bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of collagenase and elastase on arteriolar vasomotor responses
Authors: Clifford Philip S. , Ella Srikanth R. , Yang Yan , Davis Michael J. , Meininger Gerald A. , Hill Michael A.
Published: June 2021

The extracellular matrix contains a number of proteins including collagen and elastin. In addition to providing an extracellular scaffold, it is involved in physiological processes such as cell growth, differentiation, migration, and regulation of vasomotor tone. We hypothesized that breakdown of collagen or elastin in the intact vascular wall would cause arteriolar vasodilation. Rat cremaster 1A arterioles (n=29) were cannulated with glass micropipettes, pressurized, and warmed to 34°C. Internal diameter was monitored with an electronic video caliper. Exposure to type II collagenase (30 units/ml) for 5 min resulted in a transient 34.1±4.0% increase in diameter. Exposure to elastase (5 units/ml) for 5 min had no effect on diameter, but resulted in a 36.3±1.8% increase in length. Both collagenase and elastase treatments abolished myogenic responses, but preserved steady‐state dilation to acetylcholine and constriction to phenylephrine. Collagenase had no significant effect on vascular compliance, but elastase caused a significant leftward shift in the passive pressure/diameter curve. The results show that exposure of arterioles to collagenase resulted in transient vasodilation whereas elastase treatment caused irreversible lengthening of the vessel segment. These findings suggest differences in coupling of extracellular matrix proteins to vascular smooth muscle cells.
Effect of Multiparity on Vascular Compliance and Collagen Content
Authors: Tawfik Huda , Ali Mohammad , Schulz Richard , Kaufman Susan
Published: June 2021

We have shown that vascular compliance is decreased in multiparous rats. When the balance of collagen synthesis/breakdown changes, the functional characteristics of the vessels are altered. Collagen degradation is regulated by matrix metalloproteinases (MMPs). We have previously shown that multiparity increases oxidative stress and peroxynitrite formation. Peroxynitrite can change MMP levels in vascular tissues and thus alter collagen content. We examined the effect of multiparity on collagen content of aorta and mesenteric arteries by Trichrome Masson staining of paraffin imbedded vascular sections, by total vascular collagen and by hydroxyproline. MMP2 activity in vascular tissues was measured by gelatin zymography. Functional changes in vascular compliance were assessed by pressure myography. Data demonstrate that multiparity is associated with decreased MMP2 activity in aorta (870±229 vs 4797±1116) and mesenteric arteries (544±113 vs 2294±777). This was accompanied by increased total collagen (aorta: 536±37 vs 259±32; mesentery: 426±24 vs 206±12) and hydroxyproline (aorta: 149.5±3 vs 64±7; mesentery: 142.8±12.8 vs 74.0±11). Vascular compliance was decreased in vessels from multiparous rats. These data suggest that multiparity decreases vascular compliance via mechanisms involving MMP‐regulated collagen degradation which is downstream of the peroxynitrite signalling pathway.
Effect of High Salt Diet on Response of Rat Mesenteric Resistance Arteries to Angiotensin (1‐7)
Authors: Raffai Gabor , Lombard Julian H.
Published: June 2021

This study determined the vasoactive effects of acetylcholine (ACh) and angiotensin 1‐7 (ANG 1‐7) on isolated mesenteric resistance arteries (100‐300 μm) from male Sprague‐Dawley rats fed either normal salt (NS, 0.4% NaCl) or high salt (HS, 4% NaCl) diet, with water ad libitum. Diameter changes from pretreatment control (Δ μm) were recorded in NE‐pre‐constricted vessels maintained at 75 mmHg in a tissue culture myograph system. Dilation (Δ μm) to ACh was suppressed in HS (19±14 μm) vs. NS (44±4 μm) vessels, while ANG 1‐7 dilated arteries from rats fed HS diet (32±6 μm) but not NS diet (‐1±7 μm). ANG (1‐7) dilations were abolished by the mas‐receptor antagonist d‐Ala‐angiotensin (1‐7) (7±7 μm) and endothelium removal (1±10 μm). Inhibition of nitric oxide synthase with L‐NAME (4±5 μm) and cyclooxygenase inhibition with indomethacin (11±7 μm) also inhibited vasodilator responses to ANG (1‐7) in HS vessels. These results indicate that ANG (1‐7) causes endothelium dependent vasodilation via the mas receptor in rats fed HS diet, but not during NS diet. (NIH #HL‐65289 and #HL‐72920).
12‐ and 15‐Hydroxyeicosatetraenoic Acids (HETEs) Function as Endothelium‐Derived Hyperpolarizing Factors (EDHFs) in Human Coronary Arterioles
Authors: Larsen Brandon T , Miura Hiroto , Campbell William B , Gutterman David D
Published: June 2021

12‐ and 15‐HETEs are lipoxygenase (LO)‐derived arachidonic acid metabolites that are produced abundantly by human coronary arterioles (HCAs); however, their biological role is unknown. We hypothesized that 12‐ and 15‐HETEs function as EDHFs. HCAs were isolated from atrial appendages and studied by immunohistochemistry (IHC), membrane potential (Em) recording, and videomicroscopy. IHC revealed endothelial expression of 12‐ and 15‐LO. In endothelium‐denuded vessels, 1 μM 12‐HETE induced smooth muscle hyperpolarization (Em ‐54±2 vs ‐43±1 mV at baseline, n=4, p<0.05) that was attenuated by charybdotoxin + apamin (K+ channel inhibitors that block EDHFs; ‐43±1 vs ‐37±1 with inhibitors alone, n=4, p<0.05 vs. 12‐HETE alone). 12‐ or 15‐HETE elicited concentration‐dependent vasodilation (68±5% and 67±6% at 10 μM, respectively, n=11 each) that was inhibited by 40 mM KCl (3±6% and 1±4%, respectively, n=5, p<0.05) but not by endothelial denudation (65±17% and 64±6%, respectively, n=3). 12‐ and 15‐HETE‐induced dilations were reduced by charybdotoxin + apamin (16±6% and 6±5%, respectively, n=4, p<0.05), but not by iberiotoxin (a specific inhibitor of large‐conductance Ca2+‐activated K+ channels; 92±8% and 87±2%, respectively, n=2). We conclude that 12‐ and 15‐HETEs elicit endothelium‐independent dilation and hyperpolarization of HCAs by a mechanism requiring K+ channels. These findings support a role for LO‐derived eicosanoids as EDHFs and suggest that these HETEs modulate vasomotor tone in the human heart.
Responses of protein expression in rat brain to deafferentation
Authors: Kawano Fuminori , Nakajima Yoshie , Nakai Naoya , Terada Masahiro , Ohira Takashi , Oke Yoshihiko , Nomura Sachiko , Ohira Yoshinobu
Published: June 2021

Effects of deafferentation on the protein expression in the brain of adult rats were studied. The dorsal roots at the L4‐6 segmental levels of the spinal cord were bilaterally transected in order to inhibit the afferent neural input from the hindlimb muscles. After 3 weeks of ambulation, the thalamus including hypothalamus region was sampled, frozen in liquid nitrogen and powdered in a liquid nitrogen‐filled mortar. The powdered tissue was dissolved in lysis buffer and two‐dimensional electrophoresis was performed using the soluble fraction. Subsequently, the spots with different expression were picked up and the protein was identified using a peptide mass fingerprinting method. As the results, 11 spots with lower density were found in the deafferentated compared with sham‐operated rats. These proteins were identified as beta‐synuclein, histidine triad nucleotide binding protein 1, cofilin 2, and some glycolitic enzymes. According to the previous studies, the decreased levels of these proteins were closely associated with the degeneration and/or dysfunction of neurons. Therefore, it was suggested that the loss of afferent feedback form muscle affected the thalamic function.
Sex differences in neuronal nitric oxide synthase (nNOS) expression in the paraventricular nucleus (PVN) and subfornical organ (SFO) in angiotensin II (ANG II)‐induced hypertension
Authors: Singh Minati , Xue Baojian , Hay Meredith , Johnson Alan Kim
Published: June 2021

Previous studies from our laboratory have shown that there are sex differences in ANG II‐induced hypertension. ICV infusion of L‐NAME augments the pressor effects of long‐term infusions of ANG II in female mice but not in males. However, the mechanisms underlying these sex differences in ANG II‐induced hypertension are not well understood. The present study tested the hypothesis that there is differential expression of nNOS in response to systemic infusions of ANG II in male vs female mice and that this may contribute to sex differences in ANG II‐induced hypertension. We used both intact and gonadectomized male and female mice that were either treated with ANG II (800 ng/kg/min; sc) or vehicle. Following 7 days of ANG II infusion, total cellular protein was isolated from tissue punches of the PVN and SFO and analyzed for the protein nNOS. Western blot analysis of total nNOS protein expression indicated significantly higher levels of nNOS in the PVN and SFO in intact females with or without ANG II treatment when compared to intact males. Under basal conditions, nNOS protein was reduced by gonadectomy in females and increased by gonadectomy in males. Gonadectomized animals did not increase nNOS after ANG II treatment. These results suggest that increased nNOS protein associated with the presence of female gonadal hormones plays a protective role against ANG II‐induced hypertension in female mice.

(Support: NIH HL‐59676, HL‐62261, HL‐14388, & DK‐66086)
Effects of inducible nitric oxide synthase (iNOS) blockade within the periaqueductal gray on cardiovascular responses during mechanical, heat, and cold nociception
Authors: Ally Ahmmed , Tedesco Anthony , Chaitoff Kevin A
Published: June 2021

We examined the role of inducible nitric oxide synthase (iNOS) within the periaqueductal gray mater (PAG) on cardiovascular responses and glutamate and GABA concentrations during mechanical, thermal, and cold nociception in anesthetized rats. Mechanical stimulus was applied by bilateral hindpaw pinch (10 sec) that increased mean arterial pressure (MAP) and heart rate (HR). Bilateral microdialysis of a selective aminoguanidine (AGN; 10 μM) into the PAG for 30 min potentiated MAP and HR during a mechanical stimulation. Cardiovascular responses recovered following discontinuation of the drug. Heat stimulus was generated by immersing both hindpaw metatarsus in a water‐bath at 52°C for 10 sec. MAP and HR were increased, but administration of AGN into the PAG also augmented cardiovascular responses during the heat stimulus. Cardiovascular responses recovered following discontinuation of the drug. In contrast, application of a cold stimulus by immersing both hindpaw at 10°C for 10 sec resulted in decreases of MAP and HR. However, following microdialysis of AGN into the PAG, a cold stimulus had no effect on the cardiovascular responses. Results demonstrate that iNOS within the PAG plays a differential role in modulating cardiovascular responses during mechanical‐, heat‐, and cold‐mediated nociception.
Exercise training normalizes ACE and ACE2 in the brain of rabbits with pacing induced chronic heart failure
Authors: Kar Sumit , Gao Lie , Zucker Irving H.
Published: June 2021

Activation of the brain renin‐angiotensin system (RAS) and elevated Angiotensin II (Ang II) increase sympathetic nerve activity (SNA) in chronic heart failure (CHF). Exercise training (EX) normalizes SNA and plasma Ang II. The mechanisms are unclear, but EX may modulate components of the RAS such as Angiotensin‐converting enzyme (ACE) and ACE2. This study investigated the effect of EX on the regulation of ACE and ACE2 in the brain with a pacing CHF+EX model. We hypothesized that the ratio of ACE to ACE2 would increase in CHF to mediate increased SNA and be normalized by EX. Experiments were performed on four groups of rabbits: normal, normal+EX, CHF, and CHF+EX (n=4‐5/group). We analyzed the cortex, cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM by Western blotting, PCR, and double immunofluorescence (IF). ACE protein and mRNA expression in the cerebellum, medulla, hypothalamus, PVN, NTS, and RVLM were significantly upregulated in CHF (0.3±0.03 to 0.8±0.1, RVLM, P<0.05). EX normalized this in all areas (0.8±0.1 to 0.4±0.1, RVLM). ACE2 protein and mRNA significantly decreased in CHF and also normalized with EX. IF showed ACE2 is present in the cytoplasm of neurons and ACE in endothelial cells. This suggests RAS activation involves an imbalance of ACE and ACE2 and may mediate changes in Ang II and Ang 1‐7 in regions of the brain that regulate autonomic function and suggests EX as a therapy to normalize SNA in CHF.
Effects of Stanniocalcin in the Nucleus of the Solitary Tract (NTS) on Arterial Pressure (AP)
Authors: Ciriello John , Moreau Jason M. , Oiamo Tor , Wagner Graham F.
Published: June 2021

Stanniocalcin‐1 (STC‐1) is a glycoprotein hormone that decreases cytosolic Ca++ in neurons. A decrease in Ca++ availability to NTS neurons decreases AP. Therefore, the possibility exists that STC‐1 may function in NTS to regulate Ca++ availability of neurons involved in AP regulation. In the first series of experiments, the distribution of STC‐1 receptors in the dorsal vagal complex (DVC) was investigated by in situ ligand binding using a STC‐1‐alkaline phosphatase fusion protein. STC‐1 binding sites were found on neurons throughout the DVC, although most were on neurons of the medial NTS (mNTS). In the second series, microinjection of STC‐1 (1.76‐176 nM; 20 nl) into mNTS in anesthetized rats elicited a dose‐related decrease in AP. On the other hand, neither STC‐1 given iv nor injections into NTS of STC‐1 conjugated to GTP elicited AP responses. The STC‐1 evoked depressor response was due to sympathoinhibition as it was blocked by iv hexamethonium bromide. Finally, microinjection of an STC‐1 primary antibody (100 nl) bilaterally into STC‐1 responsive sites in NTS not only blocked the STC‐1 induced depressor response but also elicited a long lasting increase in AP. Taken together these data suggest that STC‐1, through receptor mediated effects, is involved in altering the excitability of NTS neurons that function as components of neuronal systems controlling AP.

Supported by HSFO and CIHR
Humanized Mouse Models for HIV Research
Authors: Garcia‐Martinez J. Victor
Published: June 2021

HIV is the causative agent of AIDS. Currently, the AIDS epidemic expands to all corners of the world and there are an approximately 35,000,000 infected individuals. HIV is mostly transmitted after mucosal exposure and women represent the fastest group of newly infected individuals. Despite the increased availability of antiretroviral therapy, there have not been any declines in the rates of new infections. In fact, revised estimates for 2006 have increased the number of new HIV infections in the US to 56,300. Currently, the prospects of a preventative vaccine are not encouraging. One of the most significant issues preventing the advancement of the field is the fact that HIV's tropism is limited to humans and chimpanzees. Therefore in vivo analysis of HIV is severely restricted. The recent development of humanized mice reconstituted with human hematopoietic stem cells represents a significant advance that permits the in vivo analysis of HIV. Humanized mice can be infected by HIV in the same manner as humans and HIV infection results in a sequel that mimics to a significant extent what is normally seen in infected patients. We have taken advantage of humanized mice to study HIV transmission and to develop novel approaches to prevent the further spread of AIDS.
The Isoc Channel is a Critical Determinant of Interendothelial Gap Formation
Authors: Barry Christina Jean , Cioffi Donna , Stevens Troy
Published: June 2021

Inflammatory agonists deplete Ca2+ from the endoplasmic reticulum and activate Ca2+ entry in a process referred to as store‐operated Ca2+ entry (SOC). Activation of a Ca2+‐selective SOC current, Isoc, increases permeability and induces intercellular gap formation in pulmonary artery endothelial cells. However, the mechanism by which Ca2+ entry through the ISOC leads to barrier disruption is unclear. Endothelial cells express the transient receptor potential canonical (TRPC) homologues 1 and 4 that contribute to the molecular basis of ISOC. TRPC4 specifically links the Isoc channel to the membrane skeleton via interaction with protein 4.1. We therefore sought to determine whether inhibition of the TRPC4‐4.1 interaction is sufficient to prevent thapsigargin from inducing gaps. Thapsigargin activates SOC entry and induces a large rise in cytosolic Ca2+. Introduction of a competitive peptide that disrupts the TRPC4‐4.1 interaction reduces the global Ca2+ response to thapsigargin and abolishes Isoc. Moreover, peptide inhibition of the TRPC4‐4.1 interaction nearly abolished thapsigargin‐induced gap formation, and increased the rate of gap resealing. Thus, Ca2+ entry through the Isoc channel is critical to intercellular gap formation. Supported by HL60024.
Endothelium‐dependent contraction induced by acetylcholine in the chicken ductus arteriosus: involvement of a cyclooxygenase‐derived TP receptor agonist
Authors: Villamor Eduardo , Schuurman Marijn J , Zoer Bea , Cogolludo Angel L , Perez‐Vizcaino Francisco
Published: June 2021

In numerous vascular beds, acetylcholine (ACh) evokes the simultaneous release of endothelium‐derived relaxing and contracting factors (EDRF and EDCF, respectively). We aimed to determine whether ACh evokes the release of an EDCF in the chicken ductus arteriosus (DA) and to identify its nature. Isolated rings of the DA from 19‐d chicken fetuses (total incubation: 21‐d) were mounted in a myograph. Low concentrations of ACh (30 nM‐ 1 µM) elicited a relaxation, which was followed by a contraction at higher concentrations (3 μM ‐0.1 mM). Both relaxation and contraction were abolished by removal of endothelium and were sensitive to the antimuscarinic agents atropine and 4‐DAMP (M3‐receptor antagonist). ACh‐induced relaxation was impaired in the presence of the nitric oxide (NO) synthase inhibitor L‐NAME and the soluble guanylate cyclase (sGC) inhibitor ODQ. ACh‐induced contraction was impaired in the presence of the nonselective inhibitor of cyclooxygenase (COX) indomethacin, the selective COX‐1 inhibitor valeryl salicylate, and the thromboxane A2 (TP) receptor blocker SQ‐29458, whereas the response was not affected by the selective COX‐2 inhibitor nimesulide, the thromboxane synthase inhibitor furegrelate, or the H2O2 scavenger PEG‐catalase. We conclude that in the chicken DA, stimulation of muscarinic receptors by ACh induces an endothelium‐dependent relaxation followed by an endothelium‐dependent contraction. The relaxation involves the NO‐sGC pathway, whereas the contraction involves a COX‐1 product that stimulates TP receptors.

Supported by "Fundaci ónde Investigación Médica Mutua Madrileña".
Genetic Evidence for the negative Regulation of Endothelial Cell Proliferation by Caveolin‐2.
Authors: Xie Leike , Frank Philippe G , Lisanti Michael P , Sowa Grzegorz
Published: June 2021

Caveolae are small flask‐shaped plasma membrane invaginations, which participate in endocytosis, membrane sorting and signal transduction. Caveolin proteins are the major components of caveolae. Unlike the multifaceted role of caveolin‐1 (Cav‐1), the function of Cav‐2 is poorly characterized. The goal of this study was to determine if Cav‐2 may play a role in regulating endothelial cell (EC) proliferation in vitro. To realize this goal, we isolated and purified mouse lung ECs (MLECs) from wild type (WT) and Cav‐2 KO mice using magnetic beads coated with antibody against EC specific marker PECAM‐1. This yielded over 95% pure populations of WT and Cav‐2 KO MLECs as judged by immunofluorescent labeling with antibodies against EC marker proteins (such as VE‐Cadherin and von Willebrand Factor). Using three independent proliferation assays: i. Cell counts; ii Colorimetric measurement of MTT to formazan conversion; and iii. Immunofluorescent labeling of Brdu incorporating (S‐phase associated) cells, we have determined that Cav‐2 KO MLECs proliferate by ca. 2‐fold faster than their WT counterparts. Finally, immunobloting with cyclin‐specific antibodies determined upregulation of S phase‐ and mitosis‐associated cyclins A and B. Overall, our data suggest that Cav‐2 may negatively regulate EC proliferation.
Knockdown of MT1‐MMP expression in Human Umbilical Vein Endothelial Cell Inhibits MMP‐2 and TIMP‐2 expression
Authors: LIN PO‐WEI , Lee Hsinyu
Published: June 2021

Matrix‐Metalloproteinases (MMPs) play important roles in regulating the interaction between cells and extracellular matrix (ECM) via degradation of various matrix proteins. In vertebrate, more than twenty MMPs targeting different substrates have been identified. Membrane Type 1‐Metalloproteinase (MT1‐MMP) not only degrades ECM protein but also activates Metalloproteinase‐2 (MMP‐2, Gelatinase A), which is important to endothelial cell migration. In addition, Tissue Inhibitor of Metalloproteinase‐2 (TIMP‐2) also regulates MMP‐2 activation. However, the relationship between MT1‐MMP and MMP‐2/TIMP‐2 expression remains unclear. In this study, we intend to investigate whether MT1‐MMP regulates the expression of MMP‐2 and TIMP‐2 by introducing MT1‐MMP siRNA in human umbilical vein endothelial cells (HUVECs). Our results indicated that MMP‐2 expression was decreased in the presence of MTI‐MMP siRNA as detected by real‐time PCR and western blotting analysis. Moreover, similar results were also observed in TIMP‐2. These results suggested that MT1‐MMP knockdown attenuated MMP‐2 and TIMP‐2 expression in HUVECs. Our study demonstrated that MT1‐MMP not only regulates MMP‐2 activity but also modulates the expression of MMP‐2 and TIMP‐2.
Effects of Bile Duct Ligation (BDL) and Enalapril on Angiotensin receptors in the Subfornical Organ (SFO) in Rats
Authors: Walch Joseph D , Ji Lisa , Carreno Flavia R. , Hinojosa‐Laborde Carmen , Cunningham J. Thomas
Published: June 2021

Inappropriate vasopressin (AVP) release causes fluid retention, ascites, and dilutional hyponatremia associated with cirrhosis. Although the central molecular mechanisms that mediate inappropriate AVP release are not clear, plasma angiotensin II (AngII) has been implicated as a factor in the pathogenesis of dilutional hyponatremia. To better understand the relationship between the CNS effects of circulating AngII and increased AVP release during cirrhosis, we examined the effects of BDL with treatment of enalapril on AT1R expression in the SFO. Starting the 21st day after BDL surgery BDL rats were either treated with enalapril dissolved in tap water (0.25mg/mL) or with normal tap water. On the 28th day (after 7 days of enalapril treatment), the rats were anesthetized, decapitated and brain punches were taken of the SFO for quantification of AT1R. Western Blot analysis showed an increase in AT1R in the SFO of untreated BDL rats as compared to sham ligated controls. In enalapril treated BDL rats, AT1R decreased compared to drug naïve BDL rats. Plasma osmolality and hematocrit also showed a trend towards normalization in enalapril treated BDL rats. These results are consistent with the hypothesis that AngII may increase excitatory drive in the SFO through upregulation of its AT1R thus leading to a decompensation of sodium balance and hyponatremia. (R01 HL062576)
Neural control of the kidney in a thyroxine induced model of heart hypertrophy (HH): impact of Nitric Oxide Synthase (NOS) blockade
Authors: Buckley Maria M , Johns Edward J
Published: June 2021

This study investigated the how the cardiopulmonary mediated reflex inhibition of renal sympathetic nerve activity (RSNA) in normal rats and those with HH induced by thyroxine administration might be modulated by nitric oxide (NO). Male Wistar rats were anaesthetized with chloralose/urethane and HH was induced by thyroxine administration (1 mg/kg/day, sc) for 1 week. All animals were subjected to 2 periods of volume expansion (VE) at 0.25% body weight/min for 30 min in the absence then presence of the NOS inhibitor L‐NAME (10µg/min/kg). Data, means ± SEM, were subjected to ANOVA and significance taken at P<0.05. Blood pressure (BP) was 93±6mmHg and 89±5mmHg in normal and HH groups, respectively. VE in normal rats decreased RSNA by 30% (P<0.05) in the absence and 60% in the presence of L‐NAME. In the HH group, RSNA did not change during the first VE while in the presence of L‐NAME it decreased by 40% (P<0.005). These findings demonstrate that the inhibition of RSNA in response to VE is tonically suppressed by NO at some stage along the reflex arc. Moreover, in the thyroxine induced HH model, it appears that the influence of NO is enhanced suppressing the renal sympatho‐inhibition to VE.
A Role For TNF‐α in Hypertension During Systemic Lupus Erythematosus
Authors: Venegas‐Pont Marcia , Glover Porter H. , LaMarca Babbette D. , Jones Allison V. , Ryan Michael J.
Published: June 2021

Systemic Lupus Erythematosus (SLE) is an independent risk factor for developing hypertension. The inflammatory cytokine TNF‐α has been implicated in the pathology of hypertension and SLE, perhaps through increased oxidative stress. We tested whether the TNF‐α antagonist etanercept (ETAN) reduces mean arterial pressure (MAP) and protects the kidney in a female mouse model of SLE (NZBWF1). Thirty week old SLE and control mice (NZW/LacJ) received ETAN (0.8mg/kg SC weekly x 4 weeks) or vehicle. Mice with SLE were hypertensive compared to controls. Treatment with ETAN reduced MAP in SLE but not control mice. Treatment with ETAN also reduced the albuminuria associated with SLE. Renal cortex NADPH oxidase activity was higher in SLE mice compared to controls and lowered in SLE mice treated with ETAN. Interestingly, the pathognomonic Anti‐dsDNA antibodies were increased in ETAN treated SLE mice. These data suggest that TNF‐α, through increasing renal oxidative stress, is an important factor in the development of SLE hypertension. (Supported by HL 08597 MJR)

*p< 0.05 vs. Ctrl †p<0.05 vs. SLE+Veh
Osmotic water permeabilities in specific segments of rat inner medullary thin limbs of Henle's loops
Authors: Dantzler William H. , Evans Kristen K. , Pannabecker Thomas L.
Published: June 2021

Three‐dimensional reconstructions of thin limbs of Henle's loops in rat renal inner medulla (IM) involving antibody to constitutive water channel aquaporin 1 (AQP1) have revealed that descending thin limbs (DTLs) of loops extending deeper than 1.0 mm express AQP1 for first 40% of their length, but fail to express it for remaining 60%. Ascending thin limbs (ATLs) and prebend segments do not express AQP1. Therefore, we examined water permeabilities in isolated perfused segments of DTLs from specific regions of IM and in ATLs. We perfused DTL segments from two IM regions: 1) final 2.0‐2.5 mm, where all DTLs lack AQP1, and 2) initial 2.5 mm. In initial 2.5 mm, we chose DTLs extending beyond first 1 mm because all of these should contain AQP1. DTLs from AQP1‐null region showed no osmotic water permeability (Pf = ‐292 ± 166 μm/s; mean ± SE; n=4). ATLs also showed no osmotic water permeability (Pf = 69 ± 69 μm/s; mean ± SE; n = 10). In contrast, DTL segments from initial 2.5 mm had mean osmotic water permeability of 1670 ± 169 μm/s (mean ± SE; n = 6). However, values for individual tubule segments varied from about 1200 to 2100 μm/s. Since there is no way to determine if segments perfused expressed AQP1 for their entire length, it seems likely that segments with lower values had regions that did not express AQP1. These data indicate that presence or absence of AQP1 assessed by immunocytochemistry accurately reflects differences in water permeability. DK16294
Effects of the diabetes mellitus in the metabolism of the catecholamines in rat mesangial cells
Authors: Moreira Roseli Peres , Andrade Maria Claudina Camardo , Ronchi Fernanda Aparecida , Arita Danielle Yuri , Casarini Dulce Elena
Published: June 2021

The aim of this study is to analyze the catecholamines (CA) profile in mesangial cells (MC) from normal and diabetic animals. The diabetes mellitus has been induced in Wistar‐Hannover by injection of streptozotocin (50mg/kg). Three groups of study ‐ Control (CT), Hyperglycemic (HG) and treated Diabetic (TD), were evaluated during 60 days. MCs were obtained through sieve fractionation and collagenase‐treatment, and collected in the third subculture. CA concentrations were determined by high performance liquid chromatography. CA concentrations in the MC extracellular medium (EM) and intracellular compartment (IC) of the CT group expressed in pg/mg of cellular protein were respectively: Norepinephrine (NE), 18.1±1.1; Epinephrine (EP), 10.0±0.6; Dopamine (DE), and NE, 117.3±10.1; AD, 62.6±9.4; DE, 39.1±7.7. The levels of CA detected for HG in the EM were NE, 43.5±5.8; EP, 149.0±33.6; DE, 57.9±11.2 and in the IC were NE, 109.4±41.3; EP, 269.6±22,3; DE, 189.1±48.9. The comparison between groups demonstrated statistically significant differences in the CA levels of TD‐HG and for CT‐HG (TD and CT had a similar behavior). Thus, we can suggest that besides the renal damage induced directly by high blood glucose levels, DM could disturb CA metabolism and contribute to the development of diabetic nephropathy. Supported by FAPESP 02/13290‐2.
Effects of diabetes over RAS enzymes and peptides of non‐obese diabetic mice mesangial cells
Authors: Colucci Juliana Almada , Candido Georgia Orsi , Pacheco‐Silva Alvaro , Irigoyen Maria Claudia , Casarini Dulce Elena
Published: June 2021

Aim

We studied the balance between different components of the RAS in NOD mice mesangial cells.

Methods

primary culture of mesangial cells, ACE and NEP activity, western blotting, IHC, angiotensins and bradykinin measurement and blood pressure and heart rate analysis.

Results and conclusions

ACE activity was higher in the diabetic group comparing to normoglycemic (0.23 vs 0.11 μM/min.mg). On the other hand NEP activity was diminished (0.09 vs 0.22 μM/min.mg), maybe indicating a prevalence of constriction mechanisms. Through IHC we observed an intense ACE stain on diabetic group kidney slices, in opposite to what is described in literature for other diabetic mouse models. The angiotensin peptides levels were not significantly altered between both groups, but the BK levels were higher on diabetic animals (3.6 vs 1.7 nmol/mL). An interesting finding was that MBP (83.70 vs 110.81 mmHg) and HR (346.02 vs 591.38 bpm) of diabetic mice was lower than that of non diabetic littermates. On this mouse model, the RAS seem to account for a constrictor effect which may lead to kidney damage associated to diabetes. The most surprising data was the decreased blood pressure, despite the ACE activity augmentation. We suggest that an alternative path may be acting in order to avoid blood pressure raise, which could probably be ACE2, yet to be fully characterized by us on this mouse model. Fapesp (04/15875‐3)
Dietary protein stimulates pancreatic growth via CCK‐independent mTOR activation
Authors: Crozier Stephen James , Williams John A
Published: June 2021

Dietary protein can stimulate pancreatic growth in the absence of CCK release, but there is little data on the regulation of CCK‐independent growth. To identify mechanisms whereby protein stimulates pancreatic growth, male C57BL/6 and CCK‐null mice were fed control (20% protein) or protein‐enriched (75% protein) chow for 7 days. The weight of the pancreas increased by 32% in C57BL/6 mice and 36% in CCK‐null mice. Changes in pancreatic weight in C57BL/6 mice were due primarily to cell hypertrophy; with a 68% increase in protein content and a 24% increase in DNA content. Parallel changes were observed in CCK‐null mice. ERK, calcineurin, and mTOR are activated in models of CCK‐induced growth, but there were no differences in ERK or calcineurin activation between fasted and fed CCK‐null mice. In contrast, mTOR activation was increased after feeding and the duration of activation was prolonged in mice fed protein‐enriched chow compared to control chow. Changes in pancreatic weight and DNA content were completely inhibited, and changes in protein content were partially abated, when the mTOR inhibitor rapamycin was administered during protein‐enriched chow feeding. Thus, dietary protein stimulates pancreatic growth in the absence of CCK via prolonged mTOR activation.
P2X7 Expression is Greater in Red Blood Cell Membranes of Humans with Type 2 Diabetes than Healthy Humans.
Authors: Thuet Kelly Marie , Ford David A , Bowles Elizabeth A , Ellsworth Mary L , Sprague Randy S , Stephenson Alan H
Published: June 2021

Expression of P2X7 purinergic receptors on red blood cell (RBC) membranes has been reported. ATP, released from RBCs has been proposed to stimulate the P2X7 receptor in an autocrine fashion to induce the production and release of epoxyeicosatrienoic acids (EETs). However, RBCs from humans with type 2 diabetes (DM2) have been reported to release less ATP than healthy humans in response to physiological stimuli. To examine whether DM2 also alters RBC expression of the P2X7 receptor we examined its expression in humans with DM2 and healthy humans. We also compared RBC P2X7 expression from an animal model of DM2, the Zucker Diabetic Fatty rat (ZDF) to its control, the lean ZDF rat. Blood was obtained from healthy humans, humans with type 2 diabetes and from 12 and 18 week control and DM2 ZDF rats. RBCs were separated and membranes were isolated. Western immunoblotting was conducted on the membranes and P2X7 expression was measured by densitometry using beta‐actin as a loading control. Expression of P2X7 was 3 fold higher in RBC membranes from humans with DM2 than in healthy humans (p<0.0002). RBC membranes of fatty ZDF rats at 12 and 18 weeks did not express greater P2X7 than lean ZDF rats. These results suggest that the greater P2X7 expression in humans with DM2 may act as a compensatory mechanism in the presence of reduced ATP release and that the ZDF rat may not accurately model this aspect of human DM2.
Short‐term Caloric Restriction Improves Glucose Tolerance in Older Mice Independent of Changes in SIRT‐1
Authors: Rippe Catarina , Lesniewski Lisa A , Donato Anthony J , Seals Douglas R
Published: June 2021

In rodents, life‐long caloric restriction (CR) preserves in vivo glucose‐insulin function with aging, possibly by increasing the protein deacetylase sirtuin 1 (SIRT‐1). Because life‐long CR is not feasible in humans, we determined if short‐term CR could improve glucose‐insulin function in old mice and the role of SIRT‐1. Old (30 mo) male B6D2F1‐mice were fed either ad libitum (AL, n=4‐6) or were restricted to 70% of AL for 5 weeks after 2 weeks of progressive restriction (CR, n=4‐6). At baseline, no differences were observed in the area under the curve (AUC) for glucose or insulin during an i.p glucose tolerance test between AL and CR. The AUC for glucose was not significantly different after vs. before CR (10,085±211 vs. 11,844±1371 mg/dl*min, P=0.1) or AL (12,720±1343 vs. 12,486±1085, P=0.9). However, the AUC for insulin was 77% lower after CR (34±8 vs. 149±22 ng/ml*min, P<0.05), but was 77% greater after AL (234±34 vs. 132±12, P<0.05). CR did not affect SIRT‐1 protein expression in either skeletal muscle or white adipose tissue (WAT) (P=0.9 and P=0.8, post CR vs. AL), but increased expression of insulin receptor substrate‐1 (IRS‐1) by 190% in muscle (2.8±0.6 vs. 1.0±0.2, P<0.05) and by 150% in WAT (2.5±1.0 vs. 1.0±0.4, P<0.005). Our findings indicate that short‐term CR markedly improves insulin sensitivity in old mice independent of changes in peripheral SIRT‐1 expression. The CR‐induced improvement in insulin sensitivity is associated with robust increases in IRS‐1 expression in both skeletal muscle and WAT.

NIH AG006537, AG015897, AG029337, AG000279, The Swedish Medical Research Council
Effects of fatty acids on insulin secretion
Authors: Reeves Valerie , Smart Eric J
Published: June 2021

Caveolin‐1 and cdc42 are involved in insulin granule fusion at the membrane. Normally, caveolin‐1 binds cdc42 and acts as a guanylyl nucleotide inhibitor thereby preventing insulin granule fusion. Glucose stimulates the release of caveolin‐1 from cdc42 allowing insulin granules to fuse with the plasma membrane. Fatty acids prevent the secretion of insulin. The goal of this study is to determine if fatty acids alter the interaction of caveolin‐1 and cdc42. MIN6 cells, a murine pancreatic insulinoma cell line, were treated with 10 μM stearic, linoleic, or palmitic acid for 10 min. In addition, another set of cells were treated with the fatty acids and 20 mM glucose for 10 min. The cells were then immunostained for caveolin‐1, cdc42 and insulin and visualized by immunofluorescence. In untreated cells, caveolin‐1, cdc42, and insulin co‐localized on vesicles. Treatment with glucose caused a decrease in insulin signal and caveolin‐1 and cdc42 no longer co‐localized. In the presence of linoleic or palmitic acid, caveolin‐1 and cdc42 co‐localized and glucose stimulated insulin secretion was inhibited. In the presence of stearic acid, caveolin‐1 and cdc2 did not co‐localize and glucose stimulated insulin release. These data show that palmitic and linoleic acid prevent insulin release by allowing caveolin‐1 to remain bound to cdc42 whereas stearic acid does not interfere with glucose stimulate insulin secretion.
Intermittent Hypoxia Alters Small Mesenteric Artery Vasoreactivity in Mice: Role of NFATc3
Authors: Sanchez Lilliana , de Frutos Sergio , Friedman Jessica , Kanagy Nancy , Gonzalez Bosc Laura
Published: June 2021

We previously found that intermittent hypoxia (IH)‐induced hypertension in mice requires NFATc3 activation through endothelin 1 signaling and that IH induces NFATc3‐dependent small mesenteric arterial (SMA) remodeling with upregulation of a‐actin. Therefore, we propose that NFATc3 is required for IH‐induced increase in SMA constrictor sensitivity to depolarizing stimuli. SMA from air exposed NFATc3 wild type (A WT) and knockout (A KO), and 7 days IH WT and KO mice were cannulated, endothelium denuded, and pressurized to 60 mmHg. SMA were superfused with increasing concentrations of KCl (15 to 100 mM) and changes in internal diameter recorded. Unexpectedly, IH WT SMA constricted less to KCl than A WT while IH and A KO SMA were not different from A WT (? diameter from Ca2+‐free at 30 mM KCl: A WT= 47.9±3.8, A KO= 43.2±8.2, IH WT= 26.9±2.6*, IH KO= 40.3±8.5, n=6, *p<0.05 vs. A WT). Since NFATc3 regulates expression of Kv1.5, 2.1, BK and β, INCREASED K CHANNEL EXPRESSION COULD CAUSE THE REDUCED DEPOLARIZATION‐INDUCED CONSTRICTION. MRNA FOR THESE CHANNEL SUBUNITS WAS MEASURED BY REAL TIME PCR. NO DIFFERENCES WERE OBSERVED FOR KV 2.1, BK andβ. However, Kv1.5 mRNA was significantly increased in IH WT compared to A WT, IH KO and A KO. These data suggest IH induces a reduction in membrane depolarization‐mediated SMA constriction due to increased Kv1.5 expression that is dependent on NFATc3. Supported by AHA SDG 535347 and PREP NIH.
Investigations on the role of inflammatory cytokines in EMT and cell migration in alveolar epithelial cells
Authors: Buckley Stephen T , Medina Carlos , Ehrhardt Carsten
Published: June 2021

Epithelial‐mesenchymal transition (EMT), a process through which epithelial cells lose their polarity and become motile mesenchymal cells, is a contributory factor in fibrosis. Growth factors and inflammatory cytokines are purported to orchestrate this process. Here the ability of TGF‐β1 and inflammatory cytokines to induce EMT and their effect on cell migration was assessed. Alveolar epithelial cells (A549) were stimulated with TGF‐β1 and/or cytomix [CM; IL‐1β + TNF‐α + IFN‐γ]. Alterations in cell morphology and changes in expression of EMT markers were measured. The influence of TGF‐β1, IL‐1β, TNF‐α and IFN‐γ (alone and in combinations) on cell migration was determined by wound closing assay. Control cells exhibited an epithelial morphology with expression of E‐cadherin and an absence of α‐SMA. Treatment with TGF‐β1 induced EMT with cells adopting a spindle‐shaped phenotype. This was coupled with a down regulation of E‐cadherin expression and an up‐regulation of α‐SMA. CM, either alone or in combination with TGF‐β1, was found to induce EMT to a greater extent than TGF‐β1. CM, IL‐1β + TNF‐α and IL‐1β alone were all found to significantly (P<0.001) enhance cell migration. TGF‐β1 had little effect, either alone or when added to different combinations of cytokines. Our results suggest that inflammatory cytokines together with TGF‐β1 may play an important role in the development of fibrosis via the mechanism of EMT.
Cloning and Expression of Aquaporin in the Antennal Gland of Crayfish, Procambarus Clarkii
Authors: Gao Yang , Gao Yongping , Wheatly Michele , Krane Carissa
Published: June 2021

Aquaporins (AQPs) , water transport proteins, have been cloned from a wide variety of species, however, little is known in crustaceans. The objective of this study is to clone the AQP from crayfish and examine the mRNA expression, both temporally (molting cycle) and spatially (tissue‐specific). A pair of degenerate primers was used in amplifying a 367bp fragment of cDNA from crayfish antennal gland. The deduced amino acid sequence showed homology with AQPs from mosquito (54%), drosophila (52%), rat (42%) and human (43%). The AQP mRNA expression in the antennal gland was compared with that of other epithelia tissues: gill and hepatopancreas and non‐epithelia tissues: cardiac muscle and tail muscle, by using real‐time PCR. Compared with antennal gland, the expression was lower in gill and higher in cardiac muscle and hepatopancreas respectively. No significant difference was found in tail muscle. Also, the relative mRNA expression of AQP in antennal gland is lower in the premolt when compared to inter‐ and post‐ molt stages. These preliminary observations suggest that crayfish AQP does play an important role in transferring water, regulating nutrients and salt conservation in multiple tissues and the molting stages. Future study will be focused on the expression of this aquaporin in the different tissues of crayfish during molting. (Supported by Grants IBN 0445202 to MGW, YG and CMG).
Alcohol Intoxication Inhibits Sca‐1 Expression by Bone Marrow‐Derived Gr‐1+ Cells During Bacteremia
Authors: Melvan John Nicholas , Raasch Caroline , Bagby Gregory J , Nelson Steve , Zhang Ping
Published: June 2021

We have shown that enhanced Sca‐1 expression by lineage negative hematopoietic precursors is a key component of the granulopoietic response to bacterial infection, which is impaired by alcohol intoxication. Currently, no knowledge exists about the effects of alcohol on the Sca‐1 response in cells committed to the granulocyte lineage (Gr‐1+). This study investigated Sca‐1 expression by Gr‐1+ cells during bacteremia and the effect of alcohol on this response. Acute alcohol intoxication was induced via intraperitoneal (i.p.) injection of 20% alcohol (5g ethanol/kg) in Balb/c mice. Thirty minutes after i.p. injection, bacteremia was initiated by intravenous injection of E. coli (5x107 CFU in 100 µL saline/mouse). Animals were sacrificed 12 and 24hrs thereafter. Bone marrow cells were analyzed by flow cytometry. Bacteremia increased the percentage of Gr‐1low and Gr‐1high cells coexpressing Sca‐1 at both time points. Alcohol intoxication suppressed these responses. These data show that alcohol suppresses Sca‐1 expression in cells committed to granulocyte lineage development during bacteremia, which may serve as a mechanism underlying alcohol induced immune suppression.
Influences of pregnancy on the core temperature response to ip TNF[alpha] in rats
Authors: Fewell James Edwin , Liu‐Tertil Limin
Published: June 2021

Pregnant (P) rats exhibit hypothermia in response to doses of E. coli LPS that elicit fever in nonpregnant (NP) rats. In NP rats, LPS elicits significant increases in plasma concentrations of IL‐1β, IL‐6, IL‐1ra and TNFα whereas in P rats, LPS elicits significant increases in antipyretic/cryogenic cytokines (i.e., IL‐1ra & TNFα) but not in pyrogenic cytokines (i.e., IL‐1β & IL‐6). The present experiments were carried out on 58 NP and 56 near‐term P chronically‐instrumented and conscious rats to investigate the influence of pregnancy on the core temperature (Tc) responses to i.p. TNFa.

In NP rats, doses of TNFa ranging from 0.1 to 1000 µg/kg produced significant increases in Tc (i.e., fever). In P rats, however, TNFa did not elicit fever but produced hypothermia in doses of 10µg/kg and greater. Thus, our data provide insight into the possible role that TNFα plays in mediating the differential Tc response to LPS in NP and P rats.
Thermal tolerance and Hsp70 expression in the Western fence lizard,Sceloporus occidentalis: Geographic variation in measures of acute stress and their role in selection on sprint speeds.
Authors: McMillan David Michael , Rees Bernard , Irschick Duncan J
Published: June 2021

The paradigm of local adaptation to temperature has been extensively studied in the laboratory as well as in the field. However, many studies have exclusively examined invertebrate species which are often restricted to thermally isolated microhabitats. In this study, we examined the potential for temperature adaptation in the western fence lizard, Sceloporus occidentalis, which occurs in the western United States across thermally heterogeneous habitats. We collected lizards from sites paired in altitude and latitude (four sites total) and measured thermal tolerance (CTMax and CTMin) as well as expression of Hsp70, a molecular chaperone produced in response to thermal stress. In addition, we measured sprint speed of lizards before and after exposure to an experimental heat stress (or control treatment). We found that CTMax increased at all sites during the course of the summer season and CTMin decreased during the same time period but only at some sites. We also found that all lizards exposed to a heat treatment suffered reduced sprint speeds relative to control animals but the magnitude of the decrease depended upon the original site of capture. Finally, across all sites, increased expression of Hsp70 was correlated with reduced sprint speeds. These results indicate the potential for temperature adaptation to act within these populations and may lead to locally adapted populations of S. occidentalis.
Phosphoinositide‐mediated arterial contractions in mice
Authors: Andresen Jon , Silswal Neerupma , Qu Cheng‐Kui
Published: June 2021

Phosphoinositides are important signaling molecules in the vasculature. Recently, it was demonstrated in skeletal muscle that PI(3,5)P2 stimulates ryanodine receptors (RyR) resulting in increased cytosolic calcium (Ca2+) levels and enhanced contraction. Using isolated mouse aortas held under isometric tension, we examined the effect of PI(3,5)P2 on vascular smooth muscle. Exogenous PI(3,5)P2 (10−8 M to 3x10−6 M) elicited concentration‐dependent contraction of the mouse aorta. Unlike PI(3,5)P2, the isomers PI(4,5)P2 and PI(3)P, did not cause contraction. Blockade of RyR with 10−5 M ryanodine significantly reduced contraction to PI(3,5)P2. Depletion of sarcoplasmic (SR) Ca2+ with 10−6 M thapsigargin alone, or in combination with ryanodine and caffeine (20 mM) completely abrogated contraction to PI(3,5,)P2. Preincubation with 10−5 M verapamil, however, blocked the sustained, but not the initial phase, of the contraction to PI(3,5)P2. It appears that activation of RyR by PI(3,5)P2 releases Ca2+ from the SR, which elicits a transient contraction of smooth muscle. Maintained contraction to PI(3,5)P2, however, evidently requires extracellular Ca2+ entry via VDCC. These data suggests that PI(3,5)P2 may regulate vascular tone through the modulation of cytosolic Ca2+ levels. This research was supported by an AHA SDG (0735053N) and UMKC start‐up funds.