout Dina , Brands Michael W. , Banes‐Berceli Amy K.L.
Published: June 2021

Angiotensin II (ANG II) utilizes activation of JAK2 signaling pathways to cause hypertension. It is unknown if NE is dependent on JAK2 pathway activation to increase in blood pressure. We hypothesized that NE requires JAK2 activation to increase blood pressure. Male Sprague‐Dawley rats were catheterized via abdominal aorta and femoral vein to chronically measure blood pressure and infuse drugs. NE and ANG II were infused for 18 days and tissues harvested for western blot analysis. Mean arterial pressure (MAP) increased significantly in the ANG II and NE infused rats. There was a significant increase in phosphorylation levels of JAK2 as measured by Western blot analysis in the ANG II (10 ng/kg/min) and NE (2.8 mg/kg/day) treated rats than the saline controls. Infusion of JAK2 inhibitor AG490 (10 ng/kg/min) did not inhibit initial NE‐induced increases in blood pressure but did reduce NE's ability to maintain sustained increases in MAP. To determine if NE also requires a vascular component that involves JAK2 activation we utilized ex vivo myograph recordings. Using the thoracic aorta from naïve male rats we tested whether NE requires JAK2 activation for vascular contraction. We found that NE‐induced contraction was not significantly reduced by treatment with the JAK2 inhibitor AG490. These data suggest that NE and ANG II both increase and utilize JAK2 activation but via different mechanisms. (ABB NHLBI R00; MWB NHLBI R01).
The relationship between blood pressure and arterial characteristics in clinically normotensive children
Authors: Phillips Aaron A , Peralta‐Huertas Jose , Fitzgibbon Laura K , Wade Terrance , Cairney Jon , O'Leary Deborah
Published: June 2021

Atherosclerosis is the major cause of cardiovascular disease. In adults, elevated blood pressure (BP) is recognized as a major risk factor for atherosclerotic progression. In children the relationship between elevated BP and atherosclerosis progression is less understood. The objective of this study was to examine differences in central arterial distensibility (DIST) and intima‐media thickness (IMT) as well as systemic pulse transit time (PTT) in normal blood pressure (NBP) and elevated blood pressure (EBP) children. The sample consisted of 104 children (21 EBP, 83 NBP) aged 11–14 years. Blood pressure was clinically normal (on two separate occasions); however, the EBP had a significantly elevated BP in comparison to their NBP counterparts. We assessed PTT, IMT, and DIST using applanation tonometry, ultrasonography of the common carotid artery, electrocardiography, and toe pulse oximetry. No significant differences were found between NBP and EBP for IMT (0.42(0.06) vs. 0.43(0.05) mm, respectively) or DIST (65.3(27.6) kPa−1x10−3 vs. 62.7(21.2) kPa−1x10−3, respectively). PTT was significantly elevated in the EBP group (341(23) ms) vs. the NBP (321(25) ms) group. Children 11–14 years of age with elevated BP do not show central arterial changes yet systemic vascular tone appears to be elevated. Heart and Stroke Foundation of Canada
Pregnancy and Pseudopregnancy: Physiological States with High Endogenous Ouabain, Coexistent Suppression of Maternal Arterial Myocyte NCX and α2 Na+ Pumps, and Resistance to Ouabain‐Induced Hypertension
Authors: Jacobs Brandiese E , Liu Yong , Zulian Alessandra M , Golovina Vera A , Hamlyn John M
Published: June 2021

Endogenous ouabain (EO) is elevated and correlates with blood pressure (BP) in ~50% of patients with essential hypertension. “Ouabain‐like” inhibitors are increased in pre‐eclampsia (PE) and correlate with BP. EO and ouabain inhibit the α2‐Na+ pump isoform in arterial myocytes, enhance Ca2+ entry via NCX1.3, and raise BP. We explored whether EO per se is elevated in pregnancy and pseudopregnancy (PSP) and determined whether exogenous ouabain induces PE in otherwise normal pregnant rats. We implanted mini‐osmotic pumps containing ouabain in pregnant (E10), non‐pregnant, and PSP rats and measured BP by tail cuff. At E20 or equivalent, blood, adrenals, and aortas were collected to determine ouabain (RIA), ouabain‐like materials (RRA), NCX, and α2‐Na+ pump expression (Western blot). Plasma EO increased in pregnant (4.7 ± 1.9 nM plasma equiv) vs non‐pregnant (0.3 ± 0.02 nM plasma equiv, p<0.001) rats and was confirmed by HPLC‐RIA and mass spectrometry. Adrenal EO content was lower in pregnant (12.3 ± 5.0 nmoles/kg) vs non‐pregnant rats (32.5 ± 15.3 nmoles/kg, p<0.01). NCX and α2‐Na+ pump expression decreased in pregnancy. Ouabain infusion raised BP in non‐pregnant rats (+21.3 mmHg, p<0.01), but had no effect on BP in their pregnant or PSP counterparts. These data show: 1) that the pregnant and PSP conditions are physiologically high EO states; 2) both conditions are associated with the development of a physiological resistance to ouabain that prevents BP from increasing. 3) The mechanism of the ouabain resistance is exclusively maternal, and likely related to decreased expression of, and reduced Ca2+ entry by, vascular NCX. Failure of the maternal circulation to develop resistance to EO may underlie hypertension in PE.
Endothelin‐1 and the Metalloproteases ECE and NEP in DOCA‐Salt Hypertensive Rats
Authors: Nelissen Jelly , Lemkens Pieter , Debets Jacques , van Essen Helma , Bost Jet , Schiffers Paul , De Mey Jo , Janssen Ben
Published: June 2021

Endothelin‐1 (ET‐1) is thought to be a key player in DOCA‐salt induced hypertension. We hypothesized that the bioavailability of ET‐1 is controlled by tissue specific expression of the metalloproteases endothelin converting enzyme (ECE) and neutral endopeptidase (NEP). To test this hypothesis we measured ECE and NEP protein content in kidneys and hearts of DOCA‐salt treated rats 3 and 6 weeks after implantation of a 100 mg DOCA pellet combined with high salt drinking water. In addition, ET‐1 content was determined in these organs by RIA. During DOCA‐salt treatment, blood pressure was significantly elevated in association with increased kidney and heart weight. Despite the occurrence of structural damage in these organs (verified by histology), ECE, NEP, and ET‐1 content were not significantly altered 3 and 6 weeks after DOCA treatment (n=6/organ/group). These data indicate that both ET‐1 and metalloprotease content in the heart and kidneys do not contribute to the maintenance of hypertension in the DOCA‐salt rat model.

This research was performed within the framework of project T2‐108 of the Dutch Top Institute Pharma.
Preventive effects of exercise training on dexamethasone‐induced hypertension, oxidative stress and peripheral insulin resistance
Authors: Amaral Sandra Lia , Perez Otavio Andre Brogin , Barel Matheus , Bechara Luiz Roberto , Tanaka Leonardo Y , Dionísio Thiago José , de Andrade Louzada Juliana Cavalcante , Dionísio Evandro José , Viscelli Bruno Alvares , Martuscelli Aline Mio , Bosqueiro José Roberto , Santos Carlos Ferreira , Ramires Paulo Rizzo
Published: June 2021

This study investigated whether exercise training (T) could prevent the insulin resistance, oxidative stress and hypertension induced by dexamethasone (Dexa) treatment. Rats underwent a T period where they were either submitted to a running protocol (8weeks) or kept sedentary. After this T period, the animals underwent a Dexa treatment (1mg/kg/day, i.p., 10days), concomitant with training. An ipGTT was performed at the end of the experimental period and the area under the glucose curve (AUC) was calculated. Arterial pressure (AP) was measured. Reduced/oxidized glutathione ratio (GSH/GSSG) and lipid peroxidation were also determined in the skeletal muscle. Western blot analysis was performed to identify PKCα protein expression in the tibialis anterior (TA) muscle. Dexa increased AP (23 mmHg) and T attenuated this increase by 11%. Dexa decreased the GSH/GSSG ratio by 44% and increased the lipid peroxidation by 55% in sedentary groups, and T blocked these alterations. Dexa reduced the PKC‐α protein expression by 45%, which was prevented by T. Accordingly, T attenuated the increases in the AUC, whose values were 39% higher in sedentary ones. Therefore T is effective in regulating physiological antioxidant systems and glucose uptake, which could contribute to prevent hypertension and insulin resistance induced by Dexa.
Development of auditory deficits in a rat model of placental insufficiency
Authors: Gilbert Jeffrey S. , Soldner Emma L.B. , Fitzakerley Janet L.
Published: June 2021

Growth restricted infants are at increased risk of auditory dysfunction, although the cause of these acoustic deficits is not understood. In the present study, a well‐established surgical model of chronic placental insufficiency was used to test the hypothesis that prenatal growth restriction causes cochlear and/or central auditory system dysfunction. The hearing of the offspring of normal pregnant (NP) and reduced uterine perfusion pressure (RUPP) rats was evaluated at 4 and 8 postnatal weeks of age using the auditory brainstem response (ABR) technique. 4‐week‐old RUPP and NP offspring had similar thresholds at all frequencies tested between 1 to 32 kHz. At 8 weeks of age, male RUPP rats developed a mild to moderate hearing loss (15–30 dB) at several frequencies, while the thresholds of female RUPP rats were unchanged. Analysis of interpeak intervals did not indicate any peripheral or central processing changes in the RUPP animals at either age. The delay in the onset of the cochlear deficits may indicate that the hearing loss observed in male RUPP rats is subsequent to other perinatal changes, such as the development of hypertension or glucose intolerance.
The effect of renal perfusion pressure on renal injury in hyperglycemic AngII hypertensive rats
Authors: Jin Chunhua , Polichnowski Aaron , Ohsaki Yusuke , Cowley Allen W.
Published: June 2021

Hypertension and diabetes often co‐exist and are the leading causes of end stage renal disease. The contribution of renal perfusion pressure (RPP) to diabetic nephropathy was explored in the present study by comparing the left kidney, with RPP chronically servocontrolled (SC), to the right uncontrolled (UC) kidney. Diabetes was induced with a single i.p. injection of streptozotocin (STZ; 65 mg/kg) in male Sprague Dawley rats at 7 wks of age. At 12 wks of age, rats (n=4) were surgically prepared and hypertension produced by i.v. AngII (25 ng/kg/min) + 4% NaCl diet for 14 days then anesthetized for bilateral ureteral urine collection. Over the 14 days of AngII infusion, RPP averaged 173±7 mmHg to the UC kidney and 112±3 mmHg to the SC kidney with plasma glucose 353±45 mg/dL; cholesterol 76±6 mg/dL; triglycerides 146±11 mg/dL. Urinary albumin and protein excretion were significantly greater in the UC kidneys compared to SC kidneys (61.6±14 vs 7.9±5 ug/min; 200±50 vs 49±20 ug/min p<0.05). In a group of non‐STZ AngII + 4% NaCl diet treated rats which reached the same levels of hypertension, albumin excretion averaged 15.4±1.7 ug/min from the UC kidney vs 5.3±0.9 ug/min from the SC kidney. Together, these data indicate that hyperglycemia significantly enhances the susceptibility of the kidney to pressure‐induced injury. (HL‐081091)
Caveolin‐1 Phosphorylation Negatively Regulates eNOS Activity in Endothelial Cells
Authors: Chen Zhenlong , Liu Guoquan , Bakhshi Farnaz , Place Aaron T , Sharma Tiffany , Skidgel Randal A , Malik Asrar B , Minshall Richard D
Published: June 2021

Endothelial nitric oxide synthase (eNOS) activity is regulated by phosphorylation, Ca2+ ‐calmodulin, and interaction with caveolin‐1 (Cav‐1). However, the mechanism by which Cav‐1 negatively regulates eNOS activity following stimulation is not clear. Here, we examined the kinetics of eNOS activation (phosphorylation and NO production) and binding to Cav‐1 in endothelial cells and mouse lung homogenates following addition of Ca2+ ionophore A23187. Ca2+increased Src, Akt, Cav‐1, and eNOS phosphorylation and NO production within minutes which returned to baseline levels within 30 min. We observed an increase in eNOS association (co‐IP) with Cav‐1 following stimulation which was blocked by NOS inhibitor L‐NAME and Src inhibitor PP2. Furthermore, siRNA‐mediated knockdown of Cav‐1 potentiated eNOS phosphorylation whereas depletion of eNOS blocked the increase in Src activity and Cav‐1 phosphorylation. FRET between Cav‐1‐YFP and eNOS‐CFP in co‐transfected CHO cells significantly increased after stimulation. Moreover, in co‐transfected HEK293 cells, WT and phospho‐mimicking S1179D‐ eNOS but not phospho‐defective S1179A‐ eNOS co‐IP'd with WT and phospho‐mimicking Y14D‐Cav‐1 but not phospho‐defective Y14F‐Cav‐1 mutant. These data suggest that Ca2+‐dependent activation of eNOS induces Cav‐1 phosphorylation via Src which in turn binds to eNOS and negatively regulates its activity. Supported by NIH HL071626 and HL060678.
Macrophage Nitrite Uptake and Release
Authors: Kaimootil Sunil , Newell Clayton , Tran Tuan , Rodriguez Juan
Published: June 2021

Activated macrophages are known to produce large concentrations of nitrite from the synthesis of nitric oxide. With the recent discovery that nitrite is a biologically active substance, one question that gains more significance is how macrophages hold the nitrite they produce. In this respect, the question of how quickly macrophages internalize or externalize the anion becomes of relevance. Here we report on experiments that characterize the kinetics of nitrite uptake into, and release from the murine J774 macrophage cell line. Cells were exposed to 100 μM of 15N‐labeled nitrite and intracellular or extracellular concentrations measured as a function of time using a mass spectrometric method developed in our lab. Our results show that transport of the nitrite in and out of the cells is on the order of a minute of less, and that the ensuing intracellular and extracellular concentrations of the anion are similar. The results suggest that macrophage nitrite uptake and release is dominated by passive transport.
Role of p38 MAPK in PMA‐induced NETs release from human neutrophils
Authors: Keshari Ravi Shankar , Kumar Sachin , Jyoti Anupam , Patel Satyananda , Barthwal Manoj Kumar , Verma Anupam , Bajpai Virendra K , Dikshit Madhu
Published: June 2021

Neutrophil extracellular traps (NETs) are produced by the neutrophils to bind and kill the extracellular microorganisms. It is composed of chromatin decorated with granular proteins such as elastase, myeloperoxidase, cathepsin G, pentraxin, lactoferrin etc. In the present study we have investigated the role of p38 MAPK in PMA induced reactive oxygen species (ROS) generation and NETs formation. Human neutrophils treated with PMA induced free radical generation and NETs release in a time (1–3 hrs.) and concentration (10 – 50 nM) dependent manner as demonstrated by DCF response and elastase/DNA release respectively. Since NETs release is dependent on free radicals effect of SB 202190 (50 μM, p38MAPK inhibitor) was investigated on PMA induced free radical formation as assessed by DCF and NBT, which was not significantly reduced. NETs formation was however inhibited by SB 202190 or DPI (10 μM, NADPH oxidase inhibitor), suggesting that p38 MAPK play a role downstream to the NADPH oxidase in PMA induced NETs release. NETs are made up of DNA and elastase, their release was also inhibited by DPI and SB 202190. PMA treatment induced the phosphorylation of p38 MAPK, which was inhibited by DPI. The results of the present study suggest that PMA induced NETs formation was mediated through the activation of NADPH oxidase and p38 MAPK.
Kinetics of nitrosation of GSH using NO donors
Authors: Madrasi Kumpal Jagdish , Joshi Mahesh S , Tsoukias Nikolaos
Published: June 2021

Modification of critical thiols by reactive nitrogen species (RNS) act as regulators of protein mediated signaling. Nitrosothiols are also believed to act as vasodilators which can release NO and activate the heme protein guanylate cyclase, thus initiating the mechanism for vasodilation. Nitrosation is thought to be mediated by dinitrogen trioxide (N2O3). Combination of NO with thiyl radical (GS.) following oxidation of the thiyl anion by NO2 has also been suggested as a mechanism for nitrosothiol formation. We investigate the kinetic mechanism of GSH (0–20mM) nitrosation in vitro by NO donor PAPA/NO (0.5 – 10 mM). GSNO formation was assessed spectrophotometrically at 338 nm. Results suggest a Michaelis‐Menten dependence of the rate on GSH concentration with a half‐maximum constant (Km,GSH) that depends on NO concentration. If there were solely one mechanism of nitrosation, one would expect a constant Km GSH value. Increasing [NO] up to 10 fold showed a proportional increase in Km,GSH. Pseudo steady state approximation yields the following kinetic rate for GS mediated nitrosation:
urn:x-wiley:08926638:media:fsb2fasebj241supplement98422:fsb2fasebj241supplement98422-math-0001

The reported increase in Km,GSH with NO concentration suggest a significant contribution of GS.mediated nitrosation in the formation of GSNO.

(Supported by NIH SC1HL95101)
Vasodilatation induced by a new NO donor involve KCa channels activation and cell membrane hyperpolarization
Authors: Lunardi Claure N , Pereira Amanda C , Biazzotto Juliana C , da Silva Roberto S , Bendhack Lusiane M
Published: June 2021

This study aimed to investigate the role of K+ channels activation induced by the new NO donor cis‐[Ru(bpy)2(py)NO2](PF6) (PY). Vascular reactivity was studied in denuded rat aortic rings and efficacy (ME) and potency (pD2) of PY were analyzed. Concentration‐effect curves for the NO donor were constructed in phenylephrine (Phe) or 60 mM KCl‐contracted arteries. Efficacy and potency values were lower in KCl (ME 60.2±3.5%, pD2 5.86±0.09, n=6) than in Phe‐contracted aortic rings (ME 104.4±1.0%, pD2 6.54±0.07, n=5). TEA (1mM), which is considered selective for KCa channels inhibited the relaxation to PY in Phe‐contracted arteries (ME 93.1±1.9%, pD2 5.83±0.13, n=6). Only Ca2+‐activated (KCa) blocker Apamin (1μM) reduced the Hill slope of the curve to PY from 2.66±0.37 (n=5) to 1.33±0.15 (n=6), but it did not alter the potency or efficacy of PY. The blockade of K+ channels voltage‐dependent (Kv) and ATP‐sensitive (KATP) had no effect on the relaxation induced by PY. In order to assess the effect of PY on the membrane potential of smooth muscle cells we used Di‐4‐Anepps probe and laser scanning confocalmicroscopy. The addition of PY decreased the ratio of fluorescence of the dye, suggesting that PY causes aortic cells membrane hyperpolarization. In conclusion, the vasorelaxation induced by the new NO donor involves KCa channels activation and cell membrane hyperpolarization. Supported by FAPESP, CAPES and CNPq.
Role of NO and the NADPH Oxidase in Hyperhomocysteinemia induced Cardiac Metabolic Dysfunction in Pregnancy
Authors: Song Su , Huang Harer , Martin‐Fernandez Beatriz , Ojaimi Caroline , Kaminski Pavel , Zhang Suhua , Wang Ziping , Wolin Michael , Hintze Thomas
Published: June 2021

Hyperhomocysteinemia (HHcy) causes cardiovascular dysfunction and is associated with many complications during pregnancy related to reduced NO bioactivity. This study assessed the mechanisms of HHcy on the NO‐dependent control of cardiac O2 consumption in pregnant rats. Rats were mated 3 times and were fed Met in drinking water to increase plasma Hcy (3.0±1.2 to 14.6±8.9 uM at the end of 1st pregnancy). This caused significant increase of heart wt/body wt (0.24±0.01 to 0.27±0.01 g/100g) and LV wt (0.69±0.03 to 0.78±0.01 g). The ability of bradykinin and carbachol to reduce myocardial O2 consumption in vitro was impaired by 59% and 66% in heart. The response was restored by ascorbic acid, tempol or apocynin. O2− production was increased by 2.5 folds in Hcy heart using lucigenin chemiluminescence. After the 1st pregnancy, HHcy caused a 51% increase in the p47phox subunit of NADPH oxidase, an increase in Rac‐1(67%) and increased nitrotyrosine (21%). Microarray analysis from heart tissues revealed 853 genes to be differentially regulated (P<0.05). There were no changes in the NADPH oxidase subunits or NOS. However, there were up regulation of some structural genes (Myosin, MMP3) and metabolic genes (Aldehyde DH, pyruvate DH and NADH DH). HHcy increases O2− production by NADPH oxidase and reduces the ability of NO to regulate myocardial O2 consumption in pregnancy. Supported by PO‐43023, HL 50142
Downrgulation of L‐type calcium channels increase media thicknes and change smooth muscle phenotype in rat resistance arteries in vivo.
Authors: Aalkjær Christian , Møller Kate , Matchkov Vladimir
Published: June 2021

The voltage‐gated L‐type Ca channels (LTCCs) are important in smooth muscle excitation‐transcription coupling. We have developed a method for downregulation of the LTCC in rat small mesenteric arteries in vivo by the use of siRNA. Transfection with specific siRNA decreased LTCC expression by ~75%. The downregulated arteries developed reduced wall stress in response to K‐induced depolarization which resembled control arteriess pre‐treated with nifedipine. Changes in wall stress correlated with smaller changes in [Ca2+]i in the arteries downregulated for LTCC. Similar changes were seen with noradrenalin and vasopressin.

In contrast to the effect of LTCC blocker chronic treatment, the LTCC downregulated arteries had increase in media thickness in comparison to the controls. The remodeling was associated with phenotypic changes of the VSMCs: contractile phenotype markers (myocardin, tropomyosin 4, smooth muscle myosin heavy chain, smooth muscle α‐actin and h‐caldesmon) were significantly downregulated, while marker genes for the proliferative phenotype (l‐caldesmon and ki‐67) were significantly upregulated. In α‐toxin permeabilized arteries downregulated for LTCC the force development to calcium was reduced compared to control arteries. We conclude that LTCC expression and/or activity are crucial in determining VSMC phenotype as well as the structure of small resistance arteries.
Augmented endothelin‐1 constriction in pudendal arteries from ETB receptor‐deficient rats: linking hypertension and female sexual dysfunction.
Authors: Allahdadi Kyan J , Hannan Johanna L , Boesen Erika I , Webb R. Clinton
Published: June 2021

Female sexual dysfunction (FSD) afflicts 40% of women, reduces quality of life and is linked with hypertension. One potential commonality between these disorders is augmented vascular sensitivity to endothelin‐1 (ET‐1). The pudendal artery is critical in blood delivery to female genitalia. We hypothesize that pudendal arteries from hypertensive ETB receptor‐deficient (ETBR‐) rats have elevated constrictor sensitivity to ET‐1. Pudendal arteries from ETBR‐ and wild type (WT) rats were mounted in wire myographs and submitted to increasing concentrations of ET‐1 in the presence or absence of ETAR (Atrasentan; 10–9 and 10–8 M) and rho‐kinase (Y‐27632; 10–6M) antagonists. Western blot measured protein levels of the ETAR. Pudendal arteries from ETBR‐ rats display increased ET‐1‐mediated constriction (pD2=9.8±0.1) compared to WT rats (pD2=8.8±0.1). ETAR antagonism reduced ET‐1‐mediated constriction in pudendal arteries from ETBR‐ (pD2=8.7±0.1); however no antagonism was observed in WT. Rho‐kinase inhibition did not reduce ET‐1‐mediated constriction in pudendal arteries from ETBR‐ or WT rats. PE‐mediated contraction and ACh‐mediated relaxation were not different between groups. ETAR protein was increased in ETBR‐ pudendal arteries. Pudendal arteries from ETBR‐ rats show greater sensitivity and elevated ETAR expression, suggesting a common abnormality in hypertension and FSD. (NIH HL066993‐06)
The effect of electroacupuncture on bladder dysfunction caused by streptozotocin‐induced diabetes
Authors: Liao Jiuan‐Miaw , Lin Tzer‐Bin , Liou Ying‐Ming
Published: June 2021

Diabetic bladder dysfunction (DBD) is a most common complication of diabetes mellitus (DM) with poorly effective treatment options. This study examined the effect in bladder function following 1, 3, and 5 wk electro‐acupuncture (Ea) treated at Zusanli (ST‐36) acupoints (100 Hz, 0.5mA, 1.5–2ms, under conscious condition, 30 min per day) of streptozotocin (STZ) induced‐DM rats compared with that in age‐matched non‐Ea treated diabetic group, and shamed‐treated control group. Urodynamic parameters were assessed by cystometry in addition to the measurements of urinary functional changes. Diabetes resulted in decreased body weight. Bladder weight, voiding volume, and bladder capacity increased in both the non‐Ea and Ea‐treated DM groups. Peak voiding pressure (PVP) and the intercontraction interval (ICI) increased in the non‐Ea‐ and 1wk Ea‐treated DM groups. However, in 3 and 5wk Ea‐treated DM groups, PVP was showed no significantly different compared with the sham‐treated control group. Similar changes in the compliance and emptying ability of the bladder were seen in the 3 and 5wk Ea treated groups. Long‐term Ea‐treated at Zusanli acupoints effectively ameliorated a few funtional changes in bladder function. These results suggest that such a therapeutic technique has potential in clinical practice.
Improvement of relaxation in Type II diabetic mice corpus cavernosum by PhTx2‐6 toxin from Phoneutria nigriventer spider
Authors: Nunes Kenia Pedrosa , Tostes Rita , de Lima Maria Elena , Webb R. Clinton
Published: June 2021

Type II diabetes mellitus (T2DM) is a major risk factor for development of erectile dysfunction (ED). Mechanisms causing ED in T2DM are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. The toxin PhTx2‐6 purified from this venom inhibits sodium (Na+) channel inactivation in neuronal‐type Na+ channels. We hypothesize that PhTx2‐6 potentiates cavernosal relaxation in T2DM mice. Cavernosal strips were pre‐contracted with phenylephrine (10−5 M) and relaxed by electrical field stimulation (EFS) in the presence or absence of PnTx2‐6 (10−8 M). cGMP levels from mice tissue were determined after stimulation with PhTx2‐6 in presence or absence of L‐NAME (10−4M). Results showed that PhTx2‐6 enhanced cavernosal relaxation in diabetic mice (± 65%) and the toxin was not able to reverse the inhibition by L‐NAME. cGMP levels are increased by PhTx2‐6 (± 1.57 vs. basal ±0.86), however L‐NAME abolished this enhancement. We conclude that PhTx2‐6 facilitates penile relaxation in diabetic mice probably via increasing NO release and consequently cGMP production. This toxin may be an important tool for development of new therapeutic approaches to treat ED in T2DM.