bluuebunny/crossref_metadata_2025 huggingface dataset:
Network of Paraventricular Nucleus Gene Expression in Angiotensin‐salt Hypertension
Authors: Yuan Lihui , Li Xia , Sved Judith , Sved Alan , Liu Li , Raizada Mohan
Published: June 2021

Neurons in the hypothalamic paraventricular nucleus (PVN) are critical in control of cardiovascular function and their altered activity may contribute to the pathogenesis of hypertension. In this study we used gene expression profiling to identify PVN genes that may contribute to hypertension produced by the interaction of angiotensin II (AngII) and high dietary salt. Hypertension was established in male Sprague Dawley rats by infusion of AngII (150 ng/kg/hr, sc) in combination with a 2% NaCl diet; control rats were fed a 0.1 % NaCl diet and did not receive AngII. After 13 days of treatment, brains were collected and the PVN isolated and analyzed using Agilent gene array chips. Significantly regulated genes (ANOVA, p<0.05) were evaluated using PathwayStudio® software to reveal functional network relationships. A gene network revealed transcriptional regulation patterns centered around tumor necrosis factor (TNF). PVN expression of TNF was decreased in hypertensive rats and is positioned in a pivotal role that directly or indirectly regulates expression of other hypertension‐related genes. This network also involves several transcriptional factors (MLLT1, TLX2) and receptors (ITGAM, HTR1 A). Our data suggest that decreased PVN expression of TNF plays a hub role in network of changes in gene expression associated with hypertension. (Supported by HL76083)
Longitudinal analysis of AT‐1 angiotensin receptor binding in the rostral and caudal ventrolateral medulla and nucleus of the solitary tract in the rat brainstem
Authors: Bourassa Erick A. , Speth Robert C.
Published: June 2021

The solitary tract nucleus (NTS), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM) are brainstem structures that have been identified as central sites at which blood pressure is controlled. Each are reported to be responsive to angiotensin II (Ang II) and to contain type 1 Ang II receptors (AT1R). This study used quantitative densitometric saturation autoradiography to determine the density of AT1R in the RVLM and CVLM of the female rat. Results showed that specific binding of 125I‐Sarcosine1, Isoleucine8 Ang II in the NTS had high density (1207 ± 99.8 fmol/g wet weight) and high affinity (KD of 328 ± 12.3 pM). Specific binding was much lower in the CVLM and RVLM. The CVLM (−14.24 to −13.76 mm relative to Bregma) contained 379 ± 63.9 fmol/g wet weight. The KD (178 ± 27.2 pM) was significantly lower than NTS (p<0.01). The density of AT1R in the RVLM (−13.64 to −12.08 mm relative to Bregma) was 275 ± 38.1 fmol/g wet weight with KD=164 ± 20.4 pM (p<0.01 versus NTS). These results indicate that there is a low density, but high affinity population of AT1Rs in the CVLM and RVLM that are the likely mediators of the effects of locally acting and exogenously applied Ang II on blood pressure in rats. This study was supported by the Peptide Radioiodination Service Center, University of Mississippi.
Stimulation of adenosine A1 receptors in the nucleus of the solitary tract (NTS) triggers the release of vasopressin into the circulation
Authors: McClure Joseph M , Rossi Noreen F , Chen Haiping , O'Leary Donal S , Scislo Tadeusz J
Published: June 2021

Our previous study showed that stimulation of NTS A1 adenosine receptors exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and β‐adrenergic vasodilation vs. vasoconstriction mediated by neural and unknown humoral factors (Am J Physiol 289:H2536‐H2542, 2005). The follow up study identified vasopressin as a potential humoral factor involved: systemic blockade of V1 vasopressin receptors abolished/reversed iliac vasoconstriction (FASEB 20: A363, 2006). Stimulation of NTS A1 adenosine receptors attenuates the baroreflex (FASEB 21: A466, 2007), thus may increase circulating vasopressin. Therefore in the present study we compared circulating vasopressin levels before and after microinjections into the NTS of selective A1 adenosine receptor agonist N6cyclopentyladenosine (CPA, 330 pmol/50nl) or vehicle (artificial cerebrospinal fluid, ACF, 50 nl), using the same experimental model as previously (chloralose/urethane anesthetized rats). CPA increased circulating vasopressin over 4‐fold (117.0±19.0 vs. 26.4±10.4 pg/ml, P=0.0006, n=8) whereas ACF did not increase vasopressin level significantly (50.9±11.3 vs. 25.0±7.2 pg/ml, P=0.083, n=5). We conclude that stimulation of NTS A1 adenosine receptors increases circulating vasopressin probably via inhibition of NTS baroreflex mechanisms. NIH HL‐67814
Post‐synaptic effects of Group II metabotropic glutamate receptors (mGluRs) on second‐order baroreceptor neurons of the nucleus tractus solitarius (NTS) in rats
Authors: Sekizawa Shin‐ichi , Hoffman Matthew , Bonham Ann C.
Published: June 2021

In vivo and in vitro studies have suggested that Group II mGluRs have complex presynaptic effects on baroreceptor signal transmission in the NTS, heterosynaptically depressing both glutamate and GABA release. Here, we tested the effects of postsynaptic Group II mGluR activation by agonists (L‐CCG‐1, 10 μ M or LY379268,100 nM) and by endogenous glutamate release (TS stimulation) on functionally‐identified second‐order baroreceptor neurons in a brainstem slice with blockade of ionotropic GluRs (NBQX, 10 μ M & AP5, 50 μM) and GABA‐A‐Rs (bicuculline, 10 μ M). At concentrations selective for mGluR Group II activation as shown by blockade by the mGuR, antagonist, LY341495 (0.2 – 1 μM), the agonists, LY379268 and L‐CCG‐1 (10 μM) hyperpolarized the membrane potential (−10 ± 1.2 mV, n = 24, and −6.5 ± 1.0 mV, n = 16, respectively). Under these same conditions, TS stimulation (6 paired stimuli/s) also induced a hyperpolarization (−5.1 ± 0.5 mV; n = 3). LY379268‐induced hyperpolarization was decreased with a nonhydrolyzableGDP analog, GDP‐β‐S in the patch pipette by 72 ± 6.9 % (p < 0.05, n = 7), indicating G‐protein participation. The data suggest that mGluR Group II receptors may modulate baroreceptor signal transmission by postsynaptic effects in addition to presynaptic effects on glutamate and GABA release at the first synapses in the baroreceptor central network.
Glucocorticoids Enhance Expression of Angiotensin II Type 1 Receptors in the Dorsal Hindbrain
Authors: Su Ye , Daubert Daisy L. , Sumners Colin , Speth Robert , Li Hongwei , Scheuer Deborah A.
Published: June 2021

We have shown that increases in dorsal hindbrain (DHB) corticosterone (Cort) enhance the blood pressure response to acute stress. Stress activates brain angiotensin type I receptors (AT1R), and glucocorticoids can increase AT1R expression. Thus, we hypothesized that chronic increases in DHB Cort elevate AT1R expression and binding within the nucleus of solitary tract (NTS). Male Sprague‐Dawley rats were treated with sham, 50% or 100% Cort DHB pellets, or 100% Cort pellets subcutaneously (sc) for 6 days. AT1R mRNA was analyzed by quantitative real‐time PCR, and the binding of AT1R was assessed by autoradiography and quantitative densitometry in the NTS and the rostral ventral lateral medulla (RVLM). Data were analyzed by ANOVA. In the caudal NTS, AT1R binding density was significantly increased (P<0.05) in 50% and 100% DHB Cort‐treated rats (1527±54 and 1436±42 fmol/g) relative to DHB sham‐treated rats (1215±57 fmol/g). However, in the rostral NTS and RVLM, there were no effects of DHB Cort treatment on AT1R binding. AT1R mRNA in the NTS was significantly (P<0.05) increased in DHB 50% Cort‐treated rats relative to DHB Sham‐ and sc‐treated rats (1.77±0.13 versus 0.73±0.1 and 0.97±0.1 arbitrary units). The increase in mRNA in 100% DHB Cort‐treated rats was not significant. We conclude that chronic elevations in DHB Cort increase AT1R mRNA and binding within the NTS. Funded by NIH grant number HL 076807.
Direct Activation of Mouse Bronchopulmonary C‐fibers by Protease Activated Receptor‐1 (PAR1) stimulation
Authors: Kwong Kevin , Nassenstein Christina , Undem Bradley J
Published: June 2021

The chemical mediators most involved in bronchopulmonary C‐fiber activation at sites of airway inflammation are poorly understood. We hypothesize that stimulators of certain protease activated receptor (PARs) may be important in this regard. A single cell RT‐PCR analysis of mouse lung‐specific neurons that express the capsaicin receptor, TRPV1 (C‐fiber marker), revealed that 9 of 9 neurons also expressed PAR1 mRNA and 8 of 9 neurons express PAR3 mRNA. Neither PAR2 nor PAR4 were expressed in the C‐fiber neurons. Strong expression of all 4 PARs was found in the ganglion tissue per se (positive control). Using an ex vivo isolated lung‐nerve preparation, we found that thrombin (1 μM), which activates PAR1, PAR3 and PAR4, evoked action potential discharge in lung C‐fibers (peak frequency = 7 ± 1 Hz, n = 7). This was mimicked by TFLLR (10 μM), a selective PAR1 activating peptide, whereas SLIGRL, a selective PAR2 activating peptide, was ineffective. Consistent with the ex vivo lung preparation, TFLLR, but not SLIGRL, elicited robust inward currents (−29.8 ± 13.2 pA/pF; n = 6) in whole cell voltage clamped capsaicin‐sensitive vagal neurons. These results indicate the PAR1 is the most relevant PAR in overt activation of bronchopulmonary C fibers in mouse lungs. This has relevance to inflammatory airway disease in that PAR1 is potently activated by thrombin a peptidase known to be elevated at the site of inflammation, including allergic inflammation of the lungs. Funded by NIH.
Enhancement of the hypoxic ventilatory reponse in adult rats subjected to neonatal maternal separation is not affected by sleep‐wake states
Authors: Kinkead Richard , Montandon Gaspard , Lajeunesse Yves , Bairam Aida , Horner Richard
Published: June 2021

Neonatal maternal separation (NMS) disrupts respiratory control development. In the awake male rat, the hypoxic ventilatory response of adults previously subjected to NMS is ~25% greater than controls (Genest et al., 2004; 2007). Because changes in arousal state affect respiratory control, we tested the hypothesis that NMS‐related enhancement of the hypoxic ventilatory response occurs during sleep also. Experiments were performed on two groups of rats (n = 5 per group): Pups subjected to NMS were placed in an incubator 3h/day for 10 consecutive days (P3 to P12). Controls were undisturbed. Rats were then reared normally until adulthood (8 – 10 weeks) when EEG and neck EMG electrodes were implanted for sleep‐wake state monitoring by telemetry. Following recovery (14 days) baseline ventilatory activity was measured using plethysmography for 5 h. During each sleep‐wake state, rats were exposed to a brief hypoxic period (12% O2 for 90 sec). Sleep‐wake states were scored visually. Minute ventilation changed across sleep‐wake states (p = 0.03). The hypoxic ventilatory response of NMS rats was greater than controls during wakefulness, REM, and non‐REM sleep (34, 47 and 53%, respectively; p ≤ 0.05). Since excessive hypoxic ventilatory response can lead to respiratory instability during sleep, these data suggest that early life stress exposure could predispose to respiratory disease in the adult. Supported by the CIHR.
RAPHE OBSCURUS (ROb) MODULATES THE VENTILATORY RESPONSE TO FOCAL ACIDIFICATION OF THE RETROTRAPEZOID NUCLEUS (RTN)
Authors: Dias Mirela Barros , Li Aihua , Branco Luiz Guilherme , Nattie Eugene E
Published: June 2021

Recent data suggest that the caudal medullary raphe (MR) does not contribute to chemoreception directly but modulates breathing by interaction with the RTN (Li et al., J Physiol 2006). To investigate this hypothesis, we dialyzed artificial cerebrospinal fluid equilibrated with 25% CO2 into RTN and ROb simultaneously in male rats in a whole body plethysmograph at a temperature within the rat thermoneutral zone (30°C). We compared data from animals with both probes correctly placed to data from rats with both probes misplaced. Simultaneous acidification of RTN and ROb increased VE up to 23%, compared with the baseline and up to 28% compared to the control group, since focal acidification in the control group decreased VE. The responses were similar in wakefulness and sleep. For single site dialysis experiments, both probes received 25% CO2 dialysis, but just one of the probes was correctly placed. The 25% CO2 dialysis into RTN caused a small increase in VE (∼8%; n=05), and this effect was due to an increased f. The focal acidification of the ROb had no effect on VE (N=04). Although there was no response to CO2 dialysis in the ROb, the simultaneous dialysis into the RTN and ROb potentiated the effect of the focal acidification of RTN alone. These results indicate a modulatory role of caudal MR with respect to central chemoreception at the RTN. (Supported by FAPESP and R37 HL 28066)
Exercise Combined with Hypoxia Enhances Phagocytosis and Oxidative Burst of Neutrophils to E.coli
Authors: chou ya‐ting , Wang Jong‐shyan
Published: June 2021

This study investigates how exercise combined with hypoxia affects phagocytosis and oxidative burst of neutrophil to E.coli. Eight sedentary men engaged six conditions in a normobaric hypoxia chamber for 1 hour: two normoxic exercise [i.e., strenuous exercise (SE, up to VO2max) and moderate exercise (ME, 50%VO2max) in 21% O2], two hypoxic exercise (i.e., ME in 12% and 15% O2), and two hypoxic conditions (i.e., 12% and 15% O2). Our results showed that SE induced higher phagocyotic and oxidative burst activities of neutrophils, as well as, CD11b and CD62L expressions on neutrophils than ME. However, combined with 12% or 15% O2 intervention, ME promoted the extent of oxidative burst activities of neutrophils, accompanied by augmenting the increases of neutrophil CD11a, CD11b, CD62L, and CD16b expressions by this exercise. Additionally, normoxic SE and ME increased neutrophil CD88 expression and decreased binding of C1q to neutrophil, which changes were similar to two hypoxic ME. Therefore, we conclude that ME combined with 12% or 15% O2 intervention enhanced E.coli.‐induced oxidative burst of neutrophils, possibly by up‐regulating expressions of adhesion molecules on neutrophils.
Exercise Combined with Hypoxia Modulates Cytotoxicity of Natural Killer Cells to Nasopharyngeal Carcinoma Cells
Authors: Wu Chia‐Kuan , Wang Jong‐shyan
Published: June 2021

This study investigated how exercise combined with hypoxia affects the cytotoxicity of natural killer cells (NK) to nasopharyngeal carcinoma cells (NPC) and its underlying mechanism. Eight sedentary men engaged six conditions in a normobaric hypoxia chamber for 1 hour: two normoxic exercise [i.e., strenuous exercise (SE, up to VO2max) and moderate exercise (ME, 50%VO2max) in 21% O2], two hypoxic exercise (i.e., ME in 12% and 15% O2), and two hypoxic conditions (i.e., 12% and 15% O2). Our results showed that SE induced higher NK cell count and perforin/granzyme levels and NK‐induced apoptosis of NPC than ME. However, combined with 15% O2 intervention, ME promoted extent of NK‐induced apoptosis of NPC, accompanied by augmenting the increases of NK cell count and perforin level by this exercise. Although ME combined with 12% O2 intervention increased NK count and perforin level, no significant change occurred in cytotoxicity of NK to NPC following this intervention. Therefore, we conclude that ME combined with 15% O2, but not 12% O2, intervention enhances NK count and perforin level and subsequently promotes the cytotoxicity of NK to NPC.
Modulation of Pulmonary Vascular Smooth Muscle Cell Phenotype in Hypoxia: Role of Myocardin and ELK‐1
Authors: Zhou Weilin , Raj J Usha
Published: June 2021

Hypoxia triggers pulmonary vascular remodeling, which is associated with vascular smooth muscle cell (SMC) phenotype modulation from a contractile to a synthetic state. Altered expression of cGMP‐dependent protein kinase (PKG) after hypoxia is implicated in SMC phenotype modulation. But the mechanism of SMC phenotype modulation after hypoxia at a gene transcriptional level is not clear. In this study, we found that in pulmonary vascular SMC, hypoxia (24h) significantly down‐regulated protein and mRNA expression of Myocardin, but up‐regulated mRNA expression of E‐26‐like protein 1 (Elk‐1). Acute hypoxia (1h) also induced phosphorylation of ELK‐1. Data from chromatin immunoprecipitation assay showed that under hypoxia conditions, less Myocardin and more ELK‐1 was bound to serum response factor (SRF), the transcription factor that is involved in SMC specific gene transcription. These data indicate that hypoxia activates ELK‐1 and inactivates Myocardin, thus inhibiting SRF function, suppressing SMC specific gene transcription and promoting SMC modulation to a de‐differentiated, synthetic state. Inhibition of PKG by PKG siRNA down‐regulated the expression of Myocardin, but up‐regulated the expression of ELK‐1, indicating that PKG may modulate SMC phenotype via regulation of Myocardin and ELK‐1.
Hypoxia‐regulated phenylethanolamine N‐methyltransferase expression
Authors: Wong Dona Lee , Tai T.C. , Wong‐Faull David C. , Claycomb Robert
Published: June 2021

Hypoxia is being used to identify mechanisms for stress‐induction of the epinephrine synthetic enzyme, phenylethanolamine N‐methyltransferase (PNMT). When PC12 cells transfected with a PNMT promoter‐luciferase reporter gene construct are exposed to hypoxia (5% oxygen), luciferase activity rapidly rises (6 hr) and is sustained for 72 hr. Signaling pathway inhibitor pre‐treatment prior to oxygen reduction abrogates induction and identifies two major signaling pathways, cAMP and phospholipase C (PLC). Downstream hypoxia inducible factor‐1α (HIF‐1α) and PNMT activators Egr‐1 and Sp1 are elevated. HIF‐1α co‐transfection or HIF‐1α activation by CoCl2 and desferoxamine stimulate the PNMT promoter. While HIF‐1α is constitutively expressed, inclusion of the RNA synthesis inhibitor actinomycin D and protein synthesis inhibitor cycloheximide inhibit their induction. Hypoxia elevates Egr‐1 and Sp1 mRNA, protein and protein‐DNA binding complex formation as well. Stimulation is rapid but transient for Egr‐1 while sustained for Sp1 (24 hr). Egr‐1 binding site mutation in the PNMT promoter prevents activation by hypoxia. However, hypoxia generates long and short forms of PNMT mRNA, which produce inactive and active enzyme respectively. Thus, hypoxic stress transcriptionally activates PNMT but post‐transcriptional controls limit expression of functional enzyme.
Cardiovascular improvements after lung transplantation in patients with cystic fibrosis
Authors: Layton Aimee M , Rebmann Rebecca A , De Meersman Ronald E , Wilt Jessie S , Bartels Matthew N
Published: June 2021

Due to a lack of published data, we investigated the effects of lung transplantation on cardiopulmonary parameters for patients with cystic fibrosis (CF). METHOD: Pre‐ and post‐lung transplantation data from a traditional progressive cycle ergometer cardiopulmonary exercise test were retrospectively analyzed for 5 males and 5 females with cystic fibrosis. Most recent exercise tests were used for post‐transplant data, unless rejection was suspected at time of test. Last test prior to suspected rejection was used for these patients. RESULTS: Post‐transplant, patients showed significant improvements (p<0.05) at rest in heart rate (HR), respiratory rate (RR), minute ventilation (VE), energy expenditure per kilogram (REE), and body mass index (BMI). Oxygen consumption per workload was significantly decreased during peak exercise post‐transplant. CONCLUSION: Patients with CF who undergo lung transplant show improved cardiopulmonary function. The observed decrease in REE may have resulted from improved respiratory efficiency as shown by the decreased HR, RR, and VE at rest, and may contribute to nutritional status improvement as evidenced by increased BMI. Supported by VIDDA foundation.
Apical secretion of collagen II from endothelial cells precedes blood vessel formation during postnatal vasculogenesis
Authors: Alvarez Diego F. , Robinson Daniel , ElZarrad M. Khair , Stevens Troy , King Judy A.
Published: June 2021

Postnatal vasculogenesis requires lumen formation during vessel development. Conflicting studies suggest that endothelial vacuolization or inside‐out matrix deposition initiates lumen formation. Since adult pulmonary microvascular endothelial cells (PMVECs) are highly vasculogenic, we sought to determine the mechanism resulting in lumen formation during PMVEC‐mediated postnatal vasculogenesis. GFP‐transfected PMVECs were mixed with matrix gel, injected subcutaneously in rats, excised within 10 days, and processed for microscopy (n=5 each). After 2 days post‐implantation, PMVECs formed clusters of 4 to 6 cells (stage 1) with some clusters displaying apical matrix deposits (stage 2). After 4 days, lumens contained either matrix (stage 3) or blood cells (stage 4). After 10 days, a predominance of blood vessels (stage 4) was observed. There was no evidence of endothelial vacuolization at any time point. By transmission microscopy, the apical matrix deposits were identified as collagen. Liposomal transfection of quantum dots in PMVECs revealed apical secretion of the dots by this phenotype during stage 2. GFP‐PMVECs within the gel were involved in all stages described, and collagen II was identified as the isoform present in the apical structures during stages 2 and 3. We conclude that endothelial apical secretion of collagen II precedes blood vessel formation during postnatal vasculogenesis. HL66299.
Receptor Dimerization with EPCR Redirects PAR‐1 Signaling in Endothelial Cells
Authors: McLaughlin Joseph N , Malik Asrar B
Published: June 2021

Activated protein C (APC) is the only therapeutic shown to reduce mortality in patients with severe sepsis, however its mechanism of action remains unknown. An important possibility is that APC influences the activation of the G‐protein coupled thrombin receptor (PAR‐1) in the endothelium to promote barrier function. We propose that PAR‐1 signaling is regulated by direct interactions with the single transmembrane endothelial protein C receptor (EPCR), and that this co‐factor, not the proteolytic activity of thrombin or APC per se, determine whether PAR‐1 signaling is endothelial barrier‐disruptive or barrier‐protective. We postulate that the interaction of PAR‐1 with EPCR alters the G‐protein specificity of PAR‐1 by stabilizing a discrete active conformation. Toward this end we show here that PAR‐1 constitutively interacts with EPCR. However, the PAR‐1/EPCR complex is maintained only when PAR‐1 is activated by APC and not by thrombin. Hence EPCR may function as a critical molecular switch redirecting PAR‐1 signaling from endothelial barrier‐disruptive to barrier‐protective.
Hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells depends on epidermal growth factor receptor (EGFR) tyrosine kinase activity
Authors: Toby Inimary T , Chicoine Louis G , Cui Hongmei , Nelin Leif D
Published: June 2021

The hallmark of pulmonary hypertension is vascular remodeling, which has recently been postulated to represent a type of malignant transformation. In cancer therapy, EGFR tyrosine kinase has been recognized as a therapeutic target to prevent tumor cell proliferation. We hypothesized that hypoxia would lead to human pulmonary microvascular endothelial cell (hPMVEC) proliferation via EGFR tyrosine kinase activity. hPMVEC were placed in normoxia (20% O2, 5%CO2) or hypoxia (1% O2, 5% CO2) with or without AG1478 added. Western blotting for total EGFR and PCNA, and proliferation assays were done. Protein levels of total EGFR and PCNA were increased in hypoxia compared to normoxia groups. Hypoxic hPMVEC had a 2‐fold greater level of proliferation compared to normoxic controls. This increase in proliferation was prevented by addition of AG1478. Our data demonstrate that EGFR tyrosine kinase activity is critical in promoting proliferation of hPMVEC in hypoxia. We speculate that EGFR tyrosine kinase inhibition represents a novel therapy to prevent vascular remodeling in pulmonary hypertension.
Receptor for activated C kinase1 (RACK1) binds with G beta1 and restricts focal adhesion kinase activation and restoration of endothelial barrier function
Authors: Knezevic Nebojsa , Kini Vidisha , Malik Asrar B , Mehta Dolly
Published: June 2021

We showed that sustained activation of focal adhesion kinase (FAK) is required for reversing the increase in endothelial permeability induced by inflammatory mediator thrombin. However, mechanism that sustain FAK activation is not known and therefore will help in identifying signaling cues responsible for restoring endothelial barrier function. Gβ1 and receptor for activated C kinase (RACK1) belong to large family of WD40 repeat proteins and act as scaffold proteins to orchestrate downstream signaling events. We therefore investigated the contribution of RACK1 and Gβ1 in regulating FAK activation and recovery of endothelial barrier function. We show that RACK1 is associated with Gβ1 in untreated cells. Thrombin induced a time‐dependent reversible dissociation of RACK1 from Gβ1, enabling it to interact with upstream FAK regulator Fyn. Inhibiting endogenous expression of Gβ1 had no effect on thrombin‐induced increase in endothelial permeability but markedly impaired endothelial barrier repair. Knockdown of Fyn in endothelial cells also prevented barrier recovery post‐thrombin challenge, linking Gβ1 to the regulation of Fyn function. In contrast, knock down of RACK1 increased basal phosphorylation of FAK and potentiated endothelial barrier recovery. Thus, these data demonstrate that RACK1 prevents the access of Gβ1 from forming a complex with Fyn, which is required for FAK activation and restoration of endothelial barrier function.

Supported by NIH grants: HL71794 and HL84153
Ginkgo Biloba extract, via upregulation of heme oxygenase‐1, confers protection from oxidative stress‐related apoptosis induced by cigarette smoke extract in human lung endothelial cells
Authors: Kou Yu Ru , Hsu Chiu‐Ling , Wu Yuh‐Lin , Lee Tzong‐Shyuan
Published: June 2021

We investigated the cytoprotective effects and therapeutic mechanisms of Ginkgo Biloba extract (EGb) against apoptosis of human pulmonary artery endothelial cells (HPAECs) induced by cigarette smoke extract (CSE). Challenge of CSE (160 microgram/ml) caused reduction in cell viability, increases in reactive oxygen species (ROS) and promotion of caspase‐dependant apoptosis in HPAECs, all of which were alleviated by pretreatment with EGb (100 microgram/ml). Exposure of HPAECs to EGb alone produced activation of mitogen‐activated protein kinases (MAPKs), increase in nuclear level of transcription factor Nrf2 and upregulation of heme oxygenase‐1 (HO‐1, an oxidative stress‐responsive protein). This HO‐1 upregulation was eliminated by inhibitors of MAPKs. While gene knockdown by small interfering RNAs targeting HO‐1 prevented the EGb‐induced HO‐1 upregulation, it also abolished the antioxidant, anti‐apoptotic and cytoprotective effects of EGb in HPAECs challenged with CSE. We conclude that EGb confers protection from oxidative stress–related apoptosis induced by CSE in HPAECs and its antioxidant and anti‐apoptotic effects are mediated through upregulation of HO‐1. (Supported by NSC‐95‐2320‐B‐010‐025‐MY3).
Mast Cell Chymase Induces Cardiomyocyte Anoikis by a Mechanism Involving Laminin Degradation and Disruption of Focal Adhesion
Authors: Zheng Junying , Powell Pamela C. , Pat Betty , Dell’Italia Louis A. , Killingsworth Cheryl R. , Dell’Italia Louis J.
Published: June 2021

Loss of cardiomyocytes plays a causal role in the development of heart failure. Adhesion to glycoproteins of the extracellular matrix (ECM) is necessary for survival of the cardiomyocytes in the heart. Proteases are able to degrade glycoproteins and induce anoikis of adherent cells. Mast cells contain various proteases such as chymase. In mitral regurgitation (MR) dogs, there is an increase of mast cell infiltration and degranulation. In addition, chymase activity is elevated in early MR and remains elevated throughout the course of MR. In this experiment, dog myocytes were treated with various doses of chymase (0.1, 1.0, and 10 <μ>g/ml), each at a time course of 1, 4, 12, and 24 hours. After treatment, cells showed decreased attachment to laminin with a progressive increase in chymase concentration of 0.1 to 10 <μ>g/ml. At 24 hours, 70% of cells were detached after treatment with 10 <μ>g/ml of chymase, while > 90% of untreated cardiomyocytes remained. Detection of cleaved caspase‐3 indicated that detached cells were undergoing apoptosis. Focal adhesion kinase, one of the key mediators of integrin–ECM interactions was rapidly degraded in the presence of chymase. Separate experiments demonstrated that chymase concentration dependently degraded laminin. These results suggest that chymase can mediate anoikis of dog cardiomyocytes through a mechanism involving degradation of laminin and cell/matrix interactions.
Activation of Na,K,2Cl‐cotransporter stimulates outer medullary connective tissue expression
Authors: Paliege Alexander , Mutig Kerim , Theilig Franziska , Goligorsky Michael , Bachmann Sebastian
Published: June 2021

Nitric oxide (NO) deficiency stimulates renal medullary Na,K,2Cl‐cotransporter (NKCC2) activity. Chronic NO synthase inhibition has also been shown to increase the production of extracellular matrix components, thus promoting renal fibrosis. The role of NKCC2 in this process is not fully understood. We here hypothesize that chronic activation of NKCC2 leads to increased production of extracellular matrix components despite intact NO production. To test this, male adult Brattleboro rats with central diabetes insipidus were treated with the vasopressin 2 receptor analoque desmopressin (5ng/h; 5d), via osmotic minipumps. Urinary output served as a measure of treatment efficiency. NKCC2 expression in outer medullary tissue samples was determined by TaqMan real time RT PCR. Microarray analysis was used to identify differentially regulated genes. In the treated rats 24h urine output was decreased to 6% (p=.01) and outer medullary NKCC2 expression was increased to 170% (p=.013) of controls. Microarray analysis revealed increased expression of numerous extracellular matrix components including collagen I, III–VI, XII, XV and XVIII (>2fold; p<10‐5). We conclude that the activation of NKCC2 in a setting of intact local NO synthesis is associated with increased outer medullary connective tissue production. Long term studies are needed to elucidate the pathophysiological significance of this finding.
Glycolytic Enzyme Interactions with the Caveolin Scaffolding Domain (CSD) in a Lymphocyte Model
Authors: Fischer Caitlin N. , Hardin Christopher D.
Published: June 2021

Previously, we found caveolin (CAV‐1) expressed by transfection in lymphocytes induced caveolae formation and targeted glycolytic enzymes such as aldolase, phosphofructokinase and glyceraldehyde‐3‐phosphate dehydrogenase to the plasma membrane in the CAV‐1 transfected lymphocytes. We hypothesized that if CAV‐1 with a mutant caveolin scaffolding domain (CSD) is expressed in lymphocytes then colocalization of the glycolytic enzymes with CAV‐1 will be reduced. We tested this hypothesis by comparing the colocalization of CAV‐1 with glycolytic enzymes in lymphocytes which expressed either a wild type CAV‐1 (WT) or a mutant CAV‐1 which had either one mutation (SM) or two mutations (DM) in the CSD. Colocalization analysis by confocal microscopy for CAV‐1 and ALD was 0.77 in CAV‐1 WT lymphocytes, 0.24 in CAV‐1 SM lymphocytes, and 0.59 in CAV‐1 DM lymphocytes. Analysis of colocalization of the enzymes PFK, GAPDH, and ALD with CAV‐1 averaged 0.65 for CAV‐1 WT cells, 0.49 for CAV‐1 SM cells and 0.51 for the CAV‐1 DM cells. The shift in distribution of glycolytic enzymes and CAV‐1 in the CAV‐1 WT, the CAV‐1 SM or DM CAV‐1 types indicates that a single mutation to the CSD reduces glycolytic enzyme membrane targeting, and two mutations in the CSD produces CAV‐1 retention in the cytoplasm, both suggesting that an intact CSD domain is essential for CAV‐1 targeting of glycolytic enzymes to the membrane. Support NIH DK60668.
The impact of low‐ and high‐density lipoproteins on inwardly‐rectifying K+ channels in macrophages
Authors: GURSAHANI Kunal , Singh Dev , Epshtein Yulia , Chopra Arun , Levitan Irena
Published: June 2021

Hypercholesterolemia is a major risk factor for macrophage dysfunction, including release of multiple proinflammatory cytokines. In this study, we address a hypothesis that membrane potential is one of the mechanisms contributing to lipoprotein‐induced macrophage dysfunction. Our earlier studies showed that an increase in cellular cholesterol suppresses inwardly‐rectifying K+ channels (Kir) in endothelial cells in vitro and in vivo. Here we show that Kir current is also suppressed by cholesterol in alveolar and in bone marrow‐derived macrophages. Specifically, we show that macrophage Kir are suppressed by enriching the cells with cholesterol using methyl‐b‐cyclodextrin or by exposing them to acetylated LDL. In cotrast, HDL results in a decrease in the level of cellular cholesterol and induces an increase in Kir. Furthermore, unexpectedly, exposing macrophages to oxLDL also significantly increases the amplitude of the current. In this case, however, the level of membrane cholesterol is not affected. We suggest, therefore, that the similarities between the effects of cholesterol depletion, HDL and oxLDL may be due to their effects on lipid packing. Finally, we show that blocking Kir currents results in a significant increase in the production of interleukins 1α and β suggesting that membrane depolarization contributes to the inflammatory response of the macrophages under high cholesterol conditions.
Ca2+ pulsars: spatially restricted, IP3R‐mediated Ca2+ release important for endothelial function
Authors: Ledoux Jonathan , Taylor Mark S , Bonev Adrian D , Hannah Rachael M , Tallini Yvonne N , Kotlikoff Michael I , Nelson Mark T
Published: June 2021

Intracellular Ca2+ is essential for endothelial functions such as the control of vascular tone. Using confocal microscopy, endothelial Ca2+ signaling was investigated in cut‐open mesenteric arteries from mice expressing the Ca2+ indicator GCaMP2 exclusively in the endothelium. Spontaneous and local Ca2+ release (“Ca2+ pulsars”) from endothelial IP3 receptors (IP3R) have been identified and are also present in pressurized arteries. The Ca2+ pulsars show distinct kinetics from ryanodine receptor–dependent Ca2+ sparks and from IP3R‐mediated Ca2+ puffs. The endothelium‐dependent vasodilator acetylcholine increased the frequency of Ca2+ pulsars. The Ca2+ pulsars are localized in the endothelial projections through the internal elastic lamina to the vascular smooth muscle. Immunocytochemistry also identified IP3R and KCa3.1 channels in the myoendothelial projections. While block of KCa3.1 channels with ChTX depolarized the endothelial membrane by ≈10 mV, inhibition of Ca2+ pulsars with CPA (10 μM) prevented ChTX‐induced depolarization. Moreover, exposure to CPA induced a depolarization of the endothelium similar to ChTX (≈11 mV). Our results suggest a novel IP3R‐dependent mechanism regulating endothelium‐smooth muscle communication.

Supported by NIH (HL44455 & HL63722) and CIHR.
The effect of mitochondrial substrate on secretagogues‐induced Ca2+ signaling in pancreatic acinar cell
Authors: Won Jong Hak , Yule David
Published: June 2021

We investigated the effect of cell permeable mitochondrial substrates such as dimethyl succinate (DMS) on secretagogues‐induced Ca2+ oscillations in pancreatic acinar cells. DMS did not markedly alter basal [Ca2+]i However, CCh‐induced Ca2+ oscillations in pancreatic acinar cell were inhibited in a concentration dependent, and reversible manner by DMS but not by 10 mM methypyruvate or 10 mM succinate. When cells were stimulated with 10 μM CCh or 30 μM CPA in the absence of extracelluar Ca2+, the Ca2+ release phase of the response was unaffected by 10 mM DMS but Ca2+ influx upon readmission of extracellular Ca2+ was inhibited. In addition, the sustained plateau of the calcium response stimulated by 10 μM CCh in the presence of extracellular was inhibited by 10 mM DMS. The amplitude of CCK‐induced Ca2+ oscillations was not affected by DMS but the frequency of the Ca2+ oscillations was reduced. These data indicate that DMS exposure and presumably manipulation of ATP levels inhibits Ca2+ influx in pancreatic acinar cells, ultimately resulting in a modulation of Ca2+ oscillations.
MUC1 interacts with CAML: A protein involved in Ca2+ signaling
Authors: Lillehoj Erik P. , Guang Wei
Published: June 2021

Objective: MUC1 is a transmembrane mucin glycoprotein expressed on the surface of lymphocytes and epithelial cells. Its cytoplasmic COOH‐terminus (CT) is evolutionarily conserved and mediates intracellular signaling. The objective of this study was to identify proteins involved in MUC1 CT signaling.

Methods: We searched for proteins binding to the MUC1 CT using a yeast two‐hybrid system and confirmed the results by coimmunoprecipitation (coIP) analysis.

Results: Of the positive clones encoding proteins that interacted with the MUC1 CT, the majority coded for CAML (calcium modulating cyclophilin ligand), a protein involved in Ca2+ signaling. The MUC1 CT interacted with CAML as revealed by growth on selective media and in situ X‐alpha‐galactosidase activity of yeast double transformants or progeny from matings of single transformants. Binding of the MUC1 CT to CAML was confirmed by reciprocal coIPs (MUC1 CT IP + CAML IB and CAML IP + MUC1 CT IB). By deletion mutagenesis, only the MUC1 CT and not its extracellular tandem repeat region or SEA domain interacted with CAML. Likewise, the region of CAML binding to MUC1 was localized to its NH2‐terminus.

Conclusion: Our results have identified CAML as a novel binding partner of the MUC1 CT and suggest that MUC1 plays a role in Ca2+ signaling.
Signaling processes for initiating smooth muscle contraction
Authors: Ding Hai‐Lei , Ryder Jeffrey W. , Stull James T. , Kamm Kristine E.
Published: June 2021

Relationships among signaling processes involved in Ca2+/calmodulin‐dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined to provide insights into cellular mechanisms for initiating smooth muscle contraction. A genetically encoded MLCK biosensor showing Ca2+‐dependent CaM binding by decreased fluorescence resonance energy transfer (FRET) coincident with increased kinase activity was expressed in smooth muscles of transgenic mice. Isolated bladder smooth muscle tissues were neurally stimulated for 3 s. The latencies for the onset of [Ca2+]i and FRET were 55±8 and 65±6 ms, respectively. Both increased along with RLC phosphorylation at 100 ms while force latency was 109±3 ms. Increases in [Ca2+]i, FRET and RLC phosphorylation were maximal by 1.2 s whereas force increased more slowly to a maximal value at 3 s. Phosphorylation of two myosin RLC phosphatase regulatory proteins (MYPT1 and CPI‐17) did not change during the initial contraction phase. Thus, increases in [Ca2+]i lead to a rapid MLCK activation and RLC phosphorylation showing a tightly coupled signaling complex. The delayed temporal response between RLC phosphorylation and force may be due to mechanical effects associated with myosin cross bridge attachment and expression of force through series elastic elements in the tissue. This study was supported by NIH Grant HL026043.
Rotaviruses and their components induce antiviral gene expression in intestinal epithelia
Authors: Frias Amena , Vijay‐Kumar Matam , Wu Hui‐Xia , Neish Andrew , Gewirtz Andrew
Published: June 2021

Rotavirus, the world's leading cause of severe diarrhea in children, primarily infects the epithelial cells of the small intestine. Rotavirus is typically cleared within 7 days via pathways not strictly dependent on adaptive immunity. Therefore, we hypothesize that epithelial cell antiviral gene expression is important for host defense against rotavirus and sought to define the mechanism by which epithelia detect rotavirus, particularly whether epithelial antiviral gene expression resulted from detection of a viral component or required active viral replication. Polarized model intestinal epithelia were treated with live rhesus rotavirus (RRV) (MOI 1–3) or RRV that had been UV‐irradiated (UV‐RRV) to render it non‐infectious but leave its epitopes intact. Expression of viral proteins and activation of interferon regulatory factors 3 and 7 (IRF 3/7) and STAT 1/3 were measured by western blotting. Cytokines IL‐8 and IFNB were measured by ELISA. Apical, but not basolateral, treatment of model epithelia with RRV resulted in productive infection (synthesis of viral proteins) and robust activation of all pathways measured. Interestingly, such activation of host antiviral pathways was largely recapitulated by treatment with UV‐RRV. Together, these results suggest that epithelial antiviral gene expression may result from activation of an apical receptor that detects a component of rotavirus.
Soybean product inhibits K88 enterotoxigenic Escherichia coli binding to enterocytes
Authors: Lin Jun , He Dong , Van Hout Amber , Kaushik Radhey , Francis David , Erickson Alan
Published: June 2021

Strains of enterotoxigenic Escherichia coli expressing the K88 fimbrial adhesin (K88 ETEC) induce colibacillosis by binding to enterocytes in the small intestine of newborn and weaned piglets and causing secretory diarrhea. Current vaccination protocols are ineffective at preventing K88 ETEC infections during the postweaning period. Consequently, new procedures for preventing and treating postweaning colibacillosis caused by K88 ETEC are needed. Previous studies have shown that extracts of fermented soy products have the potential to prevent ETEC disease by blocking the binding of ETEC to enterocytes. In this study, we evaluated extracts of the fermented soy product, Dochi, for its ability to block K88 ETEC binding to enterocytes. We found that Dochi extracts were able significantly inhibit binding of K88 ETEC to brush borders isolated from porcine enterocytes (50% inhibition, P < 0.05) and cultured porcine intestinal epithelial (IPEC‐J2) cells (60% inhibition, P < 0.05). The inhibitory compound in the Dochi extract was found to elute during fractionation on a Superose 12 FPLC size exclusion column at an approximate molecular weight of 1300 Da. Future studies will be focused on determining the chemical nature of the inhibitory compound and on determining if the inhibitory compound can be used to prevent or treat K88 ETEC‐induced colibacillosis in weaned piglets.

Supported by South Dakota State University Research/Scholarship Support Fund, the South Dakota Agricultural Experiment Station, and the South Dakota Center for Infectious Disease Research and Vaccinology.
PepT1 associated with lipid rafts modulates bacterial‐epithelial interactions
Authors: Nguyen Hang Thi Thu , Dalmasso Guillaume , Sitaraman Shanthi V , Merlin Didier
Published: June 2021

PepT1 is a di/tripeptide transporter normally expressed in the small intestine and induced in colon under inflammatory conditions such as inflammatory bowel disease (IBD). Our aim was to investigate the role of PepT1 in inflamed human colonic epithelial HT29‐Cl.19A cells. We found that infection of HT29‐Cl.19A cells with enteropathogenic E. coli (EPEC) induced PepT1 promoter activity. PepT1 expression in infected cells was confirmed by detected levels of mRNA and protein. Importantly, PepT1 mediates transport activity in infected cells as shown by induced lysine‐proline‐valine specific uptake. Furthermore, we showed the presence of PepT1 in lipid rafts (LR) in EPEC‐infected cells and its colocalization with lipid raft marker GM1. To assess the role of PepT1 associated with LR in host cells during EPEC infection, we transfected HT29‐Cl.19A and CHO cells with wild type PepT1 and its mutated forms that were not found in LR. Our results show that in both the cell lines, only PepT1 associated with LR reduces the attachment and internalization of EPEC. In conclusion, we demonstrated that LR‐associated PepT1 plays a role in modulating bacterial‐epithelial interactions in the colon, suggesting a novel function of this transporter in epithelial cells.
Can diet influence the expression of genes associated with control of appetite?
Authors: Paulino Gabriel , Oort Pieter J , Knotts Trina A. , Adams Sean H. , Raybould Helen E
Published: June 2021

Food intake is modulated by both humoral and neuronal pathways; there is evidence that maintenance on a high fat diet leads to hyperphagia, in part via a peripheral, Cholecystokinin (CCK)‐dependent mechanism, involving the gut‐brain axis. We tested the hypothesis that a high‐fat diet inducing hyperphagia is associated with altered expression of genes involved in the short‐term regulation of food intake in the nodose ganglion and hypothalamus. The first aim of the study was to determine metabolic changes occurring in rats fed either a low‐fat (LF) or a high‐fat diet (HF). The second aim was to determine altered expression of genes for receptors involved in the control of food intake by chronic ingestion of a HF diet. Rats fed a HF diet and prone to obesity had hyperleptinemia and hyperinsulinemia after 8 weeks. The expression profile of receptors involved in appetite was modified in the hypothalamus (cannabinoid receptor CB1 dysregulated) and in the nodose ganglia (increase in CB1, Fatty Acid Amide Hydrolase, Growth Hormone Secretagogue Receptor and Cholecystokinin 1 Receptor), leading to an increase in orexigenic signaling after chronic ingestion of a HF diet. A more systematic and integrative investigation of the hormones and receptors expression involved in humoral and neuronal pathways of appetite is needed to understand how can a dysregulation lead to obesity.
Dihydrotestosterone and progesterone relax female guinea pig gallbladder strips
Authors: Kline Loren , Karpinski Edward
Published: June 2021

We demonstrated that dihydrotestosterone (DHT) and progesterone (P) relaxed cholecystokinin octapeptide (CCK) induced tension in male guinea pig strips; however, the effects of DHT and P on female guinea pig strips has not been fully studied. An in vitro technique was used. Statistical analyses used either paired t‐test or analysis of variance. DHT, P, 17‐hydroxyprogesterone (17‐P) or 20α–hydroxyprogesterone (20‐P; 10 μM 50 μM, and 100 μM) were used to generate concentration response curves. All 4 agents induced a concentration dependent relaxation of CCK‐induced tension. There was no significant difference between the P‐ and 17‐P‐induced relaxations. 20‐P induced significantly (P<0.05) less relaxation than P. DHT produced a relaxation similar to that induced by P and 17‐P. The relaxation caused by 50 μM and 100 μM DHT were not significantly different (63.7±5.1 vs. 63.5±6.5%). The response to 100 μM DHT was significantly less (p<0.01) than 100 μM P (91.0±6.2%). To determine if the PKA/cAMP second messenger system mediated the P‐ or DHT‐induced relaxation, PKA inhibitor 14‐22 amide myristolated (PKA‐IM; 180 nM) was used. PKA‐IM caused a significant (p<0.05) decrease in P‐induced relaxation (73.5±7.9 vs. 64.2±7.0%), but had no significant effect on DHT‐induced relaxation. When the PKC inhibitors bisindolymaleimide IV (0.5 μM) and chelerythrine Cl (5 μM) were used together, a significant (P<0.05) reduction in both P‐ and DHT‐induced relaxation (P, 56.8±4.0 vs. 36.8±6.2%; DHT, 53.0±6.3 vs. 42.5±5.1%) was observed. The results suggest that both the PKA/cAMP and PKC second messenger systems mediated the P‐induced relaxation of CCK‐induced tension in female guinea pig gallbladder strips. Only the PKC system mediates the DHT‐induced relaxation.