bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization Of β2‐containing Receptors in Rat Brainstem Using [125I]A‐85380
Authors: Davila‐Garcia Martha Isabel , Kamendi Harriet , Ogunlade Blessing Ogunlade , Robinson Anastasia 
Published: November 2023

Nicotinic receptor diversity in brainstem has prompted us to determine the distribution and pharmacological characteristics of the β2‐containing. We used [125I]A‐85380 radioligand which is selective for receptors containing the β2 subunit. Our hypothesis was that β2‐containing nicotinic receptors would be present particularly in cardiorespiratory centers in the brainstem. First, we characterized the binding of [125I]A‐85380 in rat cerebral cortex (CTX). [125I]A‐85380 reached equilibrium in two hours and was found to have a Kd value of ~180pM and a Bmax of ~ 110 fmol/mg protein. In competition assays the rank order of potency for nicotinic receptor agonists was epibatidine > unlabeled A‐85380 > cytisine > nicotine > DMPP and for the antagonists the rank was DHbE > mecamylamine while upto 1mM k‐bungarotoxin did not displace any [125I]A‐85380 binding. In receptor binding and autoradiography studies where cytisine, DMPP, DHβE, epibatidine and nicotine were used to displace [125I]A‐85380, no additional binding sites were found, in all cases, binding was completely blocked by these ligands. Binding of [125I]A‐85380 was found at moderate to high levels in medial raphe areas some of which were insensitive to DHβE, and in most lateral regions of both caudal and rostral medulla.Support or Funding InformationAmerican Heart Association (MDG) 9930049N
Unexpected Negative Role of the AT1R Adaptor Protein βarrestin1 in the ERK1/2 Activation of SHR‐Derived Primary Astrocytes
Authors: Negussie Shmuel , Lymperopoulos Anastasios , Clark Michelle Alexa 
Published: November 2023

ObjectiveTo investigate the role of βarrestin1 in angiotensin II (AngII) type 1 receptor (AT1R)‐mediated extracellular signal‐regulated kinase (ERK1/2) activation in spontaneously hypertensive rat (SHR)‐derived primary astrocytes.BackgroundThe βarrestins, consisting of two isoforms, βarrestin1 and ‐2, originally discovered as terminators of G protein signaling by G protein‐coupled receptors (GPCRs), are now known to also mediate their own signaling to ERK1/2 activation independently of G proteins. The AT1R is a GPCR that can also signal to ERK1/2 via βarrestins in various cell types. However, whether it can do so in the brain has not been investigated yet. Thus herein, we sought to examine the involvement of βarrestins in the AT1R‐mediated ERK1/2 activation in SHR‐derived primary astrocytes.MethodsPrimary cultures of rat astrocytes from brainstem were isolated from the brains of 2–3 days old pups obtained from pregnant SHR. 100 nM AngII and 10 μM SII (an AngII peptide analog which is βarrestin‐biased agonist) were used to study the AT1R mediated ERK1/2 activation. The results were compared to normotensive Wistar rats. Protein analysis was done using Western blotting and mRNA measurement was done using real time PCR.ResultsOur results indicate that βarrestin1 is the major arrestin protein in astrocytes from both SHR and normotensive rats. At the mRNA level however, both βarrestin1 and βarrestin2 display comparable expression. AngII activates ERK1/2 in SHR astrocytes much more robustly than in normotensive rat ones. On the other hand, SII strongly activates ERK1/2 in normotensive rat astrocytes and only minimally in SHR ones. Importantly, AngII also upregulates βarrestin1 protein in normotensive rat astrocytes, whereas in SHR astrocytes it has no significant effect. Finally, AngII downregulates both βarrestins at the mRNA level in both SHR and normotensive rat astrocytes.ConclusionThe AngII‐AT1R‐induced ERK1/2 activation of SHR astrocytes, known to induce hypertension, appears to be almost exclusively G protein‐dependent, contrary to normotensive rat astrocytes. Since βarrestin1 protein is also upregulated by AngII in normotensive rat astrocytes and appears downregulated in SHR astrocytes, it is quite plausible that βarrestin1 may actually inhibit ERK1/2 activation in astrocytes. This might be a beneficial effect of βarrestin1 in preventing development of hypertension in the SHR condition.Support or Funding InformationFunding for this research was provided through a Nova Southeastern University Health Professions Division Grant
Calpain‐2 Activates Akt via TGFβ1‐mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells
Authors: Abeyrathna Prasanna Kumara , Kovacs Laszlo , Su Yunchao 
Published: November 2023

Calpain, a calcium‐dependent nonlysosomal neutral cysteine endopeptidase, activates an intracrine TGFβ pathway in pulmonary artery smooth muscle cells (PASMCs) and plays an important role in smooth muscle cell hyperproliferation and collagen synthesis in pulmonary vascular remodeling of pulmonary arterial hypertension (PAH). Akt is a serine‐threonine kinase involved in many cellular processes including cell proliferation and collagen synthesis. In this study, we investigated the connection between calpain activation and Akt in the collagen synthesis and cell proliferation of PASMCs caused by PDGF. We found that inhibition of calpain‐2 by calpain inhibitor, MDL28170 and calpain‐2 siRNA attenuated Akt phosphorylation at serine‐473 (S473) and threonine‐308 (T308) as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain‐2 in PASMCs induced dramatic increases in Akt phosphorylation at S473 and T308. The cell‐permeable specific TGFβ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and by overexpressed calpain‐2 in PASMCs. Moreover, SB‐431452 and knocking down ALK5 significantly reduced PDGF‐induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGFβ1 using a cell‐impermeable TGFβ1 neutralizing antibody did not affect PDGF‐induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented PDGF‐induced Akt phosphorylation at S473 and T308. Additionally, calpain inhibitor MDL28170 inhibited PDGF‐induced increase in mTORC2 activity. These data provide first evidence supporting that calpain‐2 up‐regulates PDGF‐induced Akt phosphorylation via an intracrine TGFβ1/mTORC2 mechanism.Support or Funding InformationSupported by NIH, FAMRI and Department of Veterans Affairs
Lubiprostone Stimulates HCO3− Secretion in Rat Stomach and Duodenum mediated by Different EP receptor Subtypes
Authors: Takeuchi Koji 
Published: November 2023

A recent study showed that lubiproston, a bicyclic fatty acid derived from prostaglandin (PG) E1, stimulated CFTR‐dependent duodenal HCO3− secretion in rat, and this action was mediated by the activation of EP4 receptors. In the present study, we examined the stimulatory effect of lubiprostone on HCO3− secretion in the rat stomach and duodenum, focusing on the EP receptor subtypes involved in this action. Male SD rats were used after 18 h fasting. Under urethane anesthesia, an ex‐vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3− secretion was measured using a pH stat‐method. lubiprostone significantly increased HCO3− secretion in both the stomach (>30 μM) and duodenum (>1 μM). The stimulatory action in the stomach was significantly abrogated by prior administration of the EP1 antagonist but not by the EP3/EP4 antagonists or the CFTR inhibitor, while that in the duodenum was significantly attenuated by the EP3 antagonist and the EP4 antagonist as well as the CFTR inhibitor. These results suggest that lubiprostone stimulates HCO3− secretion in the stomach and duodenum mediated by different EP receptor subtypes; the former mediated by EP1 receptors and the latter mediated by both EP3 and EP4 receptors. It is also assumed that CFTR is involved in modulating HCO3− secretion in the duodenum but not the stomachSupport or Funding InformationNone
Use of a Non‐Native Chloride Channel as a Treatment for Interstitial Cystitis
Authors: Beckel Jonathan M , Tillman Tommy S , Tang Pei , de Groat William C , Xu Yan 
Published: November 2023

Interstitial cystitis (IC) is a disease of the urinary bladder that causes increased urgency to void and pelvic pain. It is commonly thought that increased bladder sensations in IC are due to sensitization of bladder afferent nerves by inflammatory mediators. Current therapies aimed at known targets in the inflammatory or nociceptive pathways have, to date, been ineffective at reducing the symptoms in IC patients. To find potential new treatments for IC, we expressed a non‐native chloride channel (EG3RF) in bladder afferent neurons. This family of channels can be activated by either inflammatory conditions or by an exogenous pharmaceutical compound, but has no activity under normal physiological conditions. We hypothesize that successful transfection of the EG3RF chloride channel into rat bladder afferent nerves will diminish bladder hyperexcitability in the cyclophosphamide model of bladder inflammation. To transfect bladder afferent nerves, an expression plasmid containing the sequence for EG3RF was encapsulated in liposomes and instilled into the urinary bladder of Sprague Dawley rats using a transurethral catheter. Bladder activity was measured 1–2 weeks later using metabolic cages or bladder cystometry. Bladder inflammation was induced by intraperitoneal injection of cyclophosphamide (CYP, 150 mg/kg) 1 hour prior to measurement of bladder activity. Successful expression of the EG3RF channel up to 2 weeks following transfections was confirmed in the bladder smooth muscle, urothelium and dorsal root ganglia (T11–12, L1–L2 and L6‐S1 levels) using RT‐PCR, Western blot, and immunofluorescence analyses. Metabolic cage and cystometry experiments indicated no differences in voiding function between the control and non‐inflamed EG3RF‐transfected animals. However, EG3RF‐transfected rats were resistant to the excitatory effects of cyclophosphamide treatment. Treatment with cysteamine, which has been shown to activate EG3RF, decreased CYP‐induced bladder activity in both metabolic cage and cystometry experiments (31 mg/kg ip or iv, respectively) in EG3RF‐transfected rats but was ineffective in control rats. Intravesical instillation of cysteamine (5 mM) during cystometry in EG3RF‐transfected rats excited bladder reflexes while iv administration (31 mg/kg) decreased peak micturition pressure. These data suggest that expression of a non‐native chloride channel in bladder afferent nerves may be an effective treatment for painful bladder disorders such as interstitial cystitis. Further development of the technique may lead to significant advances in efficacy over existing treatments.Support or Funding InformationT32 GM075770 (Xu); K01 DK106115 (Beckel); R01 DK091253 (de Groat)
VEGF knockdown in muscle improves recovery of blood flow and arteriolargenesis after ischaemia
Authors: Machado Maria J C , Riu Federica , Benest Andrew V , Bates David O 
Published: November 2023

Vascular Endothelial Growth Factor (VEGF) signalling is essential for endothelial cell homeostasis and growth. It has thus been assumed that VEGF would be important during the revascularization process that occurs in ischaemic limbs after arterial occlusion. We used a transgenic mouse line in which VEGF is inducibly knocked out in skeletal muscle cells (ACTA1‐rtTA, tetO‐Cre, VEGF‐LoxP mice), to test the hypothesis that lack of VEGF‐A hinders the recovery of blood flow after ischaemia. In adult triple transgenic mice, homozygous for VEGF‐LoxP (ACLL mice), skeletal muscle specific VEGF‐A knockout was achieved by dosing with doxycycline (+Dox) for 10 weeks. They then underwent unilateral hindlimb ischaemia surgery under isoflurane anesthesia. Blood flow to the hindpaws was monitored by laser speckle imaging. The ratio of blood flow in the ischaemic to contralateral limb was significantly improved when compared to ACLL mice dosed with vehicle alone (0.81±0.09 +Dox vs 0.47±0.05 ‐Dox at day 21; p<0.001). Although there was no difference in capillary density (mean±SEM, mm−2) between these groups of mice (334±24 +Dox vs 261±21 ‐Dox, p>0.05), the arteriole density was significantly higher in ACLL +Dox mice (41±6 +Dox vs 20±2 ‐Dox, p<0.01). Exogenous administration of VEGF by local injection of adenovirus over‐expressing VEGF‐A165a to ACLL +Dox mice, inhibited the increase in blood flow recovery (p<0.01). Injection of a control virus (Ad.eGFP) did not affect the rate of recovery after ischaemia in ACLL +Dox mice. These results suggest that VEGF of skeletal muscle origin is detrimental, not beneficial, to arteriolargenesis (the growth of new arterioles).Support or Funding InformationMedical Research Council and British Heart Foundation
Serine 275 and serine 328 in the carboxyl‐terminus regulates phosphorylation‐dependent anti‐proliferative properties of connexin 37
Authors: Jacobsen Nicole Lynne , Taylor Samantha , Pontifex Tasha , Burt Janis 
Published: November 2023

Connexin 37 (Cx37), but not Cx40 or Cx43, suppresses the proliferation of rat insulinoma (Rin) cells, an effect requiring both its channel function and regulation mediated by the carboxyl‐terminus (CT). Additionally, data from cells expressing alanine or aspartate substituted forms of Cx37 suggest that phosphorylation at specific serine residues (275, 285, 302, 319, 321, 325, 328) within the CT directs cell survival and proliferation; “dephosphorylated” Cx37‐CT (use of alanine substitution) results in cell cycle arrest while “phosphorylation” of the Cx37‐CT (aspartate substitution) induces cell death. Interestingly, aspartate substitution at only serines 275, 319, 328 (S3D3) neither limits Rin cell growth nor induces cell death. Rather, there is no effect on the rapid Rin cell proliferation despite its expression. As the first identified full‐length form of Cx37 that retains channel functionality while supporting cell proliferation, we investigated whether phosphorylation at a single site would be sufficient to control cell cycle progression. Single point mutations with alanine and aspartate were made at each of the three sites and subsequently expressed in Rin cells. Aspartate substitution at serine 275, a putative MAPK site, induces cell death while alanine substitution does not have a significant impact on the anti‐proliferative effects of Cx37‐WT. Mutation of serine 319 to either an alanine or aspartate no longer limits Rin cell proliferation, suggesting that this site may not be critical to phenotype determination. Finally, expression of Cx37‐S328A (alanine substitution at a putative PKC site) results in cell death but Cx37‐S328D supports proliferation. Together, these data may suggest that serine 275 and 328 cooperate in modulating cell proliferation in a phosphorylation‐dependent manner.Support or Funding InformationR01HL058732 and T32HL007249
Therapeutic Transplant Efficacy of Endothelial Progenitor Cells is Impaired by NK Cells
Authors: Exner Eric C , Abel Alex C , Malarkannan Subramaniam C , Greene Andrew C 
Published: November 2023

Adult stem cells, including endothelial progenitor cells (EPCs), have long been proposed as a source of cells for therapeutic transplantation in cardiovascular (CV) disease. Studies investigating the therapeutic potential of EPCs have shown mixed results, likely due to the negative impact of chronic disease and aging on EPC function in the high CV risk patients enrolled in such studies. Using genetically controlled rats, we have established a model of EPC dysfunction in the setting of hypertension. In the SS/MCWi rat, a genetic model of salt‐sensitive hypertension, EPC function is compromised. Introgression of the 13th chromosome of the Brown Norway rat onto the SS background results in the SS‐13BN/MCWi (BN13) rat, which is protected from both hypertension and EPC dysfunction. Previously, we found increased expression of the natural killer cell receptor/ligand 2B4 (CD244) on the cell membrane of EPCs isolated from SS rats. This increase in CD244 expression was correlated with increased destruction by NK cells from both SS and BN13 rats. Here, we hypothesize that increased membrane expression of CD244 by SS EPCs leads to destruction by NK cells in vivo, which compromises their therapeutic efficacy. We first found that siRNA mediated CD244 knockdown rescued EPCs from NK cell mediated killing in vitro using an LDH release assay. We next used an NK cell depleting antibody to assess whether NK cells were responsible for EPC dysfunction in animals receiving EPCs as therapy. Finally, we transplanted EPCs treated with CD244 siRNA into naïve recipient animals to confirm that CD244 impacts EPC function in vivo. Our results suggest that increased CD244 on the SS EPC membrane leads to increased NK cell mediated killing of SS EPCs in vivo, contributing to the impaired therapeutic efficacy in our model.Support or Funding InformationNational Institutes of Health grant HL082798 awarded to A.S. Greene, PhD; T32 HL007852 PI: H.V. Forster
Differential Expansion of the Capillary Bed through Physiological Angiogenesis in Skeletal Muscle
Authors: Kissane Roger , Egginton Stuart 
Published: November 2023

A range of well‐established in vivo models of angiogenesis currently exists, involving surgical and pharmacological intervention. These approaches generally improve global measures of microvascular supply, but are rarely interrogated for their capacity to improve tissue oxygen tension. The aim of this research was to characterise a variety of muscle recruitment patterns known to induce angiogenesis, determine their effect on capillary spatial heterogeneity, and subsequently local tissue oxygen delivery.Male Wistar rats underwent seven days of angiogenic remodelling through indirect electrical stimulation of skeletal muscle (at 4, 10 or 40Hz), muscular overload (through synergist ablation) or pharmacological supplementation (Prazosin, alpha‐adrenergic blocker). Muscle samples were snap frozen and capillary content identified through Griffonia simplicifolia lectin staining, with morphometric analysis of muscle composition used as the basis for oxygen consumption modelling.We demonstrated that seven days of supramaximal indirect electrical stimulation induced significant angiogenesis (capillary to fibre ratio (C:F) increased by 59, 36 and 45% for 4, 10 and 40Hz, respectively). A similar response for muscular overload and prazosin supplementation were seen with a 42 and 27% increase, respectively. This improvement in capillarity was accompanied by a reduction in mean capillary domain size (tissue supply area), but with varying degrees of spatial heterogeneity.Skeletal muscle overload and 40Hz electrical stimulation showed a substantially more homogeneous capillary distribution, and subsequently improved tissue oxygenation at high levels of simulated exercise. Conversely, the improved capillarity associated with prazosin and 4Hz electrical stimulation showed no extensive change in capillary distribution. We therefore conclude that mechanical stimulation of angiogenesis may be accentuated by a metabolic component.
Angiogenesis and Expression of IGF2R, IGF2, uPAR, and TGFB in a Rat Obesity Model
Authors: Bayless David S , Sheldon Ryan D , Linden Melissa A. , Rector R. Scott , Laughlin M. Harold 
Published: November 2023

IntroductionOur group has previously shown that obesity decreases capillary density in skeletal muscle. Here, we tested the hypothesis that decreased capillary density in obesity is the result of increased activity of the insulin growth factor II receptor (IGF2R) in degrading urokinase‐plasminogen receptor (uPAR), activating transforming growth factor beta (TGFB), and degrading insulin growth factor II (IGF2) using an obese, diabetic rat model.MethodsAt four weeks of age, normophagic Long‐Evans Tokyushima Otsuka (LETO; n=20) and hyperphagic Otsuka Long‐Evans Tokushima Fatty (OLETF; n=20) rats were provided with either a normal, low‐fat diet (LFD, 10% kcal fat) or a high‐fat, high‐cholesterol diet (HFD, 45%kcal fat with 1% cholesterol). At 32 weeks of age, samples from rats were taken, including biceps brachii and vastus lateralis muscle, and arterial vessels. Whole tissue muscle samples were snapfrozen and cold sectioned. Slides were stained for IHC highlighting of capillaries, with manual counting of capillaries and muscle fibers in cross‐section. Vessels were homogenized in buffer and processed by way of Western blot procedure, with membranes probed for IGF2R, IGF2, TGFB, and uPAR.ResultsCapillary density in biceps brachii was reduced in OLETF LFD rats (p<0.07) compared to LETO LFD rats, but HFD increased capillary density significantly relative to LFD counterpart, especially in OLETF rats (19%, p<0.01). In addition, vastus lateralis IHC indicated a significant decrease in capillary density in OLETF LFD/HFD and LETO HFD relative to LETO LFD (p<0.05). Western blot analysis of arterial vessel samples did not indicate any significant difference in IGF2R, IGF2, TGFB, or uPAR between LETO LFD and other groups.DiscussionThese data demonstrate that capillary density is significantly downregulated in obese, type 2 diabetic OLETF rats. However, examination of arterial samples suggest that changes may not be related to changes in expression of IGF2R and associated proposed down‐regulators of vascular growth in the arterial vasculature. To complete the test of our hypothesis we will measure IGF2R, uPAR, IGF2, and TGFB expression in muscle tissue to determine whether changes in expression occur in the muscle that may mediate changes in capillarity in obesity/diabetes.Support or Funding InformationWe Acknowledge Support of this research: NIH HL36088(MHL), VHA CDA2 1BX001299 (RSR), Society of Phi Zeta, and the University of Missouri Department of Medical Pharmacology and Physiology.
Increased Cerebral Capillary Density in Aquaporin‐4 Knockout Mouse
Authors: Xu Kui , Almotah Khalil , Zhang Yifan , Radford Thomas S , Yu Xin , Boron Walter F , LaManna Joseph C 
Published: November 2023

In the brain, Aquaporin 4 (AQP4) is highly expressed in the blood–brain barrier and brain–CSF interfaces. AQP4 functions as an efficient water‐selective transporting protein and plays an important role in cerebral water balance. It has been shown that knocking out of AQP4 in mice reduces brain edema after acute water intoxication and ischemic stroke. The specific localization of AQP4 to these anatomical and cellular regions of the central nervous system may also suggest a role for AQP4 as a gas channel to facilitate oxygen transport. In this study we investigated the role of AQP4 on cerebral capillary density and cerebral blood flow using AQP4 knockout (AQP4‐KO) mice in comparison with the wild‐type (WT) mice. Cerebral blood flow was measured using arterial spin labeling (ASL) MRI in WT mice (n = 7) and AQP4‐KO mice (n = 4). Mice were then perfusion fixed and cerebral capillary density was identified by GLUT‐1 immunohistochemistry staining and was quantified from the number of the positive counts per unit area (number/mm2). Results showed that the cerebral blood flows were similar in the WT and the AQP4‐KO groups (Mean ± SD, 182 ± 30 vs. 178 ± 52, ml/min/100g tissue). The AQP4‐KO mice exhibited a 24% increase in capillary density as compared to the WT mice (Mean ± SD, 504 ± 49 vs. 406 ± 32, N/mm2). Our results showed that the deletion of AQP4 increased the capillary density without altered blood flow in mouse brain, suggesting a compensation for decreased gas transport in the KO mice.Support or Funding InformationNIH R01 NS 38632