bluuebunny/crossref_metadata_2025 huggingface dataset:
Significance of Oxidative and Nitrosative Stress in Systemic Lupus Erythematosus
Authors: Wang Gangduo , Pierangeli Silvia S. , de Martinez Elizabeth Papalardo , Ansari G.A.S. , Khan M. Firoze
Published: June 2021

Autoimmune diseases (ADs) such as systemic lupus erythematosus (SLE) and rheumatoid arthritis are chronic and life‐threatening disorders. Despite high prevalence of these diseases, molecular mechanisms underlying systemic autoimmune response remain largely unknown. Previous studies in our laboratory using an autoimmune‐prone MRL+/+ mice suggested an association between oxidative/nitrosative stress and autoimmunity. However, relevance of oxidative/nitrosative stress in disease prognosis and pathogenesis in humans is not understood. To investigate the status of oxidative/nitrosative stress in ADs, sera from 20 SLE patients and 20 age‐ and gender‐matched healthy subjects (controls) were evaluated for oxidative/nitrosative stress markers. Serum analysis showed not only higher levels of both anti‐malondialdehyde (MDA) and anti‐4‐hydroxynonenal (HNE)‐protein adduct antibodies, reflecting increased formation of lipid peroxidation‐derived aldehydes (MDA and HNE) and their conjugation to endogenous proteins. Serum Cu/Zn superoxide dismutase (an antioxidant enzyme) levels were significantly reduced in patients with SLE compared to controls, suggesting a compromised antioxidant balance. Furthermore, sera from SLE patients had significantly higher levels of iNOS and nitrotyrosine, suggesting nitrosative stress in these patients. These findings further support an association between oxidative/nitrosative stress and SLE, and suggest that oxidative/nitrosative stress markers may be useful in evaluating the prognosis of SLE as well as in elucidating the mechanisms of disease pathogenesis.
Direct mitochondria permeabilization by a new anticancer agent erucylphosphohomocholine
Authors: Lemeshko Victor V. , Kugler Wilfried
Published: June 2021

Alkylphosphocholines are a new class of anticancer agents also representing a potential source for the treatment of tropical diseases caused by Leismania and Trypanosoma parasites. Most of the reported data suggest that mitochondria might be a main target, although direct mitochondrial effects of alkylphosphocholines have never been studied so far. In this work we show that erucylphosphohomocholine, a new antineoplastic alkylphosphocholine, permeabilized the inner membrane of isolated rat liver mitochondria, causing (i) a decrease of the inner membrane potential, (ii) an increase in state 4 respiration, (iii) a significant decrease in the rate of ATP synthesis, and (iv) low amplitude swelling of mitochondria at concentrations of 25–50 uM. In the presence of cyclosporin A, it strongly inhibited mitochondrial respiration. At 100 uM, erucylphosphohomocholine caused biphasic high amplitude swelling of mitochondria. On the other hand, oligomycin or cyclosporin A partially protected certain human cancer cell lines against apoptosis induced by this drug. Our findings demonstrate that direct mitochondrial alterations might be involved in anticancer and antiprotozoal activities of alkylphosphocholines. (Supported by DIME, National University of Colombia, Medellin Branch; by a grant from the Volkswagen‐Stiftung, Germany, VWZN2047; and Genzyme Pharmaceuticals (Liestal, Switzerland).
Mathematical Model of Tissue Oxygenation in Early Sepsis
Authors: Fraser Graham Mathew , Goldman Daniel , Ellis Christopher G.
Published: June 2021

The defining characteristic of sepsis is a progressive microvascular dysfunction remote to the locus of infection. Our objective was to quantify the time course of changing tissue PO2 due to microvascular injury in early sepsis using an established mathematical model. Experimental data obtained from a rat cecal‐ligation and perforation (CLP) model of sepsis has demonstrated a progressive FCD loss at 2, 3 and 4 hours (11±4%, 15±1%, 21±5% SHAM vs 18±3%, 32±5%, 48±7% CLP respectively). Microvascular blood flow was recorded in skeletal muscle using dual wavelength intra‐vital video microscopy. Capillaries were analyzed at baseline in vivo for O2 saturations and hemodynamics. Custom vascular mapping software was used to reconstruct the real geometry (length, diameter, tissue depth and connections between vessels) of the experimental network (Volume 420 × 420 × 60 μm). A constant consumption computational model applied to the FCD conditions at each time point found mean tissue PO2 was not different between SHAM and CLP at 2 hours (34.2±4.3 vs 33.3±4.2 mmHg) or 3 hours (33.0±4.3 vs 31.2±4.5 mmHg) post. At 4 hours tissue PO2 was significantly different between SHAM and CLP (32.7±4.3 vs 25.9±6.5 mmHg, p < 0.01). This simulation suggests that a substantial FCD loss precedes a significant decrease in tissue oxygenation due to diffusional exchange between adjacent capillaries.

Supported by CIHR grant to DG and CGE.
Calcium waves along arteriolar endothelium enhance conducted vasodilation during blood flow control
Authors: Segal Steven S. , Brekke Johan F. , Shui Bo , Doran Robert M. , Tallini N. Yvonne , Kotlikoff Michael I.
Published: June 2021

Conducted vasodilation (CVD) integrates electrical and chemical signals to coordinate smooth muscle relaxation along arterioles. In vitro studies suggest that acetylcholine (ACh) initiates both signals through a local increase in endothelial cell calcium (EC Ca2+). To determine whether EC Ca2+ increases along arterioles during CVD in vivo, we studied transgenic Cx40BAC‐GCaMP2 mice with the Ca2+ sensor GCaMP2 expressed under the regulatory control of endogenous connexin40 (Cx40). Immunostaining confirmed selective coexpression of GCaMP2 and Cx40 in arteriolar EC. In the cremaster muscle of anesthetized males (9–16 wk; n =14), Ach microiontophoresis (1 μm tip, 1 μA, 1s) onto an arteriole [diameter (D)=30±2 μm; n=33] triggered a rapid rise in fluorescence at the stimulus site (t1/2=250±31 ms, ΔF/Fo=0.39±0.04; n=10). The EC Ca2+ signal propagated bidirectionally at 116±6 μm/s, decaying by 50% in 204±17 μm with oscillations up to ∼1 mm away. Dilations were biphasic: ‘Rapid’ (>2 mm/s) CVD (ΔD=10–15 μm) occurred along several mm without remote changes in EC Ca2+ and additional ‘slow’ CVD (ΔD=3–5 μm) occurred in the wake of an EC Ca2+ wave. These respective signaling pathways effectively coordinate the onset, magnitude and duration of arteriolar dilations during the local control of tissue blood flow. (Support: NIH HL41026, HL56786, HL45239, DK65992 & F2HL76999).
Cell specific expression of Sphingosine‐1‐Phosphate receptors in endothelial cells and smooth muscle cells
Authors: Hudson Nicola K , Speake Paul F , Robinson Nicola J , Baker Philip N , Taggart Michael J
Published: June 2021

Much attention has recently been given to a vasoactive role for the lipid metabolite sphingosine‐1‐phosphate (S1P). We have previously shown S1P to control net tone of human maternal and placental arteries. These data suggest S1P actions may be mediated by cell‐specific receptor expressions with potentially vasoconstrictory actions on smooth muscle cells (SMCs) and dilatory actions on endothelial cells (ECs).

In the absence of efficacious antibodies, the first step in validating this hypothesis was to establish expression of mRNA encoding S1P receptors (S1PR) 1–3 in purified cell populations. Human reference RNA was used to construct standard curves and 18s as an internal calibrator. We used human primary SMC's from myometrium and cultured umbilical vein EC's numbering from 1250 to 1 in order to assess cellular heterogeneous expression patterns, another possible contributor to net functional vasoactive action of S1P.

Results show S1PR 1–3 to be expressed in both ECs and SMCs. S1PR 1 is the predominant receptor in ECs, whereas SMCs express S1PR 3 to a greater extent than the other two sub‐types. We found similar expression patterns to be independent of cell number suggesting homogenous expression.

Future work will characterize cell‐specific expression of these receptors in native blood vessel cells in order to further elucidate the role of S1P in regulation of human vascular tone.

Research supported by Ardana PLC.
Nitric oxide synthase in the rat spinotrapezius muscle related to fiber type
Authors: Yannaccone Andrew Thomas , Shall Mary , Ratz Paul , Miner Amy , Song Bjorn , Pittman Roland
Published: June 2021

Identifying the spatial distribution of nitric oxide synthase (NOS) isoforms is important for understanding nitric oxide (NO) signaling pathways. There are gaps in the fundamental understanding of the regulation of NO production in the vascular region. We examined localization of NOS I (nNOS) in the spinotrapezius muscle of male Sprague‐Dawley rats using immunohistochemistry, muscle fiber typing and confocal microscopy. Muscles were removed and frozen in liquid N2‐chilled isopentane. Ten μm cross‐sections were cut and incubated with a NOS I antibody and a vinculin antibody to visualize plasma membranes using fluorescent secondary antibodies. Sections were counterstained with fluorescent phalloidin and 4′,6‐diamidino‐2‐phenylindole dihydrochloride to visualize F‐actin and nuclei, respectively. Muscle fiber types were determined by incubating sections in primary antibodies for four myosin heavy chain (MHC) isoforms and then in secondary antibodies using 3,3‐diaminobenzidine to visualize. Image inspection demonstrated NOS I in the sarcolemmal region and even distribution of muscle fibers expressing types I and II MHC. The exact nature of how NO exerts its effects on the microcirculation is still under investigation. A better appreciation of the spatial distribution of NO sources should improve quantitative understanding of NO concentration dispersal. Support: HL18292, HL079087, 5P30N5047463.
Significant and Sustained Impairment of Distal Limb Perfusion in Wistar Kyoto (WKY), Lean (LZR) and Obese (OZR) Zucker and Spontaneously Hypertensive Rat (SHR) Models of Hindlimb Ischemia Is Revealed by Femoral Vein Outflow During Functional Hyperemia
Authors: Ziegler Matthew A , Miller Steven J , Bills Randall G , Tune Johnathan D , Bohlen H Glenn , Murphy Michael P , Dalsing Michael C , Unthank Joseph L
Published: June 2021

Following femoral artery ligation/excision, hindlimb perfusion at rest and during contraction‐induced hyperemia was assessed by venous outflow (VO). After 2 wks, a sustained impairment at rest was observed only in WKY rats (72 ± 9.2% of control). The maximal VO increase during muscle contraction induced by electrical stimulation was greatly reduced in the ligated vs control limb (505±37% vs 119±36% in WKY). In Zucker rats, the maximal VO increase in the chronically occluded limb during muscle contraction was greater in the lean than in the obese rats (112 ± 23 vs 22 ± 10%). Administration of ramipril for 2 weeks, initiated 2 weeks after femoral artery excision, greatly improved the perfusion during muscle contraction in SHR (206 ± 14% vs 82 ± 36%). These results indicate that 1) a sustained perfusion deficit exists even at rest in WKY and skeletal muscle contraction unmasks a much greater perfusion deficit, 2) the presence of vascular disease risk factors can exacerbate the insufficiency as seen between LZR and OZR, and 3) rodent models might be useful in predicting clinical outcomes, as observed in SHR receiving ramipril therapy post‐occlusion. Contrary to the common perception, the data obtained during rest and contraction‐induced hyperemia by venous outflow demonstrate that some rodent models of hindlimb ischemia can mimic the human condition of subcritical arterial insufficiency. Supported by HL42898.
Proangiogenic role of PPARá in the mouse hind limb ischemia model
Authors: Rizvi Yasmeen , Oyekan Adebayo
Published: June 2021

Angiogenesis (ANG) is a neovascularization response to tissue ischemia, tumor growth or inflammation. PPARá is a negative regulator of inflammation but its role in ANG is controversial. PPARá WT and KO mice were treated with vehicle or clofibrate (CF; 240 mg/kg) starting from 24h after ligation of the left femoral artery. Perfusion of both limbs was measured using laser Doppler flow meter or Imager system on days 0, 3, 7, 14, 21, 28, and 35. Vascular reactivity to AII (5–250 ng), PE (0.5– 2.5 ìg), SNP (0.5–2.5 ìg) and Ach (0.5–2.5 ìg) were determined in the Kreb's perfused hind limb preparation. Following ligation, perfusion was reduced (29±7%, WT; 25±4%; KO) followed by a time‐dependent recovery that was 72±23% (WT) and 37±9% (KO) on Day 35. Except for Day 3, CF did not affect perfusion in either group of mice suggesting a pro‐angiogenic role during early neovascularization. AII‐induced increase in perfusion pressure was two‐fold greater between Days 3 and 7/14 but markedly reduced at Day 35. By contrast, PE‐induced vasoconstriction was unchanged. CF inhibited AII responses on Days 7,14 and 35 but elicited no effect on Day 3 while it inhibited PE response on Days 3 and 35. CF enhanced SNP response on Day 35 whereas Ach response remained unchanged. These data demonstrate that PPARá promotes ANG in the mouse hind limb ischemia model. In addition, vascular reactivity appears to be independent of recovery in perfusion in this model.
Dissociation of estrogen (E2) effects on the vasculature of diabetic rats
Authors: Mitchell Adam , Rogers Jennifer , Myers Adam , Mulroney Susan
Published: June 2021

Decreases in circulating estrogen seen in human female diabetics may exacerbate vascular impairment seen in the hyperglycemic state alone. E2s role as a vasoprotective agent is not clear. In normal animals, we and others have shown that E2 increases NO production while decreases sensitivity to the vasodilator ACh and increases sensitivity to the vasoconstrictor PE. To examine whether there is dysregulation of E2 affect in the diabetic (D) vasculature, diabetes was induced by STZ injection in adult female rats concurrent with ovariectomy (OVX) and E2 replacement. After 8 weeks, mesenteric arteriolar reactivity to ACh and PE was measured on a wire myograph. STZ decreases ACh sensitivity (p<0.01) vs non‐diabetic (ND) vessels. While OVX reverses this effect (p=0.04), E2 replacement was same as OVX. Similarly, while STZ increases PE reactivity (p=0.02), and OVX reverses the effect (p=0.03), E2 replacement alone was not different than OVX. In the same animals, E2 replacement prevented the STZ‐induced increases in both GSI (~30%) and TIFI (~20%), indicating E2 efficacy in the D kidney. These data confirm the vascular effects of OVX in ND rats (reported by our lab), and extend the findings by showing that the effect of E2 replacement in the D female is lost in the resistance vessels, but not kidney. This dissociation in early diabetes may predispose the female to development of diabetic vascular complications.
Cranberry juice consumption ameliorates endothelial dysfunction during estrogen deficiency: balance between NO and ROS
Authors: YUNG Lai Ming , Wong WT , Tian XY , Leung FP , Chen ZY , Yao XQ , Vanhoutte PM , Huang Y
Published: June 2021

This study examine whether consumption of cranberry juice could ameliorate endothelial dysfunction in estrogen deficiency. Vascular reactivity in aortas were studied in organ baths; protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, angiotensin converting enzyme (ACE), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R), gp91phox and nitrotyrosine were detected by Western blot. Angiotensin II level was detected by immunohistochemical staining. Relaxations were impaired in OVX rats; which was prevented by AT1R blocker. Cranberry juice consumption ameliorated such endothelial dysfunction. Phenylephrine‐induced tension was higher in OVX rats, which was attenuated upon juice treatment. Our results showed cranberry juice consumption in OVX rats (i) increased p‐eNOS level; (ii) restored protein expressions of ACE, AT1R, gp91phox and nitrotyrosine; (iii) restored angiotensin II level in the aorta. These suggest chronic oral administration of cranberry juice during estrogen deficiency augments bioavailability of NO, due to a reduced level of AT1R‐mediated oxidative stress.
Smooth muscle α1D‐adrenoreceptors mediate phenylephrine‐induced endothelial Ca2+ transients in hamster cremaster arterioles
Authors: Jackson William F. , Boerman Erika M. , Lange Erica J. , Lundback Steven S.
Published: June 2021

The α1‐adrenoreceptor (α1‐AR) agonist, phenylephrine (PE) induces Ca2+ transients in endothelial cells (EC) of arterioles, possibly due to movement of Ca2+ or IP3 from smooth muscle (SMC) into EC via gap junctions. However, the presence of α1‐AR on EC has not been excluded, and their identity in arterioles only has been inferred from pharmacology. Therefore, we determined which α1‐AR subtypes are expressed by SMC and EC, and which subtype mediates PE‐induced EC Ca2+ transients. Cell specific expression was assessed by real time RT‐PCR in samples of 50 cells from dissociated hamster cremaster arterioles using primers for α1A‐, α1B‐ and α1D‐AR with α‐SMC actin and eNOS as respective SMC and EC markers. SMC expressed α1A‐ and α1D‐AR, but not α1B‐AR, and no α1‐AR transcripts were detected in EC expressing eNOS. Western blots of vessel homogenates confirmed α1D‐AR expression, but surprisingly, did not detect α1A‐AR in up to 100 μg protein, despite positive results in heart. In cannulated vessels, the Ki for inhibition of PE‐induced constriction was 2.95 nM for the α1D‐AR antagonist, BMY 7378 and 4.11 nM for the α1A‐AR antagonist, 5‐methylurapadil, data consistent with PE acting through α1D‐AR. In vessels with Fura‐2‐loaded EC, PE‐induced constriction and EC Ca2+ transients were abolished by the α1‐AR antagonist, prazosin (30 nM). Thus, PE‐induced EC Ca2+ transients are mediated by SMC α1D‐AR. Supported by HL32469 to WFJ.
Cerebrovascular Autoregulation is Lost with Diabetes Progression
Authors: Elgebaly Mostafa Mahmoud , Mezzetti Erin , Sachidanandam Kamakshi , Dorrance Anne , Ergul Adviye
Published: June 2021

Cerebrovascular tone is critical in autoregulation of flow. Previously, we have shown remodeling of cerebrovasculature in diabetes and when ischemia / reperfusion injury was added, there was interestingly reduced infarct, yet increased hemorrhage. Hence the hypothesis behind the current study was that diabetes causes cerebrovascular remodeling leading to increased vascular tone. Aims were: to examine both the effects of diabetes and its progression on myogenic tone, vascular structure and mechanics. Pressurized middle cerebral arteries from 10 and 18 week‐old Wistar and diabetic Goto‐Kakizaki (GK) rats using the arteriograph showed that: GKs lost their myogenic tone with diabetes progression (18±3 vs. 5±3, p<0.023). At 10 weeks there was no significant remodeling. At 18 weeks GK rats had higher media: lumen ratio (0.179 ± 0.04 vs. 0.075± 0.01, p<0.0001) and lower tone (5±3 vs. 20±5, p<0.0004) compared to control. Cerebrovascular changes starts early with diabetes and worsens with disease progression thus vascular dysfunction ensues via altered mechanics and structure. This may account in part for worse hemorrhage in diabetes.
Connexin Expression in pial and penetrating cerebral arterioles of the rat
Authors: Ngai Al , Solen Joell , Nguyen Thien‐Son , Lampe Paul
Published: June 2021

Intracerebral, penetrating arterioles provide a conduit by which parenchymal dilator signals are relayed to upstream cerebral arteries. Vascular conduction likely is regulated by the expression of gap junctions, which provide the most direct pathway for intercellular coupling. However, little is known regarding gap junctional connexin expression in penetrating arterioles. In this study, we compared the expression of connexin (Cx) 37, 40, 43 and 45 in penetrating arterioles with that in pial arterioles. Arterioles were isolated from the cortices of Sprague Dawley rats; connexin expression was determined by immunofluorescence staining and confocal laser microscopy. In both types of vessels, Cx37 immunostaining was observed mainly in smooth muscle, whereas Cx40 was found in both smooth muscle and endothelium. Cx43 staining appeared to be restricted adventitially to leptomeningeal tissue, but was absent in vascular cells. In penetrating arterioles, Cx43 punctate labeling was associated with aquaporin‐4 positive processes that envelop the vessels, suggesting the presence of astrocytic end‐feet. Aquaporin‐4 staining was also found in leptomeningeal tissue associated with pial arterioles. There was no evidence of Cx45 expression in these vessels. We conclude that both pial and penetrating cerebral arterioles have a similar distribution of vascular connexin expression. Funded by AHA 0755770Z.
Neurogenic control of cerebral vasculature: potential role of P450 Epoxygenase
Authors: Iliff Jeffrey J. , Wang Ruikang , Baumann Thomas K , Alkayed Nabil J
Published: June 2021

Epoxyeicosatrieonic acids (EETs) are cerebral vasodilators formed by P450 epoxygenases (EPOX) and metabolized by soluble epoxide hydrolase (sEH). We recently reported the presence of EPOX and sEH in vasodilator nerves surrounding large cerebral arteries. In the current study, we elicited dilation of these vessels by trigeminal ganglia (TG) sensory stimulation and examined the expression of EPOX and sEH in TG. Western blot detected sEH expression in rat and mouse TG (n=2 each), and RT‐PCR identified P450 2J but not 2C EPOX isoforms in rat (n=2 from 8 animals) and mouse TG (n=1 from 3 animals). Intra‐oral sensory stimulation with capsaicin (50μl, 1–100μM) evoked vasodilation in the mouse MCA territory (14% maximal response, n=2), detected by optical micro‐angiography. These findings demonstrate that intra‐oral sensory stimulation elicits cerebral artery dilation, and that TG neurons express components of the EETs signaling system. Future studies will evaluate if EETs mediate the hyperemic response to capsaicin, and the neuroanatomic basis for such response. Supported by NS44313 to NJA and a predoctoral NRSA to JJI.
Effects of exogenous and endogenous carbon monoxide on dorsal aorta pressure and cardiac output in the rainbow trout
Authors: Achterhoff Beth , Morrterlini Roberto , Nakatsu Kanji , Olson Kenneth , Dombkowski Ryan
Published: June 2021

Carbon monoxide (CO) is an important regulator of vasomotor tone in mammals. Its endogenous effects in non‐mammals, however, are unknown. Therefore, surgeries were performed on rainbow trout (Oncorhynchus mykiss), during which the dorsal aorta and nares were canulated and a flow probe was placed around the ventral aorta. Dorsal aorta pressure, stroke volume, heart rate, and respiration rate were compared on a resting trout, trout treated with inhibitors of the endogenous CO production, and trout treated with exogenous CO (delivered via a soluble ruthenium compound, CORM‐3). Zinc protoporphyrin‐IX, and inhibitor of heme‐oxygenase, elicited a dose‐dependent (at 5, 10, and 20 μmol/kg) increase in dorsal aorta pressure (approximately 20, 40, and 50% respectively). QC‐10, a non‐specific HO inhibitor, and QC‐15, a specific inhibitor of the HO‐1 isoform, both elicited a 25% increase in dorsal aorta pressure at 3 mg/kg. QC‐15 elicited a maximal response in approximately 5 minutes, while QC‐10 responses did not reach maximum until 1 hour. Both QC‐10 and QC‐15 caused small but significant increases in cardiac output. Unexpectedly, 30 μmol/kg CORM‐3 caused dorsal aorta pressure to approximately double in minutes. The results of the inhibitors suggest that CO is constitutively produced in trout to modulate vascular resistance. The surprising result with exogenous CO delivered via CORM‐3 requires further study.
Natriuretic activity of prehypertensive Dahl salt‐sensitive (DS) and salt‐resistant (SS13BN) rats
Authors: Nakano Daisuke , Middleton Christopher L. , Socha Heather M. , Boesen Erika I. , Pollock David M.
Published: June 2021

We have recently demonstrated that activation of the ETB receptor in the renal medulla induces a natriuretic response in anesthetized rats and that sodium excretion of an acute salt load in the conscious rat requires the ETB receptor. We hypothesized that animals genetically susceptible to salt‐induced hypertension (DS) would have reduced ETB‐mediated natriuresis compared to salt‐resistant controls (SS13BN). Therefore, the aims of the current studies were to determine the natriuretic response to 1) intramedullary ETB activation and 2) an acute oral salt load in DS and SS13BN rats. Sarafotoxin 6c (S6c; 1–10 pmol/kg/min; n = 6), a selective ETB agonist, infused into the renal medullary interstitium dose‐dependently increased the sodium excretion without affecting systemic blood pressure, although no difference was observed in the response between DS and SS13BN (10 pmol/kg/min; 2.3 ± 0.2 μmol/min and 2.0 ± 0.3 μmol/min, respectively). In a separate series of experiments, conscious rats received an acute salt challenge of 900 μEq Na p.o. Both strains exhibited significant sodium excretion within the first 12 hours post challenge (DS: 1.48 ± 0.13 mEq; SS13BN: 1.68 ± 0.11 mEq). Again, there was no difference between the strains. In conclusion, these studies suggest that acute ETB‐dependent natriuretic mechanisms are intact in both salt‐resistant and salt‐sensitive strains of rats.
Acute administration of 5HT1a serotonin receptor agonist, in the dorsal raphe nuclei (DRN) or systemically, increases whereas peripheral chronic treatment inhibits sodium intake in rats
Authors: Badaue‐Passos Daniel , de Magalhães‐Nunes Ana Paula , Ventura Renato Rizo , Reis Wagner Luis , Reis Luís Carlos , Antunes‐Rodrigues José
Published: June 2021

Opposite effects have been attributed to acute or chronic activation of 5HT1a serotonin receptors. In the DRN, somatodendritic 5HT1a receptors are implicated in the auto‐modulation of serotonergic neurons’ activity. We investigated the effects of acute and chronic activation of 5HT1a receptors in spontaneous or induced sodium intake. Agonist (8‐OH‐DPAT, 7.5 nmol) or antagonist (WAY‐100635, 2 nmol) of the 5HT1a receptor was acutely microinjected into the DRN of furosemide sodium‐depleted rats. 8‐OH‐DPAT increased the sodium intake induced by low sodium diet plus depletion by furosemide (20 mg/Kg, sc) (20.1±1.1 vs 24.9±0.9 mL, at 120 min after 0.3M NaCl presentation). WAY‐100635 did not affect sodium intake in the same paradigm. Likewise, a single sc injection of 8‐OH‐DPAT (250 μg/Kg) induced a higher sodium intake (0.03±0.03 mL vs 4.5±0.5 mL, 120 min after 0.3M NaCl presentation). Conversely, chronic administration of 8‐OH‐DPAT (500 μg/Kg/day, ip, during 3 weeks) inhibited induced sodium intake (8.6±0.9 mL vs 5.8±0.4 mL, 60 min after 0.3M NaCl presentation). Water intake was not affected by the treatment. These data suggest that the time course of the 5HT1a activation may differentially affect sodium intake behaviour. It implies a 5HT1a “down‐regulation” or “desensitization” after chronic administration of the agonist. This work was supported by FAPESP # 03/00327‐8; CNPq # 478981/2004‐0, and Capes.
Elevated Arterial Perfusion Pressure Increases H2O2 in the Renal Medullary Interstitium
Authors: Hu Chunyan , Polichnowski Aaron , Kurth Terry , Mori Takefumi , Ito Sadayoshi , Cowley Allen W.
Published: June 2021

We established a direct role of arterial renal perfusion pressure (RPP) in renal injury using servo‐controlled techniques to maintain the left kidney of Sprague Dawley (SD) rats at normal RPP while exposing the right kidney to a high RPP during the development of hypertension (Hypertension 43: 752, 2004). These results showed that pressure‐induced renal injury was associated with activation of NF‐kβ and TGF–β1 in the renal outer medulla indicating oxidative stress was involved in these mechanically‐induced responses. In the present study, we determined whether medullary interstitial [H2O2], a reactive oxygen species, could be induced by a rapid increase in RPP. Left kidneys of SD rats were denervated, a linear microdialysis fiber implanted in the outer medulla, a ureteral catheter inserted, and an adjustable occluder placed around the aorta proximal and distal to the renal arteries to raise and lower RPP. Following 60 min. equilibration, dialysate was collected (2 μl/min) for 30 min and H2O2 measured at three levels of RPP (Amplex Red). As RPP was increased from 73±1 to 93±1 mmHg, medullary H2O2 was relatively constant (650±62 to 733±89 nM) but when RPP increased to 138±3, H2O2 levels increased to 1237±78 nM (P<0.05) with parallel increases in urinary H2O2 excretion. We conclude that elevations of RPP >100 mmHg rapidly and markedly increase the levels of H2O2 within the renal medulla. (Support: HL‐29587)
Real time measurement of hydrogen peroxide (H2O2) and nitric oxide (NO) release in the renal vein: The effects of Protein Kinase C beta II inhibitor (PKC β ‐) on rat kidneys exposed to extracorporeal shock wave lithotripsy (ESWL)
Authors: Krass Samuel , Zambrano Christopher , Thomas Shawn , Chen Qian , Young Lindon H
Published: June 2021

ESWL clinically treats kidney stones. However, studies have suggested that hypertension and decreased renal function are long term effects of ESWL therapy. ESWL induced renal injury results from free radical production and microvascular injury in the kidney. ESWL causes vasoconstriction, which results in renal injury analogous to an ischemia/reperfusion (I/R) injury. I/R injury is characterized by decreased endothelial nitric oxide. This is mediated by the NO scavenger, superoxide, which results in the formation of peroxynitrite, a potent oxidant. Endothelial NO synthase (eNOS) produces NO when activated. PKC is a key enzyme in the regulation of eNOS. The effects of PKC β‐ (0.055 − 0.55mg/kg) were examined in the renal veins in kidneys exposed to ESWL. PKC β‐was injected through the tail vein immediately post ESWL. NO and H2O2 were directly measured by inserting either NO or H2O2 microsensors (100 μm diameter) into the left renal vein in the anesthetized rat. Preliminary data suggests that PKC β‐attenuates H2O2 in the renal vein 20 minutes post ESWL, which may be related to an NO mechanism. In saline treated controls, NO decreases throughout 20 min post ESWL. The preliminary data thus far support our hypothesis that ESWL increases oxidative stress whereas PKC β‐attenuates oxidative stress.

This study is supported by PCOM, department of neuroscience, physiology, & pharmacology.
MicroRNA expression profiling in placenta from a mouse model of preeclampsia
Authors: Zhou Yi , Guruju Mallikarjuna R , Woods Ashley K , Sharma Ram V , Davisson Robin L
Published: June 2021

Our group discovered a genetic mouse model (BPH/5) that spontaneously develops hallmarks of preeclampsia, which can be used to investigate disease mechanisms. Emerging evidence suggests that microRNAs play an important role in embryonic development. Here we tested the hypothesis that the pathogenesis of preeclampsia is associated with changes in expression of specific miRNAs in developing placenta. Microarray technology was employed for the miRNA expression profiling in BPH/5 and C57Bl/6 fetoplacental units (day 10.5). We observed differential expression of 52 miRNAs in BPH/5 fetoplacental unit vs C57B1/6 controls of which 14 were up‐regulated, and 38 were down‐regulated (fold change >2, p<0.05, n=3). Two of the differentially expressed miRNAs, mmu‐mir‐15b, mmu‐mir‐29c, were further validated by real time stem‐loop RT‐PCR (>3 fold increase in BPH/5 vs. C57, P<0.05; n=3). To understand the contributions of these up‐ or down‐regulated miRNAs to the changes in fetoplacental development in preeclampsia, the potential target genes of miRNAs were identified using three algorithms: PicTar, TargetScan and miRBase. Through computational analysis, these two miRNAs were found to be putative regulators of VEGF, PAPPA, MMP, IGF1 and CCND2, genes that are involved in feto‐placental development. Further validation of miRNA target genes with luciferase reporter assay and miRNA function analysis are in progress.
Decreased induction of heat‐shock proteins with age in ischemia‐reperfusion injured skeletal muscle
Authors: Hammers David W , Adamo Martin , Walters Tom , Farrar Roger P
Published: June 2021

Heat shock proteins (HSPs) are molecular chaperones that are upregulated to stabilize cellular proteins in response to stress. Previously, our laboratory has demonstrated that tourniquet (TK)‐induced ischemia‐reperfusion injury (I/R) is a clinically relevant perturbation to skeletal muscle with age‐related differences in recovery. To investigate the role of HSPs in the age‐related differential recovery to I/R, 6 month‐old (Young) and 24 month‐old (Old) C57BL mice were subjected to 2 hour TK­‐induced I/R of the hind limb, and separated into either 1, 3, 5, or 7‐day recovery groups. H&E staining confirms greater damage in Old in response to I/R at days 3, 5, and 7. Western blot analysis reveals a significant upregulation of HSP 70 in both Young and Old 1 day post‐TK, however HSP 70 content continues to increase another 4‐fold by day 7 in Young, while there is no increase beyond day 1 in Old. This suggests the protective effect of HSP 70 is less in Old, allowing more damage to occur compared to Young. In addition, significantly lower phosphorylation of mTOR and FoxO proteins is demonstrated in Old 7 days post‐TK, suggesting reduced anabolic signaling coupled with elevated protein catabolism is responsible for the impaired recovery with age. This work funded in part by the U.S. Army Medical Research and Material Command grant DAMD17‐03‐1‐0735 to RPF.
Comparison of lower extremity strength, power and muscle area between healthy subjects and mobility‐limited elders
Authors: Reid Kieran F. , Doros Gheorghe , Cloutier Gregory J. , Clark David J. , Carabello Robert J. , Phillips Edward M. , Frontera Walter R. , Fielding Roger A.
Published: June 2021

This study compared muscle strength, power and cross sectional area (CSA) in healthy middle‐aged adults (mean age: 47.3 ± 5 yrs, n = 26), healthy older adults (73.8 ± 4 yrs, n = 21), and older adults with mobility limitations (77.1 ± 5 yrs, n = 21). One repetition maximum strength (1RM) and peak power output (PP) were estimated using the bilateral leg press exercise. Total thigh muscle CSA was assessed using computated tomography. Mobility‐limited elders had significantly reduced muscle strength, PP and CSA compared to both healthy groups (table). There were no significant differences for any of these measures between the healthy‐middle aged or healthy‐older participants. These data suggest that preservation of strength, power and muscle mass in healthy older adults attenuates the age‐related decline in lower extremity functional performance. Additional studies should assess the efficacy of exercise and therapeutic interventions designed to increase muscle quality in older adults with compromised physical functioning.

NIA AG‐18844 and the USDA 58‐1950‐7‐707
Treadmill training does not prevent loss of muscle mass or contractile properties in senescence
Authors: Thomas Melissa M , Betik Andrew C , Wright Kathryn J , Riel Caitlin , Hepple Russell T
Published: June 2021

With aging there is a decline in muscle mass and strength. This study was designed to examine the impact of treadmill training, beginning in late‐middle age and continuing across a 7 mo period when muscle atrophy and functional decline markedly accelerates. 29 mo old rats were divided into 2 groups: half of the animals were exercised on a treadmill for 5 (34T) or 7 mo (36T), while the others maintained normal cage activity (34C and 36C). In situ contractile properties were measured in the Gastrocnemius/plantaris (GAS) and Soleus (SOL) muscles via sciatic nerve stimulation. Mass of the GAS and SOL was lower than young adult (YA) in 34C and 36C. Training had no affect on mass in 34T but decreased mass in 36T vs 36C. GAS tetanic force was lower than YA (12.1 ± 0.9 N/g) in 34T (8.8 ± 0.9 N/g) and 34C (7.1± 1.0 N/g), with no further decline with aging. SOL tetanic force was lower than YA (14.9 ± 0.8 N/g) in 34T (5.4 ± 0.7 N/g) and 34C (5.8 ± 1.0 N/g), with no further decline at 36 mo. In addition, training did not prevent the increase in time to peak force and time to half relaxation seen with aging in controls. In conclusion, treadmill training did not attenuate declining muscle contractile function with aging and may even exacerbate muscle mass decline in senescence.

Supported by CIHR and NSERC (Canada).
Rostral ventral lateral medulla (RVLM) mediates sympathetic vasomotor response to hypoglycemia
Authors: Bardgett Megan Elyse , Stocker Sean
Published: June 2021

Acute hypoglycemia or glucodeprivation increases vasomotor and non‐vasomotor sympathetic nerve activity (SNA). Neurons in RVLM are believed to mediate the increased non‐vasomotor SNA (increased epinephrine levels) seen following 2‐DG induced glucodeprivation but the neural pathway responsible for increases in vasomotor SNA has not been established. The primary goal of this study was to determine whether the increased renal SNA observed during insulin induced hypoglycemia is mediated by the RVLM. Hypoglycemia was achieved in anaesthetized male Sprague Dawley rats (300–450g) by a bolus injection of insulin (0.25U/kg) followed by insulin infusion (7.5 mU/kg/min) for 120 minutes. Insulin decreased blood glucose from 85±5 to 40±1 mg/dl (n=8). Renal SNA significantly increased in response to insulin but not saline infusion (140±10% vs 105±9% respectively) after 75 minutes, while blood pressure did not significantly change. Then inhibition of the RVLM with bilateral injection of the GABAA agonist muscimol (500pmol/100nl) produced a greater reduction in renal SNA of rats infused with insulin vs saline (−90±10% vs −62±7% p<0.05). Injection of saline into RVLM did not affect the increase in renal SNA (137±9% at 75 minutes to 153±15% at 90 minutes). These findings indicate that RVLM neurons play a role in the increased vasomotor SNA during hypoglycemia.
Exogenous Angiotensin‐(1–12) Impairs Baroreflex Sensitivity in the Solitary Tract Nucleus in Anesthetized Sprague‐Dawley Rats
Authors: Arnold Amy C. , Chappell Mark C. , Ferrario Carlos M. , Diz Debra I.
Published: June 2021

Angiotensin (Ang)‐(1–12), a novel angiotensinogen‐derived peptide found in plasma and peripheral tissues, elicits a systemic pressor response that is blocked by either ACE inhibitors or AT1 receptor blockers (Nagata et al., 2006). Depending on the enzymes present in either the circulation or tissues (ACE, ACE2 or neprilysin), Ang‐(1–12) may serve as a precursor to Ang II or Ang‐(1–7) (Chappell et al. 2007). In addition, brain levels of Ang‐(1–12) are higher than Ang I or Ang II. Therefore, we determined baroreflex sensitivity (BRS) control for heart rate to assess whether Ang II attenuation or Ang‐(1–7) facilitation of the reflex predominates in response to solitary tract nucleus (NTS) injections of Ang‐(1–12) (144 fmol; 120 nL) in young (3–5 month‐old) anesthetized Sprague‐Dawley rats. BRS in response to increases in pressure to phenylephrine was measured before and after bilateral NTS injections of Ang‐(1–12). Injection of Ang‐(1–12) resulted in a significant decrease in BRS [n = 8; 1.01 ± 0.08 baseline versus 0.41 ± 0.08 msec/mm Hg after Ang‐(1–12); P < 0.001] which recovered to baseline levels after 120 minutes (0.87 ± 0.06 msec/mm Hg). These results suggest that within the NTS, Ang‐(1–12) modulates BRS in a manner similar to Ang II. The findings are consistent with the interpretation that processing of Ang‐(1–12) to Ang II predominates in this brain area. HL‐51952, HL‐56973
Effects of microinjection of dopamine (DA) intracisterna magna (ICM) or into caudal nucleus of tractus solitarii (cNTS) on the cardiovascular responses to chemoreflex activation in awake rats
Authors: Oliva Neto Waldyr M. , Bonagamba Leni G.H. , Mendes Ricardo A. , Machado Benedito H.
Published: June 2021

There is evidence that DA is a neuromodulator in the brainstem areas involved with the autonomic activity to the cardiovascular system. In present study we evaluated the effect of microinjection of DA ICM or into cNTS on the cardiovascular and respiratory responses to chemoreflex activation in awake rats. Guide cannulas were implanted in the direction of ICM or cNTS and femoral artery and vein catheterized. On the day of the experiments chemoreflex was activated with KCN (iv) before and after microinjection of DA ICM (n=9) or into NTS bilaterally (n=8) and mean arterial pressure (MAP) and heart rate (HR) were recorded. Microinjection of DA ICM, but not in the cNTS, produced a significant reduction in baseline MAP (98±2 vs 84±1 mmHg) and HR (374±10 vs 326±8 bpm). Microinjection of DA ICM or into cNTS produced no significant changes in the magnitude of the pressor and bradycardic responses to chemoreflex activation. Microinjection of DA into cNTS also produced no effect on the tachypneic response to chemoreflex activation. The data obtained with ICM microinjection shows that dopamine receptors in the brainstem are involved in the basal autonomic modulation to the cardiovascular system. However, DA in the cNTS does not modulate the basal autonomic activity to the cardiovascular system neither cardiovascular and respiratory responses to chemoreflex activation. Financial support: CNPq and FAPESP.
Hypoxia activates Calcium‐dependent Chloride Channels in Small Pulmonary Artery Smooth Muscle Cells
Authors: Elsen Frank P , Madden Jane A
Published: June 2021

In small pulmonary artery (SPA) smooth muscle cells (SMC), Cl−/HCO3− exchange seemed to set the level of intracellular pH (pHi) and inhibiting the exchanger prevented the pHi increase during hypoxia. We hypothesized that Ca2+ dependent Cl− channels (ClCa) channels played a role in acute hypoxic pulmonary vasoconstriction (HPV). In patch‐clamp studies using the cell‐attached‐patch configuration we recorded single ClCa channels at different patch potentials in cultured SMC from bovine SPA. Hypoxia increased ClCa channel open times, as did 20 μM caffeine. The ClCa channel antagonist, niflumic acid (100 μM) reduced open times. Using the whole‐cell patch‐clamp configuration we recorded Cl− current amplitudes evoked with a given [Ca2+]i at different test potentials. We determined Cl− as the conducting ion species by comparing the set Cl− equilibrium potential (20 mV) with measured results (∼22 mV). These single channel and whole‐cell current studies show that SPA SMC possess ClCa currents and that they can be activated by hypoxia. Funding: Med Coll WI and VA Med Ctr.
Effects of a lack of Snark on activity, heart rate, and fat and skeletal tissues in mice
Authors: Ichinoseki‐Sekine Noriko , Naito Hisashi , Tsuchihara Katsuya , Kakigi Ryo , Ogura Yuji , Kurosaka Mitsutoshi , Katamoto Shizuo , Esumi Hiroyasu
Published: June 2021

This study clarified the characteristics of Snark+/− mice in terms of their activity, resting heart rate (HR), and fat and skeletal tissues. Eleven 2‐month‐old male mice (Snark+/+: n=4, WT; Snark+/−: n=7, KO) were kept individually in stainless cages equipped with a running wheel for 4 months and given standard chow and water ad libitum. After this time, the animals were anesthetized and the hind limb muscles and adipose tissues were collected. Ten‐μm‐thick sections were cut and stained for myofibrillar ATPase at pH 4.3. At the end of the experiment, there was no difference between WT and KO in body weight or food intake. KO mice tended to have a lower HR. Interestingly, the KO mice ran twice as far as the WT mice (1420.7 ± 223.4 vs. 737.6 ± 146.7 km, p<0.01). The weight of brown adipose tissue did not differ, whereas the KO mice had significantly less white adipose tissue (14.0 ± 1.4 vs. 9.4 ± 2.6 mg/g, p<0.001). Soleus muscle weight, number of muscle fibers, and fiber type composition did not differ between WT and KO, while the fiber area in the soleus was significantly smaller in KO mice (2606.7 ± 328.6 vs. 2074.7 ± 160.7 μm2 p<0.05). Our results suggest that a lack of Snark enhanced activity, and reduced HR and fat deposition, in an environment that allowed mice to exercise. No effect on muscle weight or fiber type composition was found, although fiber area in the soleus muscle was smaller in KO.
Oxygen uptake kinetics as a determinant of very‐heavy intensity exercise tolerance
Authors: Murgatroyd Scott R , Ferguson Carrie , Ward Susan A , Whipp Brian J , Rossiter Harry B
Published: June 2021

The tolerance profile for very‐heavy intensity exercise is well described by a hyperbola with two parameters: a curvature constant (W′) and a power asymptote (CP). During cycle ergometry, where work efficiency is similar between subjects, the tolerable duration (tlim) is highly dependent on the rate at which oxidative ATP synthesis rises to meet the demand. We were therefore interested in the relationship between pulmonary oxygen uptake (VO2) kinetics and the power‐duration hyperbola. We hypothesized that CP (the upper limit for steady‐state VO2) would relate to the fundamental VO2 time‐constant (τ), and W′ to the slow component magnitude (ΔVO2sc). Eight males performed repeats of constant work‐rate (WR) cycle ergometry to tlim: 1) over a range of WRs to estimate CP and W′ and 2) with WR normalized to induce intolerance in 6 min (WR6; for τ and ΔVO2sc). VO2 was measured breath‐by‐breath using a turbine and mass spectrometer. Tolerance at WR6 averaged 365 ± 17 s. CP (range 181–289 W) was inversely correlated (R2 = 0.37) with τ (21.3‐30.5 s); and W′ (12.8‐27.3 kJ) was strongly and directly correlated (R2 = 0.97) with ΔVO2sc (370–940 ml/min). Therefore, a fast τVO2 was associated with a high CP thereby maximizing the WR for a given O2 deficit, whilst a high rate of anaerobiosis ‐ reflected in W′ ‐ resulted in a large ΔVO2sc. Together, this suggests that VO2 kinetics are a major determinant of tolerance during very‐heavy intensity exercise.
Caveolin‐1 is essential for NMDAR localization to membrane rafts and NMDAR‐mediated activation of Src and ERK1/2 in primary neurons
Authors: Head Brian Patrick , Patel Hemal H. , Tsutsumi Yasuo M. , Insel Paul A. , Roth David M. , Patel Piyush M.
Published: June 2021

N‐methyl‐D‐aspartate receptor (NMDAR) signaling is important for neuronal synaptic plasticity, learning and memory. Downstream activation of pro‐survival Src family kinases and ERK1/2 (extracellular‐regulated signal kinase) is initiated by NMDAR activation but little is known about the cellular organization of these kinases in relation to the NMDAR. We hypothesized that caveolin‐1 compartmentalizes protein complexes necessary for NMDAR signaling in discrete microdomains. We found that sub‐lethal ischemia (SLI) or pharmacological preconditioning via NMDA treatment of primary cortical neurons increases phosphorylated caveolin‐1 (P‐caveolin‐1), P‐Src, and P‐ERK1/2. Primary neurons treated with caveolin‐1 siRNA lacked NMDA‐mediated increase in P‐Src and P‐ERK and neuronal preconditioning was not observed. Basally, NMDAR2B (NR2B) was present in both buoyant fractions (BF) and heavy fractions (HF) from wild type (WT) neurons. Exposure of neurons to NMDA redistributed NR2B to HF only. In caveolin‐1−/− neurons, under basal conditions NR2B was only present in HF. Adenoviral expression of caveolin‐1 (Adv‐Cav1) in caveolin‐1−/− neurons resulted in the detection of NR2B in both BF and HF, and exposure of these neurons to NMDA redistributed NR2B to HF only. In caveolin‐1−/− neurons, NMDA treatment failed to increase P‐Src and P‐ERK. Adv‐Cav1 treated neurons from caveolin‐1−/− mice resulted in NMDA‐mediated enhancement of P‐Src and P‐ERK1/2. We conclude that caveolin‐1 protein expression is essential for localization of NR2B to membrane rafts and subsequent NMDAR‐mediated signaling in primary neurons. (Supported by NIH)