bluuebunny/crossref_metadata_2025 huggingface dataset:
Aldosterone enhances NCC protein expression via stimulating ERK1/2 phosphatase (DUSP6)‐mediated reduction of NCC ubiquitination
Authors: Feng Xiuyan , Lee Matthew , Delpire Eric , Gu Dingying , Cai Hui
Published: June 2021

The thiazide‐sensitive Na‐Cl cotransporter (NCC) localizes to the distal convoluted tubule (DCT) and is one of the key regulators of sodium balance. MAPK‐ERK 1/2 signaling pathway was reported to be mediated in NCC ubiquitination. We previously showed that ERK 1/2 knock down induced the NCC surface protein expression. We also found that aldosterone decreased the phosphorylation of ERK 1/2. We, thus, hypothesized that aldosterone upregulates the NCC surface expression via blocking ERK 1/2 induced NCC ubiquitination. To further test this hypothesis, we treated the mDCT cells with aldosterone 1 μM for 6 hours and found an increase in both NCC surface expression and DUSP6 protein expression, an ERK 1/2 specific phosphatase. Total NCC protein expression was also increased. ERK 1/2 phosphorylation and NCC ubiquitination were both decreased. When the mDCT cells were pretreated with BCI, a specific inhibitor of DUSP6, the NCC ubiquitination increased and NCC surface expression decreased. These results suggest that aldosterone induces both total and surface protein expression of NCC by inhibiting ERK 1/2 induced NCC ubiquitination via increasing ERK 1/2 phosphatase DUSP6.
Inactivation of the epithelial sodium channel (ENaC) in the aldosterone‐sensitive connecting tubule
Authors: Poulsen Søren Brandt , Praetorius Jeppe , Miller Lance , Nelson Raoul , Rossier Bernard , Hummler Edith , Christensen Birgitte Mønster
Published: June 2021

Renal ENaC‐mediated Na+ reabsorption is under control of aldosterone in the late distal convoluted tubule, connecting tubule (CNT), and collecting duct (CD). Inactivation of αENaC in CD does not disturb Na+/K+ balance in mice (Rubera et al, JCI 2003). However, αENaC deletion in both CNT and CD causes symptoms of pseudohypoaldosteronism type 1 as evidenced by disturbance in Na+/K+ balance on standard and challenging diets (Christensen et al, JASN, 2010). To further investigate the significance of αENaC in CNT, mice with Cre‐recombinase expressed under the control of the V‐ATPase B1‐subunit promoter (mice express Cre in part of the CNT cells, Miller et al, KI, 2009) were crossed with floxed Scnn1a mice (Hummler et al, Genesis, 2002). Thus, KO mice with a partial deletion of αENaC in CNT were generated. On standard diet, KO mice showed no difference in urinary Na+ and K+ excretion, urine output, or water intake compared to controls. On low Na+/high K+ diet, however, KO mice showed lower food and water intake (P<0.05), and a tendency to a lower urine output after 24h, but not after 48h. Furthermore, the relative weight loss and plasma K+ were higher in KO mice after 48h (P<0.05). In conclusion, no change in Na+/K+ homeostasis was found in KO mice partly deficient for αENaC in the CNT when fed a standard diet. On low Na+/high K+ diet, however, KO mice appeared with a phenotype. Support: the Lundbeck Foundation, and Health, Aarhus Uni.
STE20‐like kinase SPAK differentially regulates Na‐(K)‐Cl cotransporters along the distal nephron under the endocrine control of AVP
Authors: Bachmann Sebastian , Saritas Turgay , McCormick James , Ellison David , Delpire Eric , Mutig Kerim
Published: June 2021

The Na‐K‐2Cl cotransporter (NKCC2) of the thick ascending limb (TAL) and the Na‐Cl cotransporter (NCC) of the distal convoluted tubule (DCT) are critical for renal salt handling. Activation of these transporters by vasopressin (AVP) includes their N‐terminal phosphorylation. Little is currently known about the kinases that mediate this action of AVP. Two homologous Ste20‐like kinases, SPAK and OSR1, can phosphorylate the cotransporters directly. In this process, full‐length SPAK variant (FL‐SPAK) and OSR1 interact with a truncated isoform, KS‐SPAK, which has inhibitory effects. Our study tested the hypothesis that SPAK is an essential component of the AVP stimulatory pathway.

Short‐ and long‐term effects of desmopressin (dDAVP), a V2 receptor‐specific agonist, on the kinases and transporters were evaluated in wild type and SPAK‐deficient mice and in AVP‐deficient rats.

SPAK variants displayed prominent regulatory changes along TAL and DCT along with activation of the cotransporters, whereas OSR1 was less involved. The KS‐ and FL‐SPAK variants were modulated by AVP for their selective interaction with NKCC2 in control of its activation, whereas the phosphorylation of NCC was essentially governed by FL‐SPAK alone.

In sum, our data specify how SPAK may serve as a hallmark kinase in modulating Na+ reabsorption along the distal nephron under the endocrine control of AVP.
Characterization of CFTR High Expresser (CHE) cells of the small intestine
Authors: Jakab Robert L , Ameen Nadia A
Published: June 2021

The CHE cells express 8‐fold higher levels of the CFTR Clchannel compared to neighboring enterocytes, and were first identified by our laboratory (Ameen et al 1995, Gastroenterology 108:1016). We used double label immunofluorescence microscopy to further study these enigmatic epithelial cells in rat. CHE cells are found in duodenum, most frequent in proximal jejunum, and absent in ileum and colon. CFTR abundance increases in CHE cells along the crypt‐villus axis. The basolateral Cl− transporter NKCC1 was detected at similar levels in CHE cells and neighboring enterocytes at steady state. Microvilli were shorter in CHE cells, with low levels of Myosin1A ‐ a villus enterocyte specific motor that retains sucrase/isomaltase (SI) in the brush border membrane (BBM). CHE cells lacked absorptive villus enterocyte BBM proteins including: NHE3, SLC26A6 (PAT1) and SI. High levels of the vATPase proton pump were observed in the apical domain of CHE cells. Levels of Syntaxin 3, NHERF1, and Na‐K‐ATPase were similar to that of neighboring enterocytes. cAMP or carbachol stimulation robustly increased apical CFTR and basolateral NKCC1 disproportionately in CHE cells relative to neighboring enterocytes. These data strongly argue for a specialized role of CHE cells in Cl−‐mediated “high‐volume” fluid secretion in the proximal small intestine.

NIDDK Grants R01‐DK‐077065 (N.A.) and P30 DK‐34989 (Yale Liver Center).
TGF‐β1 attenuates CFTR‐mediated anion secretion across porcine vas deferens epithelia via the p38 MAPK pathway
Authors: Yi Sheng , Pierucci‐Alves Fernando , Schultz Bruce D.
Published: June 2021

This study is designed to identify effects of transforming growth factor β1 (TGF‐β1) on ion transport across vas deferens epithelia. In modified Ussing chambers, PVD9902 cells (derived from porcine vas deferens) that were exposed to TGF‐β1 exhibited attenuated forskolin‐stimulated anion secretion and reduced effects of DASU‐02, a CFTR channel blocker. RT‐PCR and western blots revealed that TGF‐β1 down‐regulates mRNA expression and protein expression of CFTR. Biotinylation assays suggested that TGF‐β1 also reduced the abundance of CFTR in the apical membrane. The effect of TGF‐β1 on anion secretion was abrogated by a TGF‐β1 receptor I inhibitor and was reduced by an inhibitor of p38 MAPK, suggesting that TGF‐β1 interacts with its cognate receptor on vas deferens epithelial cells to activate p38 MAPK signaling. This conclusion was further supported by the observation that TGF‐β1 enhances phospho‐p38 MAPK abundance. Additionally, anisomycin, a p38 MAPK activator, mimicked the effect of TGF‐β1 to reduce forskolin‐stimulated anion secretion. Men harboring profound CFTR mutations exhibit infertility and reproductive duct anomalies, whereas mild mutations have variable presentations. Our observations suggest that TGF‐β1 reduces CFTR‐mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and would compromise male fertility. [NIH HD058398 and RR017686]
Chloride channels: Role in apoptosis, cell motility and chemotherapy resistance
Authors: Hoffmann Else Kay , Jacobsen Kari S , Schwab Albrecht , Sauter Daniel , Lambert Ian H , Poulsen Kristian A
Published: June 2021

Among the pathophysiological phenotypes defining malignant growth are cell migration and evasion of programmed cell death. The talk will discuss how Cl−channels affect these processes and how changes in the expression of Cl−channels are involved in chemotherapy resistance. We investigate the role of the TMEM16 Cl− channels in cell migration in Ehrlich Lettré Ascites (ELA) cells and find that TMEM16A and TMEM16F knock‐down affects the directional migration and the rate of cell migration, respectively. Moreover we have explored the apoptotic volume decrease (AVD) in wild type (wt) and Multidrug Resistant (MDR) Ehrlich Ascites Tumor Cells (EATC). Cisplatin (5 μM) leads to AVD and apoptosis in wt EATC but essentially not in MDR cells, and the maximal capacity of the volume activated Cl− current is concomitantly strongly repressed in MDR EATC. Application of the VRAC inhibitor NS3728 prevents cisplatin‐induced AVD in wt cells and abolishes the differences in AVD and caspase‐3 activation between wt and MDR EATC suggesting that impairment of AVD via down regulation of Cl− channels protects against apoptosis in MDR EATC1. Currently, using QPCR and western blot we compare the expression of Cl− channels in wt and MDR EATC as well as in four pancreatic cancer cell lines and a normal pancreatic cell line (HPDE).

Supported by DFF, Denmark.
Role of VDAC in coronary endothelial cell dysfunction in type 1 diabetic mice
Authors: Sepassi Liliyas , Sasaki Koh , Heldak Michael , Makino Ayako
Published: June 2021

Endothelial dysfunction is a common feature of diabetic vascular complications. Rarefaction of capillaries in the heart, as a result of coronary endothelial dysfunction, represents one of the most critical mechanisms involved in cardiac ischemia. The voltage‐dependent anion channel (VDAC) plays an important role in the regulation of mitochondrial metabolic function and mitochondria‐mediated apoptosis. The objective of this study is to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in the left ventricle of diabetic mice and hyperglycemia induced significant increase in mitochondrial Ca2+ concentration and VDAC protein levels in coronary ECs. The expression of VDAC‐shRNA in diabetic MCECs not only decreased the mitochondrial Ca2+ concentration, but also attenuated mitochondrial superoxide anion (O2−) production and the activity of the mitochondrial permeability transition pore opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC protein expression in diabetic MCECs leads to the activation of pro‐apoptotic cascade and normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.

This research was supported by grant HL115578.
BKCa channel beta‐1 subunit deficiency exaggerates microcirculatory dysfunction and mortality in CLP‐induced septic mice.
Authors: Xu Hui , Garver Hannah , Fink Gregory D , Galligan James J
Published: June 2021

BKCa channels in smooth muscle cells are composed of pore forming α‐subunits and accessory beta1‐subunits that modulate Ca2+ sensitivity of α‐subunits. BKCa channels regulate arterial tone, and earlier studies in human and animals suggested that activation of BKCa channels may contribute to reduced arterial reactivity to norepinephrine in septic shock. Our recent studies suggested that activation of BKCa channels may protect against hypotension and mortality, since inhibition of BKCa channels exaggerates hypotension and mortality in endotoxemic mice. In current studies, we measured mean arterial pressure (MAP) and heart rate (HR) in cecal ligation/puncture (CLP)‐induced septic shock BKCa channel β1‐subunit knockout (KO) mice using radiotelemetry. After CLP, KO mice exhibited a more rapid fall in MAP, when compared to wild‐type (WT) mice. There was a HR‐independent shorter latency to mortality and higher mortality rate in KO compared to WT mice. 24 h after CLP, KO mice had more severe hypothermia and lower peripheral perfusion, measured in paw flow by scanning laser Doppler. Polymorphonuclear leukocyte infiltration was higher in liver tissues, but not in kidneys and intestine tissues from KO mice. KO mice demonstrated more severe small intestinal mucosal necrosis. Serum TNF‐α and IL‐6 levels in CLP‐KO mice were slightly higher than CLP‐WT mice 24 h post CLP. Our results confirmed that inhibition of BKCa channels does not protect against hypotension in septic shock. BKCa channel deficiency exaggerates mortality via microcirculatory dysfunction.
On the Potential Role of Potassium Channel KCNK5 in Human T Cell Activation
Authors: Kirkegaard Signe Skyum , Hansen Anker Jon , Gammeltoft Steen , Hoffmann Else Kay
Published: June 2021

The two‐pore domain potassium channel KCNK5 has been shown to play a role in activation of T cells from multiple sclerosis patients1. KCNK5 is also known to be important in the regulatory volume decrease (RVD) following cell swelling2. Our aim with the study is to elucidate a potential role for KCNK5 in T cell activation.

We use T cells purified from human peripheral blood mononuclear cells. Real‐time PCR and Western blotting was used to investigate mRNA and protein levels, respectively of the three potassium channels Kv1.3, KCa3.1 and KCNK5 in non‐activated and CD3/CD28 activated T cells. Furthermore volume measurements using a coulter counter were used to study RVD. We find a strong up‐regulation of KCNK5 on protein and mRNA levels in activated T cells compared to the non‐activated cells. Surprisingly, despite this up‐regulation of the potent volume regulator KCNK5 activated T cells were larger and volume regulated less efficiently than nonactivated cells. The exact role for KCNK5 in activated T cells will be examined in future studies of the increase in membrane potential expected from KCNK5 up‐regulation.
Atmospheric pressure plasma induced cell cycle arrest in human aortic endothelial cells
Authors: Shim Eunyoung , Gweon Bomi , Kim Hyeonyu , Park Sanghoo , Choe Wonho , Shin Jennifer Hyunjong
Published: June 2021

There has been a great deal of interests in application of atmospheric pressure plasma (APP) for biomedical and clinical uses such as cancer therapy and wound healing. However, most of these studies have been limited to adhesion and apoptosis of cells induced by APP in epithelial cells. In this study, we investigated the effects of a moderate APP (970 V, 50 kHz, 20 min) on the human aortic endothelial cells (HAECs). Some phenotypic changes were observed in the APP treated HAECs. Dramatic reduction of the cell population was observed which indicates the cellular cyclic arrest. To determine the involved mechanism of the changes in proliferation rate, immunofluorescence assay was used to analyze cyclin B1, p21, and p53 using the real‐time PCR. In addition to the decrease in the 2‐D migration speed, tube assay for the APP treated HAECs showed a decrease in the mean tube length when compared to that of the untreated cells. Our results support the potential use of APP to control the proliferation of the endothelial cells for anti‐angiogenic therapies for cancers.
Integrative Analysis of Transport Resistances and Free Hemoglobin on Nitric Oxide‐Red Blood Cell Interactions
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Lower consumption of nitric oxide (NO) by red blood cells (RBC) is attributed to transport resistances from membrane, intra‐ and extra‐ cellular diffusion for NO biotransport to RBC. To quantify the contribution of these transport resistances, we developed a mathematical model of NO transport to a single RBC. Free hemoglobin (Hb) is released due to intravascular hemolysis of RBC in sickle cell disease and can affect NO availability. Role of free Hb induced low NO availability in development of sickle cell disease related pulmonary hypertension has been questioned. To quantify the effect of free Hb on NO‐RBC interactions, 5% Hct was reacted with NO in presence of 10 μM Hb in a micron scale flow system. NO transport model was developed to estimate the NO concentrations in the flow system. Results from single cell transport model predict that extracellular diffusion contributed 70–90% of total transport resistance for membrane permeability (Pm) >;1 cm/s whereas membrane resistance accounts for 50–75% of total transport resistance for Pm < 0.1 cm/s. Nitrite, total NOx formations and predicted NO concentrations at the gas‐liquid interface in the flow system were lower in presence of free Hb compared to those in absence of free Hb, indicating low NO availability in presence of free Hb which may explain development of sickle cell complications of pulmonary hypertension. Grants: AHA 0530050N & R15 HL087287.
Protein Kinase G Inhibits T‐type Ca2+ Channels in Rat Cerebral Arteries
Authors: Harraz Osama F. , Welsh Donald G.
Published: June 2021

In rat cerebral arteries, T‐type Ca2+ channels are suppressed by protein kinase A (PKA) signaling. In this study, we examine whether protein kinase G (PKG), another key vasodilatory pathway, exerts a similar or distinctive influence on this conductance. Using patch clamp electrophysiology and cerebral arterial smooth muscle cells from rat, we monitored an inward Ba2+ current that was divisible into nifedipine‐sensitive and –insensitive components. The nifedipine‐insensitive conductance displayed properties reminiscent of T‐type Ca2+ channels; faster activation and steady state inactivation particularly at hyperpolarized potentials. Intriguingly, agents that target key PKG signaling proteins altered T‐type Ca2+ channel activity. In particular, activators of PKG signaling (db‐cGMP, Na nitroprusside, SNAP) suppressed T‐type currents and evoked a hyperpolarized shift in steady state inactivation. PKG inhibitors (KT5823, ODQ) masked this suppression while having no effect on basal T‐type activity. In contrast, PKG manipulations had no influence on the nifedipine‐sensitive L‐type conductance. Cumulatively, our findings support the emerging view that T‐type Ca2+ channels are a key regulatory target of vasodilatory signaling pathways (i.e. PKG and PKA).
Conformational Changes in Vinculin Measured by Fluorescence Resonance Energy Transfer (FRET) during Airway Smooth Muscle (ASM) Contraction Depend on Vinculin Phosphorylation at Tyrosine 1065
Authors: Huang Youliang , Gunst Susan J
Published: June 2021

Vinculin links integrins to actin filaments by binding to talin and actin. Vinculin can exist in a folded conformation with these binding sites masked, thus preventing its binding to actin and talin, or in an open ligand‐binding state with actin and talin binding sites exposed. ACh (acetylcholine) stimulation of ASM tissues increases the association of vinculin with actin and talin and stimulates vinculin phosphorylation at Tyr 1065 (Huang et al. JBC, 2011). We expressed non‐phosphorylatable vinculin Y1065F in ASM tissues to inhibit endogenous vinculin phosphorylation; this depressed contraction and inhibited the interaction of vinculin with talin. We constructed vinculin Wt and vinculin Y1065F FRET probes such that a FRET signal was generated when vinculin was in the closed conformation, but not in an open conformation. Probes were expressed in ASM tissues and FRET signals were analyzed in dissociated cells. Wt vinculin maintained a folded conformation in the cytoplasm of unstimulated ASM cells. ACh stimulation increased the open conformation of Wt vinculin at the membrane, but vinculin Y1065F maintained a closed conformation with or without ACh stimulation. We conclude that ACh stimulation of ASM causes a conformational change in vinculin that allows it to bind to talin and actin, and that phosphorylation at Tyr 1065 is required for vinculin to maintain an open ligand‐binding conformation. Support: NIH HL‐29289; HL074099
p110alpha subunit of PI3K is a crucial component of brain insulin receptor signaling in regulating lumbar sympathetic nerve traffic
Authors: Muta Kenjiro , Morgan Donald A. , Rahmouni Kamal
Published: June 2021

Increased regional sympathetic nerve activity (SNA) triggered by central nervous system (CNS) action of insulin has diverse physiological and pathophysiological implications. However, the molecular events underlying CNS action of insulin in mediating SNA elevation are poorly understood. Previously, we reported that pharmacological inhibition of phosphoinositide 3‐kinase (PI3K) selectively blocked the lumbar SNA response to intracerebroventricular (ICV) administration of insulin. Here, we tested the effect of genetic disruption of p110α catalytic subunit of PI3K on the sympathetic effect of CNS insulin. For this, we utilized a mouse model (p110αD933/WT) carrying a heterozygous mutation in p110α catalytic subunit of PI3K. We used multifiber sympathetic nerve recording to assess the SNA effects of insulin. In wild type mice, ICV administration of insulin (100 μU) caused a robust increase in lumbar SNA (259±37%, n=8). In contrast, ICV insulin failed to increase lumbar SNA in p110αD933/WTmice (−12±21%, n=5, P<0.05 vs. wild type mice). Our data demonstrate that p110α subunit of PI3K is an essential mediator of CNS insulin action to regulate lumbar sympathetic nerve traffic.
Optogenetic stimulation of preoptic neurons inhibits brown adipose tissue sympathetic nerve activity
Authors: Nakamura Yoshiko , Hioki Hiroyuki , Kataoka Naoya , Kaneko Takeshi , Nakamura Kazuhiro
Published: June 2021

The preoptic area (POA) is known as the center for body temperature regulation. Based on previous experimental results, we hypothesize that GABAergic projection neurons in the POA provide descending tonic inhibition to sympathoexcitatory neurons in the dorsomedial hypothalamus and in the medullary raphe and thereby, regulate the tone of sympathetic outflows to thermoregulatory effectors, such as brown adipose tissue (BAT) and cutaneous vasoconstrictors. If this is the case, stimulation of POA neurons should inhibit thermoregulatory sympathetic outflows. In this study, we examined the effect of optogenetic stimulation of POA neurons on metabolic thermogenesis in BAT in anesthetized rats. Expression of the light‐activated cation channel channelrhodopsin‐2 (ChR2) was introduced into many neurons in the POA by injection of adeno‐associated virus and an optical fiber was inserted into the brain to illuminate the POA. Skin cooling‐evoked sympathetic nerve activity in BAT and increase in heart rate were consistently inhibited only during illumination of the POA. This result supports the hypothesis that tonic descending inhibition from the POA determines the tone of sympathoexcitatory outflows to thermoregulatory effectors.
Aldosterone acts in the Nucleus Tractus Solitarius (NTS) to increase expression of vasopressin (AVP) in the Paraventricular Nucleus of the Hypothalamus (PVN)
Authors: Cho Namjik , Scheuer Deborah
Published: June 2021

Aldosterone (Aldo) is gaining attention as a stress hormone and causative factor in hypertension. We previously reported that implantation of pellets consisting of 1% Aldo to produce localized increases in Aldo within the NTS enhanced the arterial pressure response to a novel stressor in chronically stressed male Sprague‐Dawley rats. To elucidate mechanisms contributing to the enhanced stress responsiveness in these rats, quantitative real‐time PCR was performed on punches obtained from the NTS, PVN, rostral ventrolateral medulla (RVLM) and amygdala (Amyg) (Sham and 1% Aldo, n=6 each). Gene expression was analyzed statistically as ΔCt using 18S rRNA expression as the internal control. Relative fold changes in mRNA expression were normalized to Sham rats. Compared with Sham pellets, Aldo significantly (P≤0.05) increased expression for AVP in the PVN (3.8 fold) and the angiotensin type 1 receptor in the RVLM (2.8 fold), but did not affect the expression of these genes in the NTS. Aldo also significantly reduced expression of glial marker CD11b in the Amyg (0.6 fold) but not in the other regions tested. The results suggest that Aldo may act in the NTS to enhance stress responsiveness by increasing vasopressin synthesis and enhancing actions of angiotensin II in the RVLM. Supported by NIH #HL76807 and AHA 10GRNT4460047.
Developmental hyperoxia diminishes the carotid body response to hypercapnia
Authors: Bavis Ryan W , DeAngelis Kathryn J , March Ryan J , Wallace Josefine A
Published: June 2021

Rats reared in hyperoxia have smaller carotid bodies with reduced hypoxic responses. Since the carotid body (CB) also responds to changes in arterial CO2/pH, we hypothesized that developmental hyperoxia would alter CB and ventilatory responses to hypercapnia. Rats were reared in normoxia (21% O2) or hyperoxia (60% O2) until studied at 4, 6–7, or 13–14 days of age. Single‐unit chemoafferent activities were recorded in vitro from the soma of petrosal neurons with projections to the CB. Hyperoxia‐treated rats had significantly reduced carotid chemoafferent responses to hypercapnia (10% and 15% CO2) at all ages; CB CO2 sensitivity recovered within 7 days after return to room air. Ventilation was measured by plethysmography. Although steady‐state ventilatory responses to 4% and 8% CO2 were unaffected by developmental hyperoxia, ventilation increased more slowly during the normocapnia‐hypercapnia transition in hyperoxia‐treated rats. We conclude that developmental hyperoxia impairs CB chemosensitivity to both hypoxic and hypercapnic stimuli. Decreased CB CO2 sensitivity may compromise protective ventilatory reflexes during dynamic respiratory challenges. Supported by NIH grant P20 GM‐103423 (Maine INBRE).
New Theory of Respiratory Control and Regulation
Authors: Sun Xing‐Guo
Published: June 2021

We recently finished ¡°Holistic Regulation of LifeCIntegrative Physiology¡± framework as a whole. The part of respiratory control and regulation is decribed as below:

The key control of singles should be considered O2, while CO2 and [H+] act as subordinates to the core integration of breathing, circulation, metabolism, and other multi‐functional system control.

The trigger to inhale during the termination of exhale is when the level of PO2 gradually decreases (PCO2 and [H+], opposite).

The trigger to exhale during the termination of inhale is when the level of PO2 gradually increases (PCO2 and [H+], opposite), creating the cycle of breathing.

Cardiovascular function is the key factor in determining a normal breathing rhythm and frequency.

Regulation mode of breathing amplitude (depth): the next breath is induced as above to achieve a fast response through peripheral chemoreceptors. The breathing rate is exaggerated by the last breath, for example, a larger breath induces a larger breath, a smaller breath induces a smaller breath, and so on.

Regulatory control the steady breathing pattern must be integration with slow response of central chemoreceptors.
5‐HT2 receptor activation modulates phrenic motor output following chronic cervical spinal cord injury
Authors: Lee Kun‐Ze , Sandhu Milap S. , Dougherty Brendan J. , Lane Michael A. , Reier Paul J. , Fuller David D
Published: June 2021

C2 spinal hemisection (C2Hx) results in loss of ipsilateral phrenic bursting due to interruption of bulbospinal respiratory pathways. Time‐dependent return of spinal serotonin (5‐HT) innervation correlates with the partial recovery of phrenic output that is seen over weeks to months following C2Hx. Both 5‐HT2A and 2C receptors are present on phrenic motoneurons. 5‐HT2A receptors on phrenic motoneurons are upregulated following spinal cord injury, and studies in other motor pools have shown that constitutive 5‐HT2C receptor activity develops after spinal cord injury. Accordingly, we hypothesized that pharmacological blockade of these receptors would modulate phrenic motor activity after chronic C2Hx. Bilateral phrenic activity was recorded in anesthetized, vagotomized and ventilated rats. The 5‐HT2A/C receptor antagonist ketanserin (1 mg/kg, i.v.) significantly reduced phrenic burst amplitude bilaterally in C2Hx but not uninjured rats. The relative decrease of phrenic bursting was significantly greater in the ipsilateral phrenic nerve. Additionally, we tested a 5‐HT2C inverse agonist (cyproheptadine, 1 mg/kg, i.v.) and observed a slight increase of bilateral phrenic motor output in C2Hx animals. These results suggest that endogenous activation of 5‐HT2A receptors can maintain phrenic activity, while constitutively active 5‐HT2C receptors may blunt phrenic motor recovery after chronic C2Hx.
Expression of tyrosine hydroxylase within distinct populations of presympathetic‐pretomotor neurons in the rat brainstem
Authors: Nam Hyungwoo , Jackson Nateka L. , Kerman Ilan A.
Published: June 2021

Numerous motivated behaviors require simultaneous activation of somatomotor and autonomic functions. We have previously characterized the organization of central circuits that likely mediate this integration. Using recombinant strains of pseudorabies virus (PRV) that express unique reporter proteins, we delineated the distribution of presympathetic‐pretomotor neurons (PSPMNs), which send descending poly‐synaptic projections with collaterals to skeletal muscle and the adrenal gland. A significant portion of these neurons was found within brain regions known to contain catecholaminergic neurons. Thus, we hypothesized that some of the PSPMNs utilize catecholamines as neurotransmitters. To test this hypothesis, we used fluorescent immunohistochemical approach to identify co‐localizations between PSPMNs and tyrosine hydroxylase (TH). Our findings indicate that while TH‐positive PSPMNs are present throughout the brainstem, most of them are found within: the locus coeruleus (LC), subcoeruleus area (SubC), A5, and A7 cell groups. Particularly, many of these neurons are clustered within a region that spans ventral LC, dorsal SubC, and the A7 cell group. These findings indicate that specific catecholaminergic cell groups participate in motor‐autonomic integration.
Cthrc1, a Novel Hormone Involved in Regulation of Metabolism
Authors: Stohn J. Patrizia , Perreault Nicole G. , Wang Qiaozeng , Liaw Lucy , Lindner Volkhard
Published: June 2021

We discovered Cthrc1 in a screen of balloon injured arteries. It is induced in adventitial cells of injured arteries and expressed in many developing and remodeling tissues. In this study we address the role of Cthrc1 in normal adult tissues. We generated mutant mice with a novel Cthrc1 null allele, which appeared developmentally normal. As they get older, livers from Cthrc1 null mice accumulate vast quantities of lipid, leading to extensive macrovesicular steatosis. In addition, glycogen levels and cell proliferation in skeletal muscle and liver of Cthrc1 null mice were significantly increased. However, Cthrc1 expression in the adult wild‐type mouse was found in the brain and osteocytes, but not in liver or muscle. Further investigation showed Cthrc1expression predominantly in the paraventricular and supraoptic nucleus of the hypothalamus. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, in colloid‐filled follicles and the pituitary cleft. Development of a sensitive ELISA demonstrated that Cthrc1 circulates in human plasma in picogram/ml quantities. Cthrc1 is a novel circulating hormone produced by the anterior pituitary, hypothalamus and bone, and is involved in regulation of lipid storage and cellular glycogen levels.

This study was supported by HL69182, P20 RR‐15555, P20RR018789/P20GM103465 and P30RR030927/P30GM103392.
Differential bacteriostatic effects of sucralose on six species of sucralose‐tolerant environmental bacteria
Authors: Omran Arthur Phillip , Coughlin Charles Brian
Published: June 2021

Sucralose is a low‐cost, zero‐calorie, artificial sweetener marketed under the brand name Splenda as an alternative to sugar. It is very stable and due to its ability to withstand the waste water treatment process, heat, acidification, and microbial degradation it is accumulating in the environment. Our objective was to find bacterial species exhibiting sucralose metabolism. Six bacterial species were isolated from sucralose‐exposed local surface waters. These microbes were isolated on sucralose –containing media and classified using traditional methods and 16srRNA gene sequencing. These isolates were cultured in TSB media amended with progressively greater concentrations of sucralose and their growth was measured using spectrophotometery. We refuted our original hypothesis, instead observing bacteriostatic and perhaps bacteriocidal effects on all six sucralose‐tolerant isolates. The efficiency of growth inhibition appeared species specific and directly proportional to the sucralose concentration. Present experiments suggest a mechanism for the bacteriostatic effect. These results will affect how sucralose is viewed to impact naturally occurring populations of environmental microbes.
Atypical NFATc1 Regulation in Cultured Murine Intercostal Muscles
Authors: Robison Patrick , Hernández‐Ochoa Erick , Schneider Martin F.
Published: June 2021

The transcription factor NFATc1 controls the transformation of skeletal muscle fibers from fast to slow type in response to muscle activity. In the canonical pathway of NFATc1 activation, muscle activity triggers dephosphorylation of NFATc1 via the calcium dependant phosphatase calcineurin, activating NFATc1 by exposing nuclear localization sequences. Reversal of this process during periods of muscle inactivity is accomplished by constitutive kinase activity, with phosphorylation of NFAT leading to nuclear export. Using a virally expressed fluorescent NFATc1‐GFP construct, these activities can be observed as a relative accumulation or egress of NFATc1 in the nuclei of live cultured muscle fibers during electrical stimulation. Under stimulation paradigms that elicit strong nuclear influx of NFATc1 in isolated flexor digitorum brevis muscle fibers and significant influx in fibers isolated from the soleus, we have observed significant nuclear egress of NFATc1 in fibers isolated from the intercostal respiratory muscles. This suggests a possible role for activity‐inducible kinases in the intercostal muscles outside the canonical NFATc1 regulatory scheme.
Large‐strain In Vivo Lengthening Contractions Induce Mitochondrially‐mediated Apoptosis in Dysferlin‐null Muscle
Authors: Roche Joseph A , Mueller Amber L , Bloch Robert J
Published: June 2021

Background and Hypothesis

Mutations in the DYSF gene that encodes the protein, dysferlin, are implicated in late‐onset muscle wasting syndromes known as dysferlinopathies. We tested the hypothesis that dysferlin‐null myofibers undergo mitochondrially‐mediated cell death several hours after injurious large‐strain lengthening contractions.

Methods and Results

We subjected the tibialis anterior muscle (TA) of dysferlin‐null A/J and control A/WySnJ mice to large‐strain injury (LSI) by repeated lengthening contractions (20 repetitions). We measured isometric torque before LSI, and at 10min, 3hr, 6hr, 12hr, 24hr, 48hr, and 72hr after LSI, and harvested TA muscles from animals at these time points. There was no significant difference in maximum torque between A/J and A/WySnJ before LSI, and at 10min and 3hr after LSI. At 6hr and beyond, A/J muscle declined in torque, whereas A/WySnJ muscle continued to recover. Immunolabeling with antibodies to cytochrome c (cyt c) and complex IV (COX IV) showed that, whereas the intermyofibrillar mitochondrial network in A/J muscle becomes condensed at 3hr after LSI, it becomes more widespread and intensely stained in A/WySnJ muscle. At 6hr after LSI, cyt c is released from mitochondria causing its labeling to become diffused in the myoplasm and non‐coincidental with the COX IV labeling in A/J muscle, but not in A/WySnJ muscle. TUNEL staining showed a sharp increase in the number of apoptotic nuclei from 72±21 nuclei/mm2 at 6hr to 247±48 nuclei/mm2 at 12hr in A/J muscle compared to a small increase in A/WySnJ muscle from 12±7 nuclei/mm2 at 6hr to 77±28 nuclei/mm2 at 12hr.

Conclusion

Contraction‐induced injury induces mitochondrially‐mediated apoptosis in dysferlin‐null muscle.

Funded by a grant to JAR from the Jain Foundation Inc.
Low‐frequency electrical stimulation prevents muscle loss in mice with chronic kidney disease (CKD) by decreasing atrogin1 and MurF‐1 expression
Authors: Hu Li , Klein Janet D , Price Russ F , Wang Xiaonan H
Published: June 2021

Hypothesis

Low‐frequency electrical stimulation (LFES) in muscle attenuates muscle atrophy induced by CKD.

Method

CKD was induced in 20g mice by 5/6th nephrectomy. Electrode insertion points were selected using acupunture therapy guidelines. The positive needle was inserted under the fibula head near the superficial and deep fibular nerves (site name: Yang ling qian). The negative needle was inserted lateral to the knee joint under the fibula head near the fibular nerve (site name: Ho san li). The needles were connected to an SDZ‐蕅 electronic acupuncture device delivering a constant electical pulse (20Hz, 1mA) daily for 15 minutes over 4 weeks.

Results

Soleus and EDL muscle weights from uremic mice were significantly increased by LFES (P<0.05). LFES blunted the increase in CKD‐induced muscle proteolysis and improved phosphorylation of Akt (2.3‐fold, P<0.001) and FoxO3a (2.1‐fold, P<0.01). mRNAs of proteolytic atrogin‐1 and MuRF‐1 E3 ubiquitin ligases were attenuated by LFES. LFES concomitantly improved protein synthesis (increased phosphorylation of mTOR and p70S6K) and myogenesis (increased levels of MyoD, myogenin, and eMyHC) in muscles of CKD mice.

Conclusions

LEFS can prevent CKD‐induced muscle loss by a multi‐faceted mechanism affecting myogenesis, protein degradation and synthesis.

Support: NIH AR060268, and the NSF of China (30971471)
Testosterone treatment prevents spinal cord injury‐induced bone loss in male rats
Authors: Beggs Luke Allen , Beck Darren T , Conover Christine F , Ye Fan , Combs Sarah M , Miller Julie R , Balaez Alexander , Neuville Kathleen G , Wronski Thomas J , Bose Prodip , Borst Stephen E , Yarrow Joshua F
Published: June 2021

Our objective was to determine if testosterone enanthate (TE) attenuates bone loss in a rodent mid‐thoracic contusion spinal cord injury (SCI) model. Three month old male Sprague Dawley rats received Sham surgery (T8 laminectomy) + vehicle (V), 150 kdyne SCI + V (SCI 150), 250 kdyne SCI + V (SCI 250), SCI 250 + Low TE (2mg/week), or SCI 250 + High TE (7mg/week). SCI was induced via Infinite Horizons Impactor. Animals were sacrificed 3 weeks after injury. Bone structural parameters were assessed via histomorphometry at the proximal tibial metaphysis. SCI induced intensity‐dependent structural deficits, which were prevented by TE in a dose‐dependent manner. TE may provide treatment for SCI‐induced bone loss, thereby reducing the risk of fragility fractures.

Sham (a)
SCI 150 (b)
SCI 250 (c)
SCI 250 + Low T (d)
SCI 250 + High T (e)

Cancellous Bone Volume (%)
4.6 ± 0.9 c*
2.3 ± 0.3
0.8 ± 0.2 a*,e*
3.5 ± 0.5
4.6 ± 0.9 c*

Trabecular Width (μm)
14.6 ± 1.1 c
11.8 ± 1.1
10.7 ± 1.1 a
12.5 ± 0.4
13.0 ± 0.5

Trabecular Number (#/mm)
3.7 ± 0.6 c*
2.4 ± 0.2
0.9 ± 0.2 a*,d,e*
3.3 ± 0.4 c
4.1 ± 0.7 c*

Trabecular Separation (μm)
463 ± 189 c*
419 ± 36 c
1520 ± 322 a*,b,d*,e*
384 ± 106 c*
322 ± 75 c*

Values are Means ± SE of n = 4–9/group. Letters a‐e indicate differences from respectively labeled groups at p < 0.05 or * p < 0.01.

Work was supported by Veterans Department CDA‐2 to JFY.
Fibroblast Growth Factor 10 induces goblet cell hyperplasia independently from Notch signaling
Authors: Al Alam Denise , Sala Frederic G , Danopoulos Soula , Frey Mark R , Grikscheit Tracy , Bellusci Saverio
Published: June 2021

During gut adaptation, fibroblast growth factor 10 (Fgf10) is highly expressed in the ileal epithelium, where its levels are normally very low during homeostasis. In the colon, FGF10 stimulates cell proliferation and survival. However, whether it regulates small intestinal growth and homeostasis is unknown. Using a mouse model of inducible Fgf10 expression, we tested the role of this growth factor in the small intestine.

METHODS

3‐week old R26rtTA; tet(O)Fgf10 mice and littermate controls were given doxycycline food for 10 days. Cell proliferation, epithelial lineage markers, and Notch ligand expression were quantified by immunofluorescence and qPCR.

RESULTS

FGF10 induction led to increased cell proliferation and greater villus height in the ileum. We observed more goblet cells, but fewer Paneth cells, in induced mutants. Interestingly, there was no change in Dll1, Dll4 and Hes1 levels with Fgf10 induction.

CONCLUSIONS

Fgf10 induces goblet cell hyperplasia, at the expense of the Paneth cell lineage, in a Notch‐independent manner. Fibroblast growth factors may have selective trophic effects on individual intestinal cell types, which could be exploited therapeutically to improve adaptation after small bowel resection.
Integrating Geriatrics with Anatomy: An Educational Collaboration between Basic Science and Primary Care
Authors: Kleber Kara Teresa , Young Megan , Zumwalt Ann
Published: June 2021

Adapting a model introduced at Brown University, Boston University School of Medicine (BUSM) has developed a curriculum in which geriatricians and anatomists collaborate with mutual benefit. This program is a model of a productive collaboration between anatomy and a primary care specialty that benefits both faculty and students. In an innovation unique to BUSM, anatomy is taught to geriatric physicians, nurses, and students via a Grand Rounds talk on “The Anatomy of Aging.” Feedback on this session is universally positive, with respondents reporting benefits to their medical knowledge and patient care. In an adaptation of the Brown model, geriatricians then visit the gross anatomy lab when the course is 2/3 complete. The geriatricians speak informally at each lab table about clinical questions inspired by the anatomical findings in the donor, topics related to aging and disease, and issues they face as health care providers of older adults. This practice allows students to consider the donor as a person and as a patient, rather than simply an anatomical specimen. This program is well received by the students, many of whom enter BUSM with a strong inclination towards primary care.

Grant Funding Source: n/a
Integration of Histology and Pathology in a Senior Elective Course Curriculum
Authors: Cui Dongmei , Daley William
Published: June 2021

The Anat. 653 Review of Histology was a traditional basic science course offered at the University of Mississippi Medical Center, which provided a microscopic review of specimens of the tissue and organ systems to the M4 students who intend to pursue specialty training in pathology and other fields. A newly modified course not only provides a microscopic review of histology, but also involves integrated clinical cases, pathological images and discussion sessions of both basic science and clinical information. Three‐dimensional integrations include (1) integration of reading materials of both histology and pathology; (2) integration of microscopic review of histological and pathological images; (3) integration of basic science and clinical faculty in small group discussion sessions, comparing the normal cells, structures and tissues with the altered abnormal tissues and associated diseases. The students have an opportunity to consult and discuss with faculty members from both the Anatomy Department and the Pathology Department. They also have the opportunity to give a presentation of selected clinical cases during the discussion sessions. The integrated Histology and Pathology review course will be much better at preparing senior year medical students, especially those who intend to pursue specialty training in Pathology in their upcoming residency than the traditional review course.

Grant Funding Source: University of Mississippi Medical Center
Teaching surface anatomy through near‐peer instruction
Authors: Carp Shayna J , Metzger Keith , Rennie William
Published: June 2021

Study Objectives

Understanding of surface anatomy is crucial for clinical care, including physical examination, trauma management and surgery. This study evaluates the utility of a series of modules focused on surface anatomy in aiding student understanding of anatomical landmarks, as well as their ability to apply knowledge. Modules were developed in the context of larger lab sessions by an MS2, and guided by faculty, presented to MS1s. This provided opportunities for professional development of a medical student and for faculty members to mentor.

Methods

A series of modules relating surface anatomy to clinical scenarios was developed by an MS2, guided by faculty members. These were presented to MS1s at Hofstra North Shore‐LIJ SOM on a weekly basis. Each of the stations was developed around a specific goal and students were guided through activities using Socratic questioning. Effectiveness of the modules was evaluated through exam questions related to surface anatomy, and compared to the same questions from a cohort the previous year that did not have the modules.

Results

Preliminary results indicate that students who received the surface anatomy modules performed better than those who did not on questions related to surface anatomy. It is yet to be determined whether the intervention group is able to more broadly apply new knowledge by utilizing general principles of surface anatomy.
Amphibian muscle development and homologies: broader developmental and evolutionary implications, with notes on metamorphosis and terminal additions
Authors: Ziermann Janine , Diogo Rui
Published: June 2021

There has been a lot of controversy about the homologies of amphibian muscles. There is hardly any study comparing the development and adult configuration of neotenic and non‐neotenic salamanders and of frogs with direct and indirect development. Here we report our findings and explore their broader evolutionary implications, e.g. for the understanding of metamorphosis. For instance, we discuss the controversial homologies of the depressor mandibulae, levator hyoidei and branchiohyoideus, which are crucial for the understanding of amphibian biology and of the origin of amniote muscles. In three investigated salamander species, including a neotenic one, at least part of the larval depressor mandibulae derives from the larval levator hyoidei; in the two non‐neotenic salamanders some fibers of the larval depressor mandibulae become part the larval branchiohyoideus. The confusion about the homology and evolution of these and other muscles comes partly from the fact that many larval muscles disappear during metamorphosis. This contradicts the commonly accepted views that 1) during ontogeny there is a tendency to differentiate more muscles, and 2) that changes of development during evolution often refer to terminal additions. Our recent studies in modern humans have provided similar data and also contradicted these two latter views; therefore we also discuss the implications of our results for human evolution.

Grant Funding Source: start‐up package from Howard Univ. College of Medicine