bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of transcription factors in estrogen‐dependent upregulation of CYP 2C family in arteries of eNOS deficient mice
Authors: Huang An , Jacobson Azita , Yang Yangming , Ojaimi Caroline , Sun Dong , Kaley Gabor
Published: June 2021

Upregulation of cytochrome P450 (CYP) 2C29/38/40 is responsible for the EET‐mediated flow‐dependent dilation in microvessels of female mice, as a function of NO deficiency. In the present studies, we provide evidence of the correlation between transcription factors (PPAR‐γ, PPAR‐δ and RXR‐γ) and CYP 2C29/38/40 in isolated arterioles of female eNOS‐KO mice by microarray (SuperArray, OMM‐401). Five single arterioles isolated from mesentery of both genders of eNOS‐KO and WT mice were pooled and snap frozen in liquid nitrogen; ∼10 μg of total RNA were isolated, and a total of 10 arrays from 2 WT and 3 eNOS‐KO mice of each gender were performed. Microarray results indicate that associated with a specific upregulation of CYP 2C29/38/40, expression of genes for PPAR‐γ, PPAR‐δ and RXR‐γ, the nuclear receptors that function as gene regulators via specific DNA binding, was also increased (P<0.01, 0.05 and 0.03, respectively) in female eNOS‐KO compared to male eNOS‐KO vessels. These significant differences were validated by quantitative real‐time PCR and Western blot analysis. The increase in gene expression for the three transcription factors was only observed in vessels of female, but not in those of male eNOS‐KO mice, and without significant differences between vessels of WT male and WT female mice, a pattern that is similar to the upregulation of CYP 2C/29/38/40. Thus, the microarray analysis screened the global expression of genes for the transcription factors and revealed the specificity of estrogen and NO deficiency in the control of the transcription factors which may play a role in the regulation of CYP 2C expression (Supported by NIH HL‐43023, HL‐68813 and HL‐070653).
Regulation of perfusion and oxygenation by erythrocyte hemoglobin oxygen affinity
Authors: Cabrales Pedro , Villela Nivaldo R. , Tsai Amy G. , Intaglietta Marcos
Published: June 2021

Responses to exchange transfusion using red blood cells (RBCs) with modified hemoglobin (Hb) oxygen (O2) affinity were studied in the hamster window chamber model during acute anemia to determine its role on microvascular perfusion and tissue oxygenation. Allosteric effectors were introduced in the RBCs by electroporation. Inositol hexaphosphate (IHP) and 5‐hydroxymethyl‐2‐furfural (5HMF) were used to decrease and increase Hb‐O2 affinity. In vitro P50s were modified to 10, 25, 45 and 50 mmHg, normal P50 is 32 mmHg. Allosteric effectors also decreased the Hill coefficient. Anemic condition was induced by isovolemic hemodilution exchanges using 6% dextran 70 kDa to 18% hematocrit (Hct). Modified RBCs (at 18% Hct in 5% albumin solution) were infused by exchange transfusion of 35% of blood volume. Systemic parameters, microvascular perfusion, capillary perfusion (functional capillary density, FCD) and microvascular pO2 levels were measured. RBCs with P50 of 45 mmHg increased tissue pO2 and decreased O2 delivery (DO2) and extraction (VO2) and RBCs with P50 of 60 mmHg reduced FCD, microvascular flow, tissue pO2, DO2 and VO2. RBCs with increased Hb‐O2 affinity maintained hemodynamic conditions, DO2 and decreased tissue pO2. This study shows that in an anemic condition maximal tissue pO2 does not correspond to maximal DO2 and VO2. Supported by NIH BRP R24‐HL64395 and grants R01‐HL62354, R01‐HL62318 and R01‐HL76182.
Inhibition of superoxide anion‐mediated impairment of endothelium by treatment with luteolin in rat mesenteric artery
Authors: Li Yan‐fang , Ru Xiao‐chen , Wang Hui‐ping , Ma Xin , Jiang Li‐feng , Xia Qiang
Published: June 2021

This study was designed (i) to test whether endothelium‐derived hyperpolarizing factor (EDHF) and nitric oxide (NO) mediated vasorelaxation and hyperpolarization of vascular smooth muscle cell (VSMC) are impaired by superoxide anion, and (ii) to investigate the vasoprotection of luteolin against impaired endothelial function. Rat mesenteric arterial rings were isolated for isometric force recording and electrophysiological studies. Visualization of superoxide production was detected by fluorescent dye dihydroethidium. We found that 300μM pyrogallol decreased the relaxation and hyperpolarization of VSMC. Luteolin attenuated the loss of EDHF‐ and NO‐dependent vasorelaxation and the impairment of hyperpolarization to ACh induced by pyrogallol. Oxidative fluorescent microtopography showed that luteolin significantly reduced the superoxide levels induced by pyrogallol. The results suggest that exposure to superoxide anion impairs ACh‐induced relaxation and hyperpolarization of SMC in resistance arteries via impairing both EDHF‐ and NO‐mediated relaxation. Luteolin protects resistance arteries against superoxide anion caused injury, which implies that luteolin maybe effective in the therapy of vascular endothelium injury associated with oxidative stress.

This work was supported by grant Y206179 from Zhejiang Provincial National Science Foundation of China.
Separating Genetic and Hemodynamics Effects In Nrp1 Knockout Embryos
Authors: Jones Elizabeth A.V. , Yuan Li , Breant Christiane , Watts Ryan , Eichmann Anne
Published: June 2021

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. Since blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the Neuropilin‐1 (Nrp‐1) receptor, and hemodynamic defects occurring in homozygous knockout embryos. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow‐induced deficiencies from those caused by the nrp‐1 mutation, we arrested blood flow in cultured wild‐type and mutant embryos and followed their vascular development. We find that loss of nrp‐1 function rather than flow induces the altered geometry of the capillary plexus. This method allows genetic defects caused by loss‐of‐function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.
CADHERINS PLAY A ROLE IN ARTERIOLAR MYOGENIC RESPONSIVENESS
Authors: Jackson Teresa Yvonne , Martinez‐Lemus Luis A. , Hill Michael A , Meininger Gerald A
Published: June 2021

The myogenic response is characterized as a vascular phenomenon in which blood vessels constrict to an increase in intraluminal pressure and dilate in response to a decrease in intraluminal pressure. This mechanism is important for the regulation of blood flow, capillary pressure and arterial pressure. The essential molecular components and signaling pathways have not been identified. Thus, the purpose of this study was to investigate the role of cadherins, a family of calcium‐dependent cell‐cell transmembrane adhesion proteins. Since the myogenic response is intrinsic to vascular smooth muscle, N‐Cadherin, the prominent vascular smooth muscle cadherin, was selected for investigation. In the present study, isolated rat skeletal muscle arterioles were exposed to a series of step increments in intraluminal pressure (60 mmHg to 100 mmHg in increments of 20 mmHg). Diameter responses to changes in pressure were recorded in the presence and absence of a synthetic cadherin inhibitory peptide (HAV) or an inhibitory antibody both of which are known to bind to N‐cadherin. In the absence of peptide and antibody, myogenic vasoconstriction was observed as pressure was increased (60–80 and 80–100 mmHg) whereas following inhibition of N‐cadherin the myogenic responses of the arteriole were abolished. A control peptide (HGV) or a function blocking VE‐cadherin antibody had no inhibitory effect on the myogenic response. Therefore, these data suggests that cadherins are an important component of the mechanotransduction pathway supporting arteriolar myogenic responsiveness.
Biological Profiling of Drugs for Treatment of In‐stent Restenosis
Authors: Lavigne Mark C. , Queen Jillian , Kunkel Eric , Berg Ellen , Kamath Kalpana , Palasis Maria , Barry James , Eppihimer Michael J.
Published: June 2021

To elucidate the actions of drugs that are useful in treating in‐stent restenosis, we compared the activities of both Paclitaxel and Sirolimus against dexamethasone, actinomycin D, or 17β‐estradiol in a panel of human cell‐based assays that model biological processes involved in restenosis. Paclitaxel and Sirolimus each inhibited endothelial cell (EC) and vascular smooth muscle cell (VSMC) growth, while dexamethasone promoted VSMC proliferation. Sirolimus and actinomycin D each decreased tissue remodeling markers in ECs and fibroblasts. Paclitaxel had no remarkable effects, while dexamethasone enhanced PAI‐1 expression. Paclitaxel did not affect T cell activation or peripheral blood mononuclear cell (PBMC)/T cell CD40/CD40 ligand and MCP‐1 expression. Sirolimus, 17β‐estradiol, dexamethasone, and actinomycin D each inhibited one or more of these inflammatory markers. In VSMC cultures, Paclitaxel up‐regulated thrombomodulin and decreased tissue factor expression. In contrast, Sirolimus increased tissue factor expression, but had no effect on thrombomodulin in cultures containing ECs and PBMCs. Dexamethasone, 17β‐estradiol, and actinomycin D each had little or no effect on these thrombosis factors. These results suggest that drugs considered for future use in DES platforms should inhibit VSMC proliferation to afford clinical efficacy with in‐stent restenosis. Further optimization would include drug actions that are not directed at ECs so as to permit proper vascular healing following DES deployment.
Attenuation of Hydrogen Sulfide‐Induced Relaxation of Aorta from Diabetic Rats
Authors: Zangeneh Shiva , Oyekan Adebayo O , Yakubu Momoh A
Published: June 2021

Hydrogen Sulfide (H2S) is the new gasotransmitter produced from L‐Cysteine by cystathionine‐β‐synthase, and cystathionine‐γ‐lyase causing vasorelaxation via activation of K(ATP)‐channel. Information on the regulation of the H2S vascular function in disease conditions is limited. We studied the effects of diabetes on H2S relaxation of aorta from rats treated with 70 mg/kg, i.p. streptozotocin (STZ) or vehicle. Vascular responses to H2S (10−6−10−2 M) was determined in control and diabetic aorta following contraction with PE (10−7) or 5‐Hydroxytryptamine (5‐HT, 10−6 M). Tension generated by PE or 5‐HT were dose‐dependently relaxed by application of H2S with the highest concentration of H2S 10−2 M producing 66% of the 5‐HT and 40% of the PE induced tension. In the STZ, relaxation by H2S was attenuated to 41 and 32% respectively for 5‐HT and PE tensions. In this study, both 5‐HT and PE‐induced tensions were significantly enhanced in the STZ compared to the control. The enhancement was significantly higher for 5‐HT at 2.03 ± 0.03 gm compared to PE at 1.47 ± 0.025 gm.

In conclusion: H2S‐induced relaxation was selectively higher in the 5‐HT than in the PE pre‐contracted aortic rings and relaxations of aorta from STZ‐induced diabetic rats was selectively attenuated for 5‐HT‐ but not for PE‐induced tension. Thus, these selective effects could be due to the several mechanisms involved in the H2S‐induced vascular relaxation.

This work was supported by grants from NHLBI: HL03674 and HL70669.
HIV‐1 Protein Expression Results in Progressive Endothelial Dysfunction that is Ameliorated by Procysteine Treatment
Authors: Sutliff Roy L , Dikalov Sergey , Kline Erik R
Published: June 2021

Data suggest that HIV‐infected people are at a heightened risk of developing cardiovascular disease. There is increasing clinical evidence that HIV‐1 itself promotes cardiovascular risk. Endothelial dysfunction is an excellent predictor of cardiovascular disease. Here we monitor the effects of HIV‐1 proteins over time by examining endothelial function in 3 and 9 month old HIV‐1 transgenic rats. Nitric oxide was measured in serum and aortas of wild‐type and HIV‐1 transgenic rats using electron spin resonance. Relaxation studies were completed on isolated aortic rings. We observe no evidence of HIV‐1‐induced endothelial dysfunction in 3 month old rats. However, 9 month old HIV‐1 transgenic rats display less aortic tissue NO, NO‐hemoglobin, serum nitrite and S‐nitrosothiols, and impaired endothelium‐dependent relaxation than wild‐type rats. Aortas have higher levels of superoxide and 3‐nitrotyrosine. Transgenic aortas display decreased superoxide dismutase protein expression and total glutathione (GSH) levels. Administering the GSH precursor procysteine to transgenic rats decreases aortic superoxide, and restores NO‐hemoglobin and endothelium‐dependent relaxation. We conclude that HIV‐1 proteins contribute to vascular dysfunction, and ROS coupled with NO deficiency predispose the aorta to cardiovascular risk. This can be ameliorated by pharmacologically targeting endogenous antioxidants.
Accuracy of mouse aortic diameter measurement: comparison of videomicrometer and ultrasound
Authors: xiong Wanfen , Knispel rebecca A. , baxter bernard timothy
Published: June 2021

Objective: The purpose of this study was to determine the accuracy of high‐resolution ultrasound in measuring the diameter of infrarenal abdominal aorta and to compare its use to videomicrometer, which is the most commonly used method in the studies of animal models of abdominal aortic aneurysm (AAA).

Methods: Mice (C57BL/6), at 8 weeks age, underwent AAA induction by periaortic application of CaCl2. The diameter of the infrarenal aorta was measured at aneurysm induction and 8 weeks after aneurysm induction with videomicrometer and ultrasound by M‐mode ultrasonography.

Results: The mean diameter of the infrarenal abdominal aorta at aneurysm induction was 535 ± 8.3 μm by videomicrometer and 531 ± 7.9 μm by ultrasound. Eight weeks after CaCl2 aneurysm induction, the mean aortic diameter was 892 ± 59.1μm by videomicrometer and 902 ± 55.8 μm by ultrasound. The mean differences of the measured diameters by the two methods were 7.8 μm (SE=12.2 μm) at initial measurement and 10.3 μm (SE=36.9 μm) at 8 weeks after aneurysm induction. The linear correlation coefficient (r value) for the diameter after 8 weeks aneurysm induction was 0.785. Using this criteria, there was a consistent, relative narrowing calculated by both videomicrometer and ultrasound. Conclusion: The mean difference, when taken together with a good linear correlation coefficient of the diameters as measured by videomicrometer and ultrasound, makes this technique acceptable. In addition, ultrasound is suitable for keeping track of the aortic diameter changes during mouse aneurysm development and treatment without open surgery.
Expression of ETA and ETB receptor in adventitial fibroblasts
Authors: Di Wang Hui , Boyd Ryan , Forgione Andrea
Published: June 2021

Objective: The adventitia has been recognized to play important roles in vascular remodeling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin‐1 (ET‐1) in response to angiotensin II (ANG II). However, it is unclear whether the ET‐1 receptors are expressed in the adventitia. We therefore examined the expression of both the ETA/B receptors and the roles of these two types of receptors in collagen synthesis and ET‐1 clearance in adventitial fibroblasts.

Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (100 nM), the ET‐1 receptor antagonists (100 uM), BQ123 (ETA receptor) and/or BQ788 (ETB receptor). ET‐1 peptide levels were determined by ELISA, while ETA/B and collagen levels were determined by Western blot. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETA receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in time dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET‐1 peptide levels, implicating the role of the ETB receptor in the clearance of the ET‐1 peptide.

Conclusion: Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET‐1 peptide.
Role of calcium to serotonergic mediated contractility in ovine pulmonary arteries: effects of maturation and chronic hypoxia
Authors: Goyal Ravi , Loftin Matthew , Derrick Allison , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5‐HT) is implicated in idiopathic and hypoxic stress induced pulmonary hypertension (PH) in adult and newborn. However, voltage‐gated L‐type Ca2+ channel (CaL) antagonists are not effective in the treatment of PH. The hypothesis that Ca2+ dependent pulmonary arterial (PA) contractility is reduced with chronic hypoxic (CH) stress and enhanced with maturation was tested by performing wire myography on PA isolated from fetal and adult sheep that were either normoxic or maintained under CH stress. Arterial segments were exposed to blockers of L‐type calcium channels (CaL, 10 μM nifedipine), non selective cation channels (NSCC, 50 μM SKF96365) and IP3 receptors (50 μM 2‐APB) in an additive manner during 10–100 μM 5‐HT elicited contractility. The contribution of CaL and NSCC to 5‐HT elicited contraction increased with maturation and decreased with CH while IP3 mediated contractility was unchanged. As a whole, Ca2+ dependent contractility increased with maturation and decreased with CH, implicating alterations in the pathways that sensitize contractile proteins to elevations in cytosolic Ca2+. CH mediated increases in Ca2+ independent contractility were greatest in adult, possibly because the fetus is under relative hypoxic stress. These dramatic changes in 5‐HT mediated contractility with CH may contribute to vascular reactivity disorders such as PH. (Support from NIH, Sigma Xi and UM)
Disruption of vasoconstrictor responses in isolated cerebral arteries following treatment with siRNA against TRPM4
Authors: Gonzales Albert L , Crnich Rachael , Earley Scott
Published: June 2021

Membrane depolarization, Ca2+ influx, and activation of the Ca2+‐calmodulin pathway for smooth muscle contraction regulate arterial tone. The molecular mechanisms responsible for membrane depolarization in response to vasoconstrictor stimuli have not been fully characterized. We recently demonstrated that the melastatin transient receptor potential (TRP) channel TRPM4 is critical for membrane potential depolarization in response to increases in intraluminal pressure in cerebral arteries. In the current study, we further characterized the role of TRPM4 in intraluminal pressure‐ and agonist‐induced vasoconstriction using a newly developed RNA interference technique. Treatment of intact cerebral arteries with TRPM4 siRNA decreased mRNA expression by ~55% compared with controls. Using simultaneous video dimensional analysis and ratiometric Ca2+ imaging , we found that arteries treated with TRPM4 siRNA (n=7) developed less myogenic tone and had lower smooth muscle intracellular Ca2+ levels compared with controls (n=5). KCl (60 mM)‐induced constriction did not differ between groups. In addition, we found that TRPM4 siRNA‐treated arteries pressurized to 10 mmHg were less sensitive to the receptor‐dependent vasoconstrictor agonist uridine triphoshphate (UTP). These data suggest that TRPM4 plays a central role in regulating responses to vasoconstrictor stimuli in cerebral arteries. AHA0535226N
Diffuse coronary atherosclerosis in Ossabaw miniature swine with metabolic syndrome compared to Yucatan swine
Authors: Edwards Jason M , Neeb Zach , Byrd Jim , Mokelke Eric , Alloosh Mouhamad , Sturek Michael
Published: June 2021

Patients with diabetes have diffuse coronary atherosclerosis, also known as three vessel disease. The Ossabaw miniature swine is an excellent large animal model of metabolic syndrome (pre‐diabetes). Here we compare the pervasiveness of coronary atherosclerosis between the Ossabaw swine model of metabolic syndrome and the widely used Yucatan swine that do not show metabolic syndrome. Each breed was fed two separate diets which were calorie matched and divided into two groups each: control (C) and high fat/cholesterol atherogenic diet (H). Intravascular ultrasound was used to quantify atherosclerosis throughout the epicardial coronary vasculature. Ossabaw swine had increased characteristics of metabolic syndrome including: greater weight gain, glucose intolerance, and insulin resistance, compared to Yucatan. Plasma lipids and blood pressure were not different. H diet increased atherosclerosis ~3‐fold in proximal segments of coronary artery compared to C, with no breed differences. However, Ossabaw H compared to Yucatan H had ~2.5‐fold and ~4‐fold greater atherosclerosis in intermediate and distal coronary segments, respectively. Neointima of Ossabaw had less collagen, suggesting more cellular and/or lipid‐laden composition. We conclude that Ossabaw swine, not Yucatan, exhibit diffuse coronary atherosclerosis, a hallmark of patients with diabetes. Support: NIH RR013223, HL062552.
Effects of pro‐oxidant treatment on the progression of prediabetes in SHROB rats
Authors: Ernsberger Paul , Theodorou Maria , Edwards Simone , Koletsky Richard J
Published: June 2021

The spontaneously hypertensive obese (SHROB) rat is a model of prediabetes characterized by normal fasting and high postprandial glucose and insulin resistance. Oxidative stress, through the damaging effects of oxygen radicals, may contribute to the onset of diabetes. We induced oxidative stress in SHROB with a combination of a pro‐oxidant agent, hydroquinone, and a glutathione depleting agent, L‐buthionine sulfoximine (both 50 mg/kg ip). Oxidative stress is increased in untreated SHROB rats, as indicated by a 3‐fold increases in levels of peroxides in the plasma as well as in the kidney. Antioxidant antihypertensive therapy with captopril lowered oxidative stress in SHROB by about 2‐fold. Pro‐oxidant injection increased plasma peroxide levels within 1h and returned to baseline within 24h. Induction of further oxidative stress in prediabetic SHROB causes diabetes within two days of daily treatment, as indicated by morning blood glucose values averaging over 200 mg/dl versus about 100 mg/dl in control SHROB. Glucose fell to baseline within 3d, but glucose tolerance was significantly impaired. Food intake and body weight were not affected. The SHROB rat subjected to oxidative stress is a potential model to study the onset of Type 2 diabetes, and supports the hypothesis that oxidative stress may be a trigger for diabetes onset in susceptible individuals.
The Calcineurin Homologous Protein (CHP) regulates the Na+/H+ exchanger‐3 (NHE3) traffic by modulation of ezrin phosphorylation
Authors: Di Sole Francesca , Babich Victor , Jevons Mark , Moe Orson W
Published: June 2021

NHE3 is essential to the regulation of Na+ absorption in the renal and intestinal epithelium. NHE3 function is regulated in part by changes in its cell surface abundance; however, the upstream molecular signals that control NHE3 surface expression remain elusive. In opossum kidney cells, CHP over‐expression increased NHE3 activity and plasma membrane abundance, while silencing of CHP decreased cell surface NHE3 activity and protein. Consistent with a central role for ezrin dependent NHE3 trafficking, augmented CHP expression increased ezrin phosphorylation at threonine 567 without change in total ezrin. Knockdown of CHP reduces the amount of phosphorylated ezrin with no change in total ezrin. Our findings show that ezrin activation is downstream from CHP. To demonstrate that NHE3 is downstream from ezrin, we showed that over‐expression of wild type (WT) ezrin increased NHE3 activity and surface expression. This effect was enhanced by expression of the constitutively active ezrin mutant T567D, whereas the inactive non‐phosphorylatable mutant T567A did not affect NHE3. To further demonstrate the linear CHP‐ezrin‐NHE3 cascade, we showed that CHP knockdown reversed the ezrin WT mediated NHE3 activation but did not affect NHE3 up‐regulation by T567D ezrin. We conclude that CHP is a critical signal molecule that increases surface NHE3 protein and function by increasing ezrin phosphorylation and NHE3 trafficking.
INTERNALIZATION OF DCT Na‐Cl‐CO‐TRANSPORTER (NCC) DURING PRESSURE DIURESIS‐NATRIURESIS
Authors: McDonough Alicia Ann , Lee Donna H , Riquier Anne D.M. , Maunsbach Arvid B.
Published: June 2021

When blood pressure (BP) is elevated above baseline, a pressure natriuresis response ensues which is critical to volume and blood pressure homeostasis. The response involves a decrease in salt and volume reabsorption from proximal tubule and a load dependent increase in salt reabsorption in loop of Henle. The role of distal nephron is less clear. Distal convoluted tubule Na+‐Cl− cotransporter (NCC) is regulated by trafficking between apical plasma membrane (APM) and sub‐apical cytoplasmic vesicles (SCV). We aimed to determine whether NCC trafficking contributes to pressure natriuresis (increase APM NCC) or compensates for increased volume flow to the DCT (decrease APM NCC). BP was raised 50 mmHg (hiBP) for 30 min in rats by arterial constriction. Kidneys were excised and NCC subcellular distribution analyzed on sorbitol density gradients by immunoblot or kidneys were perfusion fixed and NCC analyzed by immuno‐electron microscopy. After 30 min hiBP urine output increased 11‐fold. By density gradient, 20% of the total NCC redistributed from low density APM to higher density fractions (p<0.05). By quantitative immuno‐EM pool size of APM NCC decreased 13% with a corresponding increase in the SCV pool (p<0.05). In summary, the results provide evidence for an acute decrease in APM NCC during hypertension that likely contributes to pressure diuresis‐natriuresis. HL085388, DK34316, Danish NRF.
Sodium overload modulates the electrical activity of serotonergic (5HT) neurons of the dorsal raphé nucleus (DRN)
Authors: Godino Andrea , Carrer Hugo F , Vivas Laura
Published: June 2021

Our previous results indicate that 5HT neuronal activity (as shown by Fos expression) within the DRN is modulated by body sodium status. The effect of body sodium overload on spontaneous firing frequency of the 5HT‐DRN neurons was analyzed.

Male Wistar rats were anesthetized, and a catheter was inserted in the yugular vein; subsequently, a micropipette was stereotaxically lowered into the DRN in order to record single cell extracellular action potentials. After recording basal activity for 3 min, a group of rats received a sc injection of 2M NaCl solution (1.7 ml/min), and a second group of animals received an equal volume of 0.15 M NaCl. Fifteen minutes later we determined the neuronal phenotype by iv infusion of fluoxetine. To measure plasma sodium and osmolality other rats were submitted to the same protocol but were decapitated and bled 0, 5, 10 and 15 min after NaCl infusion.

Four min after 2M NaCl infusion, neuronal activity increased significantly compared to the 0.15M NaCl group and basal recording activity. 0.15M NaCl infusion produced no change in spontaneous firing rate. Plasma sodium and osmolality significantly increased after 2M NaCl infusion compared to 0.15M NaCl group and baseline levels.

We conclude that hypernatremic systemic states increase the firing rate of 5HT‐DRN cells, possibly to modulate sodium and water intake/excretion and avoid an extracellular volume expansion.

Supported by ANPCyT CONICET.
Transport proteins in the inner medullas of diabetic kidneys are further increased by candesartan.
Authors: Blount Mitsi A. , Sands Jeff M. , Kent Kimilia J. , Smith Tekla D. , Klein Janet D.
Published: June 2021

Dilute urine is a problem in uncontrolled diabetes mellitus (DM). Angiotensin II (AngII) receptor blockers ie. candesartan slow chronic kidney disease in DM patients. We investigated UT‐A1, UT‐A3, NKCC2, and AQP2 in control +/− candesartan rats and 3‐week DM +/− candesartan rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan‐treated rats (0.34±0.14 nM) were the same. DM rats had higher aldosterone levels (1.48±0.37 nM) that were decreased by candesartan (0.97±0.26 nM). Western blot analysis showed that in inner medullary (IM) tip and base, UT‐A1 expression was increased in DM rats compared to controls (Tip: 158±13%, Base: 120±25). UT‐A3 abundance was increased in both IM tip (123±11%) and base (146±17%) of DM rats vs controls (100%). UT‐A3 was unchanged in candesartan‐treated control rats. In candesartan treated DM rats UT‐A3 increased (IM tip: 160±14%; base 210±19%). Candesartan treated DM rats had slightly higher AQP2 in IM (46%, p<0.05) vs control rats. NKCC2 was increased 45±10% in OM of DM vs control rats. While candesartan did not alter NKCC2 in controls, NKCC2 was increased a further 41% by candesartan treatment in DM rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of AngII receptor blockers in diabetes.
Contribution of Vav2 to Glomerular Injury via NADPH Oxidase Activation in Hyperhomocysteinemia
Authors: Yi Fan , Xia Min , Thomas Cindy , Li Ningjun , Li Pin‐Lan
Published: June 2021

We have demonstrated that Vav2 is a predominant isoform of GEF‐Vav subfamily and participates in homocysteine‐induced Rac1 activity and consequent activation of NADPH oxidase in rat mesangial cells. However, it remains unknown whether this Vav2/Rac1‐mediated NADPH oxidase activation contributes to glomerular injury induced by hyperhomocysteinemia (hHcys) in vivo. The present study was designed to address the role of Vav2 in the development of hHcys‐induced glomerular injury in Sprague Dawley rats. An express vector with siRNA of Vav2 (Vav2 shRNA) was introduced into the kidneys via the renal artery using ultrasound‐microbubble techniques. In Vav2 shRNA transfected rat kidneys, Vav2 expression in the renal cortex was almost completely blocked and hHcys induced by folate‐free (FF) diet had much less effect to increase Rac1 activity (33 vs. 86%) and to enhance NADPH‐dependent O2.‐production (24% vs. 65%) compared with rat kidneys receiving scrambled small RNA vectors. hHcys‐induced glomerular injury as indicated by increased urinary albumin excretion and glomerular damage index was significantly improved. In another series of experiments, we simulated the action of Hcys to stimulate Vav2. By transfection of a dominant active Vav2 variant in the rat kidneys, glomerular Vav2 expression was significantly increased by 1.5 folds, which led to 128% and 82% increase in Rac1 activity and O2.‐production, respectively. We concluded that Vav2 importantly contributes to Rac1/NADPH oxidase activation, thereby producing local oxidative stress and consequent glomerular injury associated with hHcys (Supported by NIH Grants HL57244, HL75316, DK‐54927 and HL89563).
Non‐desensitizing purinergic receptors (P2R) are responsible for sustained stimulation of vasopressin (VP) release by ATP and phenylephrine (PE)
Authors: Gomes Dayane A , Song zhilin , Sladek Celia D
Published: June 2021

ATP and norepinephrine (NE) are neurotransmitters carrying cardiovascular information to vasopressin (VP) neurons. Simultaneous exposure of hypothalamo‐neurohypophyseal system explants to ATP and PE (α1‐adrenergic receptor agonist) results in potentiation of VP release characterized by both an increase in the peak response and conversion of a transient response to a response that is sustained for hours. (J. Neurosci. 20:8868, 2000). Since the P2X2/3 and P2X7 subtypes of P2XRs do not desensitize, we hypothesized that these receptors are responsible for the sustained response to ATP. A317491 (300nM, P2X3 and P2X2/3R antag.), or 1uM BBG (P2X7R antag.) added during sustained response to ATP+PE attenuated VP release. These data supported the hypothesis that activation of the non‐desensitizing P2X2/3 and P2X7Rs is required for the sustained elevation of VP release characteristic of ATP+PE synergism. A more rapid effect of BBG suggests a prominent role for P2X7Rs. To determine if continued exposure to ATP or PE is required to sustain elevated hormone release, either ATP or PE was removed after 2hrs exposure to ATP+PE. Continued perifusion with PE alone but not ATP is required to maintain VP release. Since NE has been shown to stimulate ATP release from glia (Gorden et al Nat Neurosci 8:1078, 2005), these findings in conjunction with the ability of the P2X2/3 and P2X7R antagonists to attenuate sustained VP release support the interpretation that continuous exposure to PE is required to maintain P2X2/3 and/or P2X7R function. Supported by NIH RO1‐NS27975.
Water Conservation in Chronic Ethanol Exposure is Associated With Elevated Renal Estrogen Receptor Beta and Vasopressin V2 Receptor mRNA in the Female Rat
Authors: Huckstep Odaro John , Sato Aileen K , Hernandez Claudia A , Uyehara Catherine F T
Published: June 2021

We previously demonstrated that chronic high dose ethanol intake decreases water load excretion in female but not male rats. We thus hypothesized that sex differences in alteration of water load excretion in response to ethanol is due to changes in renal vasopressin V2 receptor (V2R) expression. Other research has identified estrogen receptor (ER) α and β in renal tissue which may affect renal fluid handling. Thus, in this study we compared renal papilla mRNA expression of V2R, ERα and ERβ between male control (n=5) and ethanol‐fed (n=15) rats, and female control (n=12) and ethanol‐fed (n=17) rats. RT‐PCR analysis revealed that the decreased water excretion observed in ethanol‐exposed females was accompanied by a 40% increase in V2R mRNA (p<0.05) and a 146% increase in ERβ mRNA (p<0.05) compared to controls. In contrast, ethanol treatment in male rats resulted in no difference in water excretion, and yielded no change to V2R or ERβ mRNA expression in the renal papilla. ERα expression was not different between males or females, nor affected by ethanol treatment. Overall, these results suggest that females can better compensate for the dehydrating effects of ethanol exposure by increasing renal responsiveness to vasopressin via upregulating renal V2R. Also, ethanol specifically upregulates the ERβ subtype in the female kidney which may modulate renal sensitivity to vasopressin. Supported by US Army Medical Command.
Detrusor muscle contractility and compliance are impacted by diet in Ossabaw miniature pigs with metabolic syndrome (MetS)
Authors: Sharma Richa , Harden Barbara , Alloosh Mouhamad , Sturek Michael , Packer C. Subah
Published: June 2021

The etiology of diabetic bladder dysfunction is not understood. Previous work suggests that MetS Ossabaw pigs have detrusor muscle that is stiffer than bladder muscle from lean pigs. The purpose of the current study was to determine if the loss of bladder muscle compliance is exacerbated in pigs manifesting robust MetS. Detrusor muscle strips from Ossabaw pigs maintained on either an excess kcal atherogenic diet or a control diet were mounted initially at slack length in muscle baths. Following equilibration, muscle strips were stimulated with supramaximal electrical field stimulation (EFS: 20 V, 60 Hz, AC, 10 sec). Following relaxation, strips were stretched in 0.5–1.5 mm increments. The cycles of incremental stretches followed by EFS‐induced contractions were repeated every 5 minutes until maximum total tension was achieved or the tissue yielded. Normalized passive and active length‐tension (L‐T) curves were generated. Due to small sample sizes (n=3/group), no statistical analyses are reported. However, both mean passive and active L‐T curves for bladder muscle from MetS pigs are elevated above the respective control curves indicating that MetS results in increased contractility in addition to decreased compliance of the bladder muscle. Stiffer bladders that are hypercontractile could explain both the incomplete voiding and increased urinary frequency experienced in diabetes which is characteristic of metabolic syndrome.

(R Sharma is an APS Undergraduate Summer Research Fellow; B Harden was an Indy Project Seed Scholar; Support: NIH RR013223, HL062552)
Melanocortin receptors mediate renal sympathetic activation to leptin in obesity
Authors: Rahmouni Kamal , Morgan Donald A
Published: June 2021

Leptin acts in the brain to decrease appetite and body weight and increase sympathetic nerve activity (SNA). Obese mice are resistant to the metabolic actions of leptin but have intact renal SNA responses to leptin. The mechanisms that mediate renal SNA response to leptin in obesity remain unknown. We have previously shown the importance of the melanocortin receptors (MCR) in mediating renal SNA response to leptin. Here, we examined the role of MCR in mediating renal sympathoactivation to leptin in obesity by testing the effect of MC3/4R blockade with SHU9119 (30 pM) on the renal SNA response to intracerebroventricular (ICV) leptin in diet‐induced obese (DIO) and agouti obese mice. DIO in C57BL/6J mice was obtained by exposure to 45% high fat diet for 20 weeks. The rise in renal SNA induced by ICV leptin (5 μg) was of the same magnitude in lean (129±10%, n=15), DIO (117±15%, n=8) and agouti obese (115±16%, n=6) mice. ICV pre‐treatment with SHU9119 inhibited (P<0.05) the increase in renal SNA induced by ICV leptin in lean (23±8%, n=10), DIO (9±11%, n=10) and agouti obese (51±14%, n=5) mice. In lean mice, ICV MTII (agonist of MC3/4R, 2 μg) increased renal SNA by 140±41%, n=4) and only 18±9% in presence of SHU9119 (n=5, P=0.003) demonstrating that blockade of renal SNA response by SHU9119 is due to inhibition of MC3/4R. Our data demonstrate the importance of the MCR as key mediators of renal sympathetic activation to leptin in obesity.
Cold Pressor Test: Medullo‐Spinal Pathways and Neurotransmitters
Authors: Nakamura Takeshi , Sapru Hreday N
Published: June 2021

The neural pathways and transmitters that mediate pressor and tachycardic responses to the cold pressor test are not firmly established. The present experiments were designed to delineate these pathways. Experiments were carried out on urethane‐anesthetized, artificially ventilated, barodenervated, male, Wistar rats. The rats were fixed in a stereotaxic instrument and placed in a plexiglass rectangular dish. Ice‐cold water was rapidly poured into the dish to immerse the ventral half of the rat's body and then removed by rapid suction. Cold water immersion for 2 min elicited an increase in mean arterial pressure (40–46 mmHg) and heart rate (60–76 bpm). Bilateral vagotomy had no significant effect on the pressor responses but significantly reduced the tachycardic responses. Bilateral blockade of ionotropic glutamate receptors (IGLURs), by injections of D‐AP7 and NBQX, in the rostral ventrolateral medullary pressor area (RVLM) or the spinal cord significantly decreased the pressor responses. HR responses remaining after the blockade of IGLURs in the spinal cord were abolished after bilateral vagotomy. These results indicate that: 1) the pressor and tachycardic responses to cold pressor test are mediated by ionotropic glutamate receptors in the RVLM and the spinal cord, and 2) inhibition of the vagal input to the heart also contributes to the tachycardic responses.

Support: NIH grants HL 024347 and HL076248.
Microinjections of alpha‐MSH into the nucleus ambiguus of the rat elicit vagally mediated bradycardia
Authors: Chitravanshi Vineet C , Sapru Hreday N
Published: June 2021

Alpha melanocyte stimulating hormone (alpha‐MSH) is one of the pro‐opiomelanocortin‐related end products. Alpha‐MSH containing neurons are found in different hypothalamic nuclei and the nucleus tractus solitarius. Since these brain regions are known to project to the nucleus ambiguus (NA), it was hypothesized that alpha‐MSH may elicit cardiac effects in the NA. Experiments were done on urethane‐anesthetized, artificially ventilated, adult, male, Wistar rats. NA was identified by microinjections (30 nl) of L‐glutamate; bradycardic responses with no changes in blood pressure (BP) were elicited. Unilateral microinjections (30 nl) of different concentrations (0.1–1.6 mM) of alpha‐MSH into the NA elicited bradycardic responses (7–44 beats/min) with no changes in BP. The bradycardic responses to maximally effective concentration of alpha‐MSH (0.8 mM) lasted for 10 min and were blocked by prior microinjections of a specific MC4 receptor antagonist. MC4 receptor antagonist by itself elicited a small decrease in heart rate. Bilateral vagotomy abolished the bradycardic responses to microinjections of alpha‐MSH into the NA. These results indicate that: 1) microinjections of alpha‐MSH into the NA elicit bradycardia, 2) the bradycardia is vagally mediated, 3) MC4 receptors mediate these actions, and 4) the melanocortin input to the NA may be tonically active.

Support: NIH grants HL024347 and HL076248.
Increases and Decreases in Arterial Pressure Result in the Activation of Phenotypically Different Populations of Neurons in the Nucleus of the Solitary Tract (NTS)
Authors: Austgen James R , Friskey Sarah A , Heesch Cheryl M , Hasser Eileen M
Published: June 2021

The NTS is the termination site of multiple visceral afferents, including the arterial baroreceptors. As expected, increasing blood pressure (BP) activates second order baroreflex neurons in the NTS. However, decreasing BP also activates NTS neurons. We hypothesized that different phenotypes of neurons are activated by increases vs. decreases in BP. Male Sprague‐Dawley rats, instrumented with arterial and venous catheters, received microinjections of the retrograde tracer Fluoro‐Gold (FG) into the caudal ventrolateral medulla. After 5d, animals were administered (i.v.) saline, phenylephrine (PE: 15 mg/kg/min), or diazoxide (DZ: 50 mg/kg, supplement 12.5 mg/kg) and monitored for 90 min. Rats were perfused, brains cut into 30 μm coronal sections, and sections immunohistochemically treated for Fos and neuronal nitric oxide synthase (nNOS) or tyrosine hydroxylase (TH). Fos expression was significantly enhanced by increases (PE, 201 ± 39) and decreases (DZ, 206 ± 41) in BP compared to saline (18 ± 6). Increased BP resulted in significantly greater activation of FG (consistent with baroreflex activation) and nNOS containing neurons compared to saline. In contrast, decreases in BP resulted in significantly greater activation of TH containing neurons compared to saline. Thus, phenotypically separate populations of NTS neurons are activated by increases vs. decreases in BP.

AHA #0710005Z (JRA); HL54669 (EMH)
Low doses of corticosterone act in the dorsal hindbrain to enhance the arterial pressure response to both acute and repeated stress
Authors: Looney Benjamin M , Daubert Daisy L , Su Ye , Scheuer Deborah A
Published: June 2021

We have shown that chronic treatment of the dorsal hindbrain (DHB) with 100% corticosterone (Cort) pellets enhances the mean arterial pressure (MAP) response to acute stress. The current experiments were performed to determine the effect of lower doses of Cort on the arterial pressure and heart rate responses to acute and repeated stress. Experiments were performed on male Sprague‐Dawley rats implanted with DHB Sham, 10%, 50% or 100% Cort pellets, n=4–6 per group. MAP and heart rate were measured via radiotelemetry (Data Science International). Rats were subjected to 1 h of restraint stress on days 6, 13, 14 and 15 days post pellet implantation. All doses of Cort significantly enhanced (P<0.05) the integrated MAP response to both acute and repeated stress relative to sham treatment. For example, on day 6 DHB sham, 10, 50 and 100% Cort treatment increased arterial pressure by 1509±496, 2736±193, 2737±499 and 2471±412 mmHg/60 min respectively, and on day 14 by 1959±328, 2952±414, 2435±301 and 2612±301 mmHg/60 min respectively. There were no significant differences (P=0.08) in baseline MAP between groups, but the 10% dose increased MAP from 103±3 on day 6 to 109±4 mmHg on day 15 (P<0.05). There were no significant effects of DHB treatment on baseline HR or the integrated HR response to stress. We conclude that even low doses of Cort enhance the arterial pressure response to acute and chronic stress.

Funded by NIH grant # HL 076807.
Endothelin‐1 (ET‐1) and ET‐A receptors mediate chronic hypoxia‐(CH)‐induced inflammation in rat carotid body
Authors: Dinger B , Liu X , He L , Fidone S
Published: June 2021

CH in rat carotid body induces an inflammatory response which is characterized by immune cell invasion and the expression of pro‐inflammatory cytokines. In the present study, we have investigated the role of ET‐1 and ET‐A receptors in the development of CH‐induced inflammation. Following 7 days of CH (380 Torr), immunocytochemical (ICC) studies demonstrated elevated levels of ET‐1 and ET‐A receptors in O2‐sensitive type I cells. In addition, double‐label ICC showed that following CH, ET‐A receptors were also expressed on invasive CD45+ immune cells distributed in tissue surrounding chemosensory cell lobules. Concurrent treatment with the ET‐A/B receptor antagonist, bosentan (200mg/kg/day), blocked ED‐1+ immune cell invasion and the up‐regulation of cytokines, including interleukin‐1β (IL‐1β), IL‐6 and tumor necrosis factorα (TNFα). Moreover, bosentan treatment blocked CH‐induced increased expression of acid sensitive ion channels (ASICs) in chemoafferent neurons in the petrosal ganglion (PG), and drug treatment significantly reduced chemoreceptor hypersensitivity. Our findings are consistent with the hypothesis that CH‐induced inflammation involves the up‐regulation and release of ET‐1 from type I cells; ET‐1 acts in an autocrine/paracrine mechanism via ET‐A receptors on chemosensory type I cells, and immune cells to promote an inflammatory response. USPHS Grants NS 12636 and NS 07938.
Mitochondrial rieske protein, are you a real hypoxic sensor in pulmonary artery smooth muscle cells?
Authors: Korde Amit S , Wang Yong‐Xiao
Published: June 2021

It is well established that hypoxia causes vasoconstriction in pulmonary arteries (HPV), which serves as an important physiological mechanism to preserve the sufficient matching of regional alveolar ventilation and pulmonary, but may also result in pulmonary hypertension when alveolar hypoxia becomes global. While the molecular mechanisms underlying HPV are incompletely understood, there is extensive evidence to indicate that mitochondria are involved in hypoxic sensing to mediate hypoxic cellular responses in pulmonary artery smooth muscle cells (PASMCs). In this study, we have found the activity of isolated mitochondrial complex III from mouse pulmonary arteries is significantly increased during hypoxic stimulation. Hypoxia results in a large increase in reactive oxygen species (ROS) generation as well in isolated mitochondrial complex III. Rieske protein knockdown by siRNAs blocks hypoxia‐induced increase in the activity and ROS formation of isolated mitochondrial complex III from mouse PASMCs, while gene overexpression of rieske protein produces an opposite effect. Interestingly, we have also shown that the iron chelator desferrioxamine blocks hypoxic increase in the activity and ROS formation of isolated complex III from mouse pulmonary arteries. Overall, our data indicate that mitochondrial complex III rieske protein may function as a major hypoxic sensor to affect ROS generation leading to hypoxic cellular responses in PASMCs.