bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho‐Kinase‐Mediated Suppression of KDR Current in Cerebral Arteries Requires an Intact Actin Cytoskeleton
Authors: Luykenaar Kevin , Radwan Rasha , Walsh Michael , Welsh Donald
Published: June 2021

This study examined the role of the actin cytoskeleton in Rho‐kinase‐mediated suppression of the delayed rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated and whole cell KDR currents monitored using conventional patch clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by uridine triphosphate (UTP; 30 µM). Consistent with past observations, KDR current suppression was blocked by the Rho‐kinase inhibitors H‐1152 (300 nM) and Y‐27632 (30 µM). Likewise, disruption of the actin cytoskeleton using cytochalasin D (10 µM) or latrunculin A (10 nM) abolished the ability of UTP to suppress KDR. In intact cerebral arteries, UTP induced actin polymerization in a Rho‐kinase‐dependent manner, suggesting that Rho‐mediated actin reorganization could play a role in the modulation of KDR current. Using pressure myography techniques, UTP‐induced depolarization and constriction of cerebral arteries were found to be significantly reduced following Rho‐kinase inhibition with H‐1152, and following the disruption of the actin cytoskeleton using cytochalasin D or latrunculin A. We conclude from our electrophysiological, biochemical, and functional observations that Rho‐kinase mediated alteration of the actin cytoskeleton likely underlies the inhibition of KDR current by UTP, ultimately contributing to the depolarization and constriction of cerebral arteries.
M‐cadherin Signaling Maintains Mitochondria Integrity of Muscle Stem Cells during Myogenic Differentiation via PI3K/Akt‐1/GSK‐3 pathway
Authors: Wang Yan , Alway Stephen E.
Published: June 2021

During muscle regeneration, the differentiation of muscle stem cells (MSC) is accompanied by apoptosis. The mechanism how the decision is made between death and differentiation is unclear. This study was to investigate the regulatory role of M‐cadherin (M‐cad) in this process. M‐cad expression was knocked down via RNA interference (RNAi) in confluent C2C12 myoblasts. Compared with control ones, myogenic differentiation was blocked and apoptosis was significantly increased in M‐cad‐RNAi cells, as determined by cell morphology, apoptotic DNA fragmentation, and caspase‐3 activation. The apoptosis is associated with mitochondrial dysfunction, as confirmed by the change in mitochondrial membrane potential via JC‐1 staining, the cytoplasmic release of cytochrome c and caspase‐9 activation. Conversely, overexpression of wild‐type M‐cad or treatment with recombinant M‐cad‐Fc chimera partially rescued the above effect and induced PI3K/Akt‐1 activation and GSK‐3ß inhibition. In addition, overexpression of either constitutively active Akt‐1 or dominant negative GSK‐3ß desensitized the cells to apoptosis induced by M‐cad RNAi. We conclude that M‐cad signaling maintains mitochondria integrity and protects against apoptosis via PI3K/Akt‐1/GSK‐3ß pathway during myogenic differentiation. It provides a novel insight in the regulation of MSC during muscle regeneration. Supported by NIH AG021530.
Myopathy and altered gastrocnemius and cardiac mTOR signaling in muscle‐specific LKB1 knockout mice
Authors: Malan Brandon B. , Mongillo Anthony D. , Thomson David M.
Published: June 2021

LKB1/AMP‐activated protein kinase (AMPK) activity is known to inhibit protein synthesis in skeletal and cardiac muscle in part through inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. However, the role of LKB1 in regulating heart and skeletal muscle size is not well understood. We have observed a progressive, age‐related myopathic phenotype in muscle specific LKB1 knockout (MLKB1‐KO) mice, which is associated with concurrent skeletal muscle atrophy and cardiac hypertrophy. The purpose of this study was to compare the phosphorylation of various components of the mTOR signaling pathway in atrophic gastrocnemius and hypertrophic heart muscles from MLKB1‐KO and littermate control (C) mice. Phosphorylation of mTOR, 4E‐BP1, and p70S6k were 65.6%, 20.3%, and 70.0% lower in MLKB1‐KO gastrocnemius muscles, respectively. Conversely, mTOR and S6k phosphorylation were 44.5% and 76.1% greater, respectively, in MLKB1‐KO hearts, although the difference was not significant for p70S6k (p = 0.19). No difference between genotypes was observed for 4E‐BP1 phosphorylation in the heart. Taken together, these findings indicate that alterations in mTOR signaling may contribute to cardiac hypertrophy and skeletal muscle atrophy in the MLKB1‐KO mouse. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR‐051928.
Involvement of NADPH oxidase‐derived superoxide anion in diabetes‐blunted aortic baroreceptor neuron excitability
Authors: Zhang Libin , Tran Thai P , Muelleman Robert L , Li Yu‐Long
Published: June 2021

NADPH oxidase‐derived superoxide has been identified as key signaling pathway in many pathophysiological processes. Our recent study has indicated that hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel function is enhanced, which contributes to the blunted aortic baroreceptor (AB) neuron excitability in diabetic state. Therefore, we investigated if NADPH oxidase‐derived superoxide mediates HCN channel activity and cell excitability of AB neurons in streptozotocin (STZ)‐induced diabetic rats. Using western blot and lucigenin chemiluminescence method, we found that protein expression of NADPH oxidase (gp91phox, p22phox, p40phox, p47phox, and p67phox) and superoxide production were higher in nodose ganglia from diabetic rats than that from sham rats. In whole cell patch‐clamp recording, action potential frequency of AB neurons in the diabetic rats was lower than that in the control rats (0.5 ± 0.5 vs. 4.9 ± 0.6 spikes/sec, p<0.05; AB neurons were identified by DiI staining). HCN current density of AB neurons was increased in the diabetic rats, compared with the control rats. In addition, apocynin (30 µM, a NADPH oxidase inhibitor) and tempol (1 mM, a superoxide dismutase mimetic) significantly reduced HCN current density and increased action potential frequency of the AB neurons in diabetic rats. These results indicate that elevation of intracellular NADPH oxidase‐derived superoxide generation contributes to the attenuated AB neuron excitability via enhancing HCN channel activation in diabetic state.
Cardiovascular and metabolic regulation in mice with Shp2 deletion in forebrain neurons
Authors: do Carmo Jussara Marcia , da Silva Alexandre A. , Hall John E.
Published: June 2021

Previous studies showed that deletion of Src homology‐2 tyrosine phosphatase (Shp2) in forebrain neurons causes early‐onset obesity. In this study we determined whether Shp2 deletion in forebrain neurons alters energy balance and if the obesity in these mice is accompanied by elevations in blood pressure (BP) and heart rate (HR). Forebrain Shp2‐/‐ mice were generated by breeding Shp2flox mice with CamKIIα‐Cre mice. BP and HR were recorded by telemetry, and motor activity, oxygen consumption (VO2), and respiratory quotient (RQ) were monitored by indirect calorimetry for 4 days in 22‐week‐old Shp2‐/‐ (n=8) and control mice (WT, n=4). Compared to WT, Shp2‐/‐ mice were heavier (46±3 vs 32±1 g), hyperglycemic (182±18 vs 170±11 mg/dl), hyperleptinemic (15±2 vs 1.5±0.3 ng/ml), hyperinsulimenic (27.6±9.0 vs 17.2±5.3 μU/ml), and consumed the same amount of food (4.2±0.4 vs 4.5±0.2 g/day). Despite features of the metabolic syndrome, Shp2‐/‐ mice had similar BP and HR than WT (112±2 vs 113±1 mmHg and 640±14 and 650±40 bpm), and exhibited increased motility (668±285 vs 420±86 cm/hr), higher RQ (0.91±0.02 vs 0.86±0.06) and similar VO2 than WT (42.4±2.8 vs 40.3±1 ml/kg/min). These results suggest that forebrain deletion of Shp2 causes obesity associated with increased lipid storage as indicated by higher RQ values. Despite obesity and many metabolic abnormalities, 22‐week‐old Shp2 mice are not hypertensive. (PO1‐HL51971)
Postural Challenge Accentuates the Autonomic Responses to an Inspiratory Apnea
Authors: Taneja Indu , Medow Marvin S. , Fadel Paul J. , Clarke Debbie A. , Stewart Julian M.
Published: June 2021

Previous studies indicate pulmonary stretch reflex produces bradycardia and hypotension. We used deep inspiratory apnea (IA) as a model to study pulmonary stretch reflex. Twelve healthy subjects (9 females, 3 males; 19‐27 yrs) performed a maximal inspiratory apnea both supine and after 10 min of head up tilt to 70 degrees (HUT). Heart rate (HR), blood pressure, (BP), respiration and microneurography (MSNA) were measured, while cardiac output (CO) and total peripheral resistance (TPR) were calculated. Oxygen saturation (PaO2) was estimated by pulse oximetry and end tidal carbon dioxide (ETCO2) was measured by capnography. HUT increased inspiratory apnea time (29.6±3sec supine vs.35.1±3.5sec HUT), MAP from 88.5±3.6 to 93±5.8 mmHg; HR from 66±2.5 to 72±3bpm; TPR from 20.2±5.3 to 30±4.6 mmHg/L/min, MSNA from 18.8±2.6 to 34.1±2.5 (bursts/min) and decreased CO from 4.9±0.5 to 3.2±0.4L/min (p<0.05). Inspiratory apnea increased ETCO2 from 40±2 to 60±2 Torr (p<0.05), PaO2 from 98±2 to 97±1.5 mmHg (p=NS) and MSNA showed three predominant phases. The first phase of quiescent MSNA was associated with a fall in MAP, HR, CO and an increase in TPR. The second phase of increased MSNA was associated with baroreflex mediated recovery of MAP, CO and peripheral resistance, HR recovered in HUT and not in supine position. During the third and final phase of increased MSNA activity, the MAP, HR, CO and TPR were maintained and the increased MSNA was likely to be due to chemoreceptor mediated stimulation of increased ETCO2. Maximal inspiratory apnea therefore results in a pulmonary stretch induced initial suppression of MSNA followed by an increased arterial baro‐chemoreflex mediated increased sympathetic response which is more pronounced during HUT. This study was supported by American Heart Association 0735603T.
The influence of age on carotid baroreflex mediated vasoconstriction in humans
Authors: Fisher James P , Young Colin N , Vianna Lauro C , Kim Areum , Deo Shekhar , Fadel Paul J
Published: June 2021

The carotid baroreflex (CBR) plays a critical role in the beat‐to‐beat regulation of arterial blood pressure (BP) primarily via reflex modulation of sympathetic nerve activity and vascular conductance. Decreased sensitivity of peripheral alpha‐adrenergic receptors has been reported in healthy older subjects however, it is unclear whether this may influence the CBR regulation of BP. To begin to address this question, mean BP, leg vascular conductance (LVC) and heart rate (HR) responses to simulated CBR hypotension were characterized in five young (22±2yr) and six older middle‐aged (63±2yr) healthy subjects using 5s pulses of neck pressure (NP, +40 Torr). Mean BP responses to NP tended to be reduced in older (+4±2 mmHg) compared to younger subjects (+9±3 mmHg), although this apparent difference did not reach statistical significance (P=0.209). Interestingly, the LVC responses to hypotension were similar in both groups (‐25±5% vs. ‐23±8%, older vs. younger, respectively; P=0.900). In contrast, HR responses were significantly attenuated in the older (+2±1 bpm) compared with the younger group (+10±3 bpm, P=0.039). In summary, these preliminary data suggest that the ability of the CBR to regulate BP in response to a hypotensive challenge appears to be attenuated in older individuals; however, alterations in sympathetically‐mediated leg vasoconstriction do not appear to contribute.

Supported by R01‐HL093167.
Arterial baroreflex control of heart rate and sympathetic nerve activity in patients with type II diabetes
Authors: Young Colin N. , Deo Shekhar H. , Fisher James P. , LeMaster Joseph W. , Hanna‐Moussa Abdullah , Khan Uzma Z. , Fadel Paul J.
Published: June 2021

Previous studies have indicated that diabetes is associated with postural hypotension and greater arterial blood pressure (BP) reactivity to acute cardiovascular stressors. However, whether impairments in arterial baroreflex (ABR) function contribute to alterations in BP control in diabetes remains unclear. To begin to address this question, muscle sympathetic nerve activity (MSNA), heart rate, and BP were continuously recorded in 8 type II diabetic patients and 4 healthy age‐matched control subjects during intravenous bolus injections of sodium nitroprusside followed 60s later by phenylephrine hydrochloride. The sensitivity (i.e., gain) of ABR‐cardiac and ABR‐MSNA control were identified from the linear relationships between RR‐interval and systolic BP and total MSNA and diastolic BP, respectively. ABR‐cardiac gain tended to be lower in diabetic patients compared to healthy controls [4.95±0.67 diabetics (n=8) vs. 9.25±3.90 controls, ms/mmHg; P=0.18]. In contrast, the ABR‐MSNA gain was similar between the two groups [‐8.32±3.03 diabetics (n=5) vs. ‐9.67±1.40 controls, units/beat/mmHg; P=0.72]. These preliminary data suggest that type II diabetic patients exhibit differential alterations in ABR function demonstrating ∼46% reductions in ABR‐cardiac control, whereas ABR‐MSNA control appears to be better preserved (∼14% reduction vs. controls).
Identifying the Neurocircuitry Involved in the Exercise Pressor Reflex during Exercise in Humans
Authors: Basnayake Shanika Deshani , Green Alexander L , Aziz Tipu Z , Paterson David J
Published: June 2021

The neurocircuitry responsible for the muscle pressor reflex in humans remains incompletely identified. Group III and IV afferents carry sensory information regarding this reflex, although central integration is still poorly defined. Emerging evidence reports that the periaqueductal grey (PAG) in humans could be a major site for controlling the cardiovascular response to exercise. We assessed the spectral changes in local field potentials (LFPs) recorded from deep brain nuclei in patients who had undergone neurosurgery for movement or pain disorders. Patients with electrodes implanted were asked to perform light handgrip exercise, followed by arterial occlusion of the exercised limb in order to maintain the pressor response. We focussed on the PAG, and compared it to control areas, i.e. the internal globus pallidus (GPi) and thalamus. The PAG showed significant increases in LFP power at frequencies from 0‐100Hz (p<0.001) during occlusion. This period was associated with maintained elevated arterial blood pressure. Cuff inflation without prior exercise resulted in a small increase in PAG activity suggesting that the inflation itself may, in part, contribute to the pressor response associated with occlusion. No change in GPi or thalamus activity was observed during occlusion. These data suggest the PAG is a part of the subcortical neurocircuitry involved in the exercise pressor reflex.
Failure of acute BCAA supplementation to delay fatigue during exercise in a hot environment
Authors: Soares Danusa Dias , Paulinelli‐Junior Reinaldo Telles , Morais Michele Macedo , Coelho Francisco Teixeira , Barcellos Luiz Alexandre Medrado
Published: June 2021

Serotonin is linked to central fatigue during exercise and its activity is enhanced in the heat. Branched‐chain amino acids (BCAA) have sought to diminish serotonergic activity and improve performance. We studied the effects of acute BCAA supplementation on physical performance and fatigue mechanisms in a hot environment (35°C and 60% rh). Ten young male participated in this study. They cycled at 40% of maximal power output (Pmax ) until fatigue in two experimental situations: PLA (placebo) and BCAA. In a double‐blind manner they ingested 4.0 mL.kg−1 b.w. of a non‐caloric artificial flavor juice, with (BCAA) or without (PLA) BCAA (0.03g BCAA.kg−1 body weight) before (twice), at the beginning and every 30 minutes during exercise. Total time of exercise (TTE), sweat rate, VO2, core temperature (Tc), mean skin temperature (Tsk), mean body temperature (Tb), heart rate (HR) and RPE were recorded. Plasma glucose, lactate and free‐fatty acids (FFA) as well as prolactin (PRL), a peripheral marker of serotonergic activity, were analyzed. Data are expressed as means ± e.p.m. BCAA didn't improve performance. At fatigue, Tc and plasma PRL augmented similarly in both conditions. There was no difference on sweat rate, Tc, Tsk and Tb between PLA and BCAA. Acute BCAA supplementation during exercise in a hot environment failed to modify fatigue induced by heat, possibly by its ineffectiveness to alter central serotonergic activity.
Delta opioid receptors (DORs) and the Autonomic Control of the Heart and Circulation
Authors: Barlow Matthew A. , Deo Shekhar H , Gonzalez Leticia , Yoshishige Darice A , Caffrey James L
Published: June 2021

The early stages of diet induced insulin resistance may involve changes in the vasomotor ganglia that regulate muscle blood flow. Delta opioid receptors (DOR) on cholinergic nerves in the heart and sympathetic ganglia actively regulate cholinergic transmission. The DOR‐1 subtype facilitates and the DOR‐2 subtype inhibits vagal transmission. The functional expression of DOR phenotypes depends on gangliosides and lipid rafts that populate local neural membranes. Oxidized lipids may disrupt membrane organization and thus alter normal DOR function. The current study tested the hypothesis that high fat feeding would increase oxidized LDL, disrupt DOR activity and produce a compensatory hyper‐adrenergic state. Body mass, insulin sensitivity, heart rate, heart rate variability, and femoral blood flow were determined in mongrel dogs fed high fat diets for six weeks. DOR receptor mediated changes in vagal control of heart rate and sympathetic control of leg blood flow were determined. High fat feeding increased heart rate and lowered muscle blood flow. Fat feeding reduced the DOR‐2 vagolytic effect in the SA node by half but left companion ganglionic sympatholytic effects unchanged. Facilitory DOR‐1 vagotonic and sympathotonic effects were weak or absent. The data support the suggestion that fat feeding alters the DOR moderation of cholinergic transmission and fosters an unopposed sympathetic overcompensation.
Parasympathetic Prejunctional Inhibition of Cardiac Sympathetic Nerves is Reduced in a Rodent Model of Heart Failure.
Authors: Donohue Timothy J , Smith Peter G
Published: June 2021

Parasympathetic prejunctional inhibition is an important mechanism regulating cardiac sympathetic tone. This study examined whether axo‐axonal 'uncoupling' contributes to reduced vagal tone in heart failure. Adult male rats underwent left coronary artery ligation; at 12‐16 weeks, ventricular ejection fraction and mean arterial pressure were reduced, consistent with heart failure. To assess prejunctional inhibition, cardiac slowing in response to right vagal nerve stimulation was compared when basal heart rate was elevated to comparable levels by β1 agonist dobutamine administration or electrical stimulation of cardiac sympathetic innervation. In sham‐ligated rats, vagal stimulation reduced heart rate to a greater degree during sympathetic stimulation than during dobutamine administration (ratio of 1.2+0.07at 8Hz). However, following infarct, vagal stimulation was less effective in reducing sympathetic‐ vs. dobutamine‐induced tachycardia (0.83+0.07 , p<0.001). These data support the conclusion that vagal prejunctional inhibition of sympathetic nerves is reduced in a rodent model of heart failure. NIH grant HL079652
Satellite cells in prevertebral ganglia of the hypertensive rat
Authors: Esfahanian Mohammad , Shah Amit H , Cao Xian , Kreulen David L
Published: June 2021

There are elevated levels of superoxide anion in non‐neuronal cells and neurons within prevertebral sympathetic ganglia of desoxycorticosterone acetate salt hypertensive (HT) rats. We hypothesized that the non‐neuronal cells were satellite cells (SCs) and we used glial fibrillary acidic protein (GFAP) as a marker for them in the celiac ganglion (CG). Using a polyclonal primary antibody for GFAP, we compared GFAP expression in the CG of the normotensive (NT) and HT rat. GFAP marked two populations of non‐neuronal cells: 1) SCs closely approximated to neurons; 2) small cells widely distributed in intercellular spaces. GFAP‐immunoreactive SCs associated with neurons were counted and their number was compared between NT and HT. The ratio of immunoreactive (IR) SCs to total SCs was greater in HT (p<.005, n=88‐130 SCs) compared to NT. The number of IR SCs per neuron was greater in HT (p<.05, n=29 neurons). There was no difference in the total number of SCs per neuron (p>.05, n=29 neurons) between NT and HT. These results show that the number of perineuronal SCs is not greater in HT but rather more SCs are expressing GFAP in ganglia from HT rats. Support: HL70687.
Identification of Rat Inferior Mesenteric Ganglion Neurons Differentially Innervating Splanchnic Veins and Arteries
Authors: Behr Ian James
Published: June 2021

The splanchnic circulation contains approximately 70% of the total body blood volume and the majority of this resides in the splanchnic veins. Splanchnic vascular resistance and capacitance can be differentially regulated by the sympathetic nervous system and in some species this is associated with separate arterial and venous innervation. We examined the innervation of mesenteric arteries and veins by neurons in the inferior mesenteric ganglion (IMG). An ex vivo preparation of the IMG attached to mesenteric vessels was placed in organotypic culture. After removing the endothelium fluorescent beads of different colors were injected separately into the arteries and veins of the mesentery. The preparation was cultured for 7 days. After 5 days a solution of fast blue was instilled around the ganglion to mark sympathetic ganglion neuron cell bodies. In 3 ganglia 90% of all neurons were filled when a single tracer was placed in both vessels. Of the neurons that innervate blood vessels 95% of them innervated either artery or vein and 5% innervated both artery and vein. These studies suggest that a majority of neurons in the IMG regulate mesenteric vascular tone and that there are separate neuronal pathways innervating artery or vein. Support:HL70687.
Multisubunit RNA Polymerases: Lessons from the Structures – Bacterial RNA Polymerase
Authors: Darst Seth Andrew
Published: June 2021

Structures of multisubunit, cellular RNA polymerases from all three kingdoms are now available. The first of three lectures highlighting these advances will focus on structures of bacterial RNA polymerase. What features are shared among all multisubunit RNAPs? What features are unique to bacterial RNA polymerase? How have the structures contributed to our understanding of the bacterial transcription cycle, its modulation by regulatory factors, and its disruption by inhibitors?
Localization of Na/HCO3 cotransporter NBCn1 (SLC4A7) in rat hippocampus
Authors: Park Hae Jeong , Rajbhandari Ira , Choi Inyeong
Published: June 2021

In an effort to facilitate the colocalization study, we developed a new NBCn1 antibody by immunizing a guinea pig with the BSA‐linked C‐terminal 14 amino acids of rat NBCn1. In membrane preparation from adult rat brain, the antibody recognized a protein of ~150 kDa, similar to NBCn1 protein detected with a rabbit polyclonal antibody against the same antigenic domain. The immunoreactive band disappeared when the antibody was preabsorbed with the antigenic peptides. The antibody labeled plasma membranes of HEK293 cells transfected with NBCn1. Immunochemistry of rat brain slices showed that NBCn1 immunofluorescence was localized in neurons of the regions including hippocampus, prefrontal cortex, neocortex, cerebellum and olfactory bulb. In the hippocampus, NBCn1 was found in the pyramidal layer of the hippocampal proper and the granular layer of the dentate gyrus. The fluorescence was also dispersed in the molecular layer (particularly stratum lacunosum‐moleculare). Double‐label immunofluorescence of NBCn1 and NMDA receptor subunit NR2A was performed to determine the colocalization of these proteins in the hippocampus. Both NBCn1 and NR2A were detected in the pyramidal layer and the granular layer, while NR2A additionally labeled subiculum. Controls without primary antibodies showed no discernible labeling. These data suggest that NBCn1 and NR2A are colocalized in some neurons of rat hippocampus.
Distribution of stanniocalcin‐1 (STC‐1) binding sites within the rat brainstem and cerebellum
Authors: Moreau Jason M. , Wagner Graham F. , Ciriello John
Published: June 2021

STC‐1 is a glycoprotein hormone that regulates calcium/phosphate homeostasis. STC‐1 has been shown within mammalian brainstem and cerebellum cells. However, whether these cells exhibit STC‐1 receptors is unknown. Thus, the distribution of STC‐1 binding sites (STC‐R) was investigated by in situ ligand binding using a STC‐1‐alkaline phosphatase fusion protein within the brainstem and cerebellum of the rat. Brains were fixed in 4% paraformaldehyde, paraffin embedded and serial, transverse sections were cut (6 μm) and processed for STC‐R. In the cerebellum, Purkinje neurons and neurons within the molecular layer of the cerebellar cortex and medial cerebellar n. exhibited STC‐R. In the brainstem, neurons exhibiting STC‐R were seen in the central grey, raphe n. (dorsal and pallidus), inferior colliculus, ventral n. of the lateral lemniscus, superior olive, lateral parabrachial n., locus coeruleus, dorsal tegmental n., parapyramidal area, external cuneate n., inferior olivary n. (medial and principal), area postrema, n. tractus solitarius, and lateral reticular n. Additionally, cells of the choroid plexus and ependymal cells lining the central canal were also labelled. These data suggest that STC‐1 may alter the function of these structures by regulating neuronal calcium/phosphate transport.

Supported by HSFO and CIHR
Locally released vasopressin increases presympathetic PVN neuronal activity in a calcium‐dependent manner
Authors: Son Sook Jin , Stern Javier E
Published: June 2021

Accumulating evidence indicates that the vasopressin (VP) is locally released within the supraoptic and paraventricular (PVN) nuclei, autoregulating VP neuronal activity. Here, we tested if intranuclear VP release also influences presympathetic PVN neuronal activity. Patch‐clamp recordings from presympathetic PVN‐RVLM neurons showed that focal application of VP (1µM) evoked an inward current (~8pA, P< 0.002), depolarized Vm (4mV, P< 0.0001), and increased PVN‐RVLM firing activity (~780%, P< 0.0001), effects blocked by a V1a receptor antagonist. The expression of V1a receptors in PVN‐RVLM neurons was confirmed immunohistochemically. V1a receptor blockade hyperpolarized Vm (~4mV, P< 0.001) and reduced ongoing firing activity of PVN‐RVLM neurons (~70%, P< 0.002). VP excitatory effect on PVN‐RVLM neurons was blocked by rapid chelation of intracellular Ca2+ (P< 0.02), but was not dependent on an influx of extracellular Ca2+. Finally, the effect of endogenously released VP was dependent on the activity of endogenous aminopeptidases. In summary, our results indicate that endogenously released VP, likely from a magnocellular neurosecretory source, tonically excites presympathetic PVN neurons, constituting a functionally relevant intercellular signaling pathway in the generation of homeostatic patterns of neuroendocrine and autonomic activities by the PVN. Supported by NIH HL68725
Effects of high‐fat diet and age on hypothalamic redox balance and central blood pressure regulation
Authors: Woods Mary E , Basgut Bilgen , Cudykier Idan , Rodriguez Michael , Whidden Melissa , Erdos Benedek , Tumer Nihal
Published: June 2021

Hypothalamic redox signaling pathways play an important role in central blood pressure (BP) regulation. Both age and a high fat (HF) diet may increase BP by elevating central levels of reactive oxygen species. Therefore, the aim of this study was to examine the effects of both age and high fat diet on hypothalamic redox balance. Young (5 mo, n=17) and old (26 mo, n=15) male F344xBN rats were fed standard or 60% HF diet for 15 wks. Young and old rats weighed 375±10 and 531±11g respectively before diet treatment, and 468±19 and 623±16g after HF diet. Initially, HF feeding was marked by an acute drop in BP and a simultaneous increase in heart rate in both young and old groups, followed by a steady increase in BP in only the young animals. Hypothalamic expression of NAD(P)H oxidase subunit p47 decreased slightly with age, but increased significantly with HF feeding in both the young and old animals, while gp91 subunit remained unaffected by both age and diet. Levels of antioxidant enzymes CuZnSOD and catalase decreased significantly with age, with a further decline in catalase due to HF diet. In addition, 4‐HNE, a marker of lipid peroxidation, increased with age but not in response to HF diet. These results suggest that both age and HF diet play important roles in altering hypothalamic redox balance.
Inhibition of PI3K survival pathway by LY294002 augments p53‐mediated myocyte apoptosis in myocardial ischemia reperfusion (MI/R)
Authors: Hock Carl E. , Xu Baohuan , Cavalieri Thomas A. , Liu Peitan
Published: June 2021

Ischemic preconditioning limits infarct size, indicating that cell‐adaptive behavior can protect cardiac cells from severe ischemic injury. This adaptive behavior can be pharmacologically modulated by activation of phosphoinositide 3‐OH kinase (PI3K) survival pathway. We have previously demonstrated that inhibition of p53 transcription with pifithrin‐alpha improves cardiac function and reduces myocyte apoptosis in aged rats following myocardial ischemia‐reperfusion (MI/R). LY294002 (an inhibitor of PI3K, 10 μM, 2 ml/kg bw, iv) or vehicle (2 ml/kg bw) were administered to male F344 young and aged rats subjected to 30 min of myocardial ischemia followed by 4 hours of reperfusion. Pretreatment of MI/R rats with LY294002 significantly increased DNA fragmentation in the area‐at‐risk, worsened cardiac output index and mean arterial blood pressure compared to MI/R rats treated with vehicle. Analysis of Western blots indicated that inhibition of PI3K by LY294002 decreased Mdm2 expression and increased expression of p53 and pospho‐p53 (Ser15). These data indicate that the PI3K survival pathway is active in MI/R in both young and aged rats and suggests that activation of this pathway may represent an important strategy to improve the recovery of cardiac function following MI/R.
Compromised cardiac performance and remodeling in collagen type VI deficient mice after myocardial infarction
Authors: Luther Daniel John , Shamhart Patricia E. , Ohanyan Vaghan , Hodnichak Cheryl M. , Miller James T. , Guo Yiru , Bolli Roberto , Meszaros J. Gary
Published: June 2021

The myocardium adapts to an ischemic insult by initiating a variety of cellular responses largely propagated by cardiac fibroblasts (CFs) and myofibroblasts, the critical mediators of myocardial remodeling and wound repair. We have reported, in addition to collagen types I and III, that type VI collagen also plays a significant role in remodeling by inducing CFs to differentiate into wound healing myofibroblasts. We hypothesize that type VI collagen is required for effective post‐myocardial infarction (MI) wound healing and repair of the myocardium. To test this hypothesis, we induced MI by LAD occlusion in collagen VI deficient mice (ColVI KO) and performed serial echocardiographic and histological studies during post‐MI remodeling. Echocardiographic studies at 4 weeks post‐MI revealed structural changes in ColVI KO mice including increases in endocardial LV volume (9% diastole; 25.6% systole), relative wall thinning (13.9%), and decreased corrected LV mass (25.1%) compared to control mice. Cardiac function was also compromised in KO mice: ejection fraction and fractional shortening decreased 11.3% and 17.4% respectively, with an increase in mitral valve deceleration time (51.5%) in KO mice. These data indicate that the absence of collagen type VI in the mouse heart causes modified remodeling of the myocardium leading to wall thinning, chamber dilation, and diminished systolic and diastolic function.
Inhibition of the IP‐receptor does not improve the cardiovascular changes in murine model of endotoxemia
Authors: Staehr Mette , Jensen Boye L , Hansen Pernille B
Published: June 2021

Selective inhibition of cyclooxygenase 2 (COX‐2) results in reduced systemic prostacyclin (PGI2) production. The levels of COX‐2 and PGI2 are increased in endotoxemia, and we therefore hypothesized that PGI2 plays an important role in the cardiovascular failure observed during sepsis.

Chronic indwelling catheters were placed in femoral artery and vein in mice and the effect of the PGI2‐receptor (IP) antagonist BR5064 (Bayer, 0.1 mg/kg) and the PGI2 analogue, Beraprost (1 μg/kg) on blood pressure was tested. Injection of Beraprost induced a significant fall in blood pressure from 103 to 77 mmHg with no effect on heart rate. BR5064 did not change resting blood pressure and heart rate, but blocked the effect of Beraprost. Next, an LPS bolus (2 mg/kg) was given which led to a significantly reduced blood pressure and heart rate (106±7 to 75±2 mmHg and 627±53 to 306±33 bpm, respectively) 8 hours after injection (n=11).

Pre‐treatment with BR5064 before LPS, did not improve the cardiovascular changes: 104±3 to 78±2 mmHg and 654±9 to 429±57 bpm respectively (n=10).

We therefore conclude that while the IP‐antagonist specifically inhibits the IP‐receptor, it does not prevent the acute deterioration of cardiovascular function in a model of endotoxemia which suggest that prostacyclin signaling is less important for the observed cardiovascular impairment.

Support: the Danish medical research council and Novo Nordisk foundation.
Localization of proteins associated with renal cystic diseases to the olfactory epithelium
Authors: Pluznick Jennifer L. , Rodriguez‐Gil Diego , Mistry Kavita , Hull Michael , Johnson Colin A. , Greer Charles A. , Caplan Michael
Published: June 2021

It has recently been reported (McEwen, et al PNAS 2007) that nephrocystin 6 (NPHP6) localizes to the olfactory epithelium (OE), and that mutations in NPHP6 can cause anosmia in addition to renal cystic disease. We demonstrate here that other proteins known to cause renal cystic disease ‐ polycystin 1 (PKD1) and meckelin (MKS3) ‐ also localize to the murine OE. MKS3 and PKD1 are both detected in the OE by RT‐PCR (products were sequenced to confirm their identity). Using previously characterized antibodies, we demonstrated that MKS3 localizes specifically to the dendritic knobs of mature olfactory neurons, whereas PKD1 localizes the cell bodies and cilia of mature olfactory neurons. The expression of MKS3 is developmentally regulated, with little or no expression detected postnatally until P7, and the strongest expression detected in adult mice. In addition, in mice null for PKD1 in the OE, MKS3 expression is reduced. In conclusion, proteins associated with renal cystic disease are expressed in the OE where they localize to the knobs and cilia of mature olfactory neurons.
Epithelia derived from human mammary tissue exhibits unique ion transport characteristics
Authors: Wang Qian , Wimberly Stephanie , Wang Lin‐hua , Schultz Bruce D.
Published: June 2021

Ion transport and barrier characteristics of mammary epithelium are poorly defined for most species, including humans. MCF10a cells, derived from non‐neoplastic human mammary tissue (Cancer Res 50: 6075, 1990), were cultured on permeable supports in typical medium (DMEM/F12) for 9‐17 days in the absence and presence of cholera toxin (Ct). At all time points tested, MCF10a cells generated an electrically tight epithelial barrier (1010±110 O cm2) that exhibited a positive short circuit current (Isc; 6.1±0.3 μA cm−2) indicative of net cation absorption or anion secretion. Cells cultured in Ct exhibited greater basal Isc and greater amiloride‐sensitive Isc (4.3 vs. 2.5 μA cm−2), indicating enhanced sodium absorption. Culturing the cells in the presence of forskolin (0.2 μM) did not replicate the Ct effect. Additionally, it was shown that the effect of Ct‐enhanced amiloride‐sensitive Isc was greatest at the shortest exposure period tested, 1 day. Regardless of culture conditions, acute exposure to forskolin (2 μM) induced a modest, slowly developing increase in Isc. Bumetanide and glibenclamide (300 μM) were associated with modest reductions in Isc (<0.7 μA cm−2). These results demonstrate that MCF10a cells provide a valuable model to identify transport mechanisms and regulatory pathways that can account for the unique composition of human milk. [NIH (P20‐RR017686), KS Ag Exp Stn, and KSU SUROP support]
Involvement of the actin and tubulin networks in the trafficking of V‐ATPase, NBCe1‐A and NBCe1‐B in a submandibular duct cell line
Authors: Oehlke Oliver , Martin Henno , Roussa Eleni
Published: June 2021

Cytoskeletal components are established players in protein trafficking in epithelial cells. The bicarbonate‐secreting epithelium of salivary ducts is equipped with several acid‐base transporters whose cellular distribution is altered after systemic acid‐base disturbances. Here we investigated the role of actin and tubulin in V‐ATPase and electrogenic NBC1 distribution under physiological and pathological conditions in vitro. Therefore, extracellular acidosis or alkalosis was induced in the presence or absence of cytochalasin D or colchicine.

Disruption of the actin network differentially influenced constitutive trafficking and adaptive redistribution after acid‐base disturbances. Following cytochalasin D treatment all transporters failed to reach the membrane. Trafficking of NBCe1‐B is impaired after disruption of the tubulin network as well, whereas localisation of NBCe1‐A was not affected by colchicine treatment during acid‐base disturbances.

The results indicate differential regulation of tubulin‐mediated trafficking for NBCe1‐A and ‐B, reflecting their differences in the direction of ion transport and localisation in a polarized epithelium in vivo.

This work was supported by the DFG.
Functional Studies of Acid Transporter in Cultured Rat Epididymal Cell
Authors: Zuo Wulin , Shan Jiajie , Li Sheng , Wong Patrick Y. D. , Zhou Wenliang
Published: June 2021

The epididymis is the site for the maturation and storage of spermatozoa. The acidic epididymal fluid, which maintains spermatozoa in a quiescent state, contributes to this process. Several transporters, including Na? exchanger (NHE) and vacuolar H+ATPase(H+ATPase), are believed to be involved in the pH regulation of epithelial fluid. In the present study, double immunofluorescense of H+ATPase and carbonic anhydrase II (CA II) in primary culture of cauda epithelial cells showed that the system was a suitable model for investigation of acid secretion by clear cells. Clear cells were pharmacologically distinguished from principal cells in acid/base transportation. The H+ATPase inhibitor, bafilomycin A1, and NHE inhibitor, amiloride, suppressed the pHi recovery by 85% and 54%, respectively. It suggested that, in addition to NHE, clear cells actively pump proton from cytoplasm into extracellular space via H+ATPase. Luminal fluid was acidified by H+ATPase, and this process could be inhibited by its inhibitors with IC50 values of 12 nM. Rat cauda sperm motility was demonstrated to be regulated by the acid fluid. The present work shows that the minority cell type‐clear cells play an important role in the pH regulation of epididymal fluid via H+ATPase.

This work was supported by Hong Kong RGC grant (No.452506) and the National Natural Science Foundation of China (No.30770817)
Slc26a3(Down‐regulated in adenoma) Cl/HCO3 exchanger is responsible for Cl absorption and HCO3 secretion in the murine large intestine
Authors: Suzuki Yuichi , Kawamata Koichi , Schweinfest CW , Soleimani M , Hayashi Hisayoshi
Published: June 2021

In the large intestine a Cl/HCO3 exchanger is suggested to be involved in Cl absorption which is closely associated with Na absorption mediated by Na/H exchanger. A Cl/HCO3 exchanger is also responsible for HCO3 secretion. Molecular identity of these Cl/HCO3 exchanger(s) was investigated using mice with gene‐targeted knockout (KO) of Cl/HCO3 exchangers: Slc26a3, down‐regulated in adenoma (Dra). Isolated cecum and distal colon were mounted in Ussing chambers. We measured transepithelial fluxes of 22Na and 36Cl, and by using a pH‐stat device a luminal alkalinization rate. Essentially similar findings were obtained for both the cecum and distal colon. In control (ddy strain) and wild type mice, mucosa‐to‐serosa flux (Jms) was larger than serosa‐to‐mucosa flux (Jsm) for both 22Na and 36Cl. In contrast, in DraKO mice Jms and Jsm for 36Cl were both markedly reduced and net 36Cl absorption was almost, if not entirely, abolished, while Jms and Jsm for 22Na were not noticeably affected. Cl‐dependent HCO3 secretion was completely abolished in DraKO mice. These results suggest that, under Ussing chamber condition, Dra is a major transporter responsible for both the absorptive and secretory transport of Cl, and for most of the net Cl absorption. Dra is also involved in Cl‐dependent HCO3 secretion.
Regulation of αENaC Expression by the Circadian Clock Protein Per1
Authors: Gumz Michelle L , Stow Lisa R , Lynch I Jeanette , Cain Brian D , Wingo Charles S
Published: June 2021

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and an important determinant of systemic blood pressure. We have previously shown that knockdown of the circadian rhythm gene Per1 prevented the aldosterone‐mediated induction of the alpha subunit of the epithelial sodium channel (αENaC) in mouse inner medullary collecting duct cells. We have extended this observation to the outer medullary and cortical collecting duct using additional in vitro cell models. Interestingly, αENaC expression was attenuated following Per1 knockdown even in the absence of aldosterone, indicating that Per1 contributes to the basal regulation of αENaC expression. Analysis of heterogeneous nuclear RNA and αENaC promoter luciferase assays showed that this regulation occurred at the level of transcription. Most importantly, expression of αENaC was attenuated in Per1 deficient mice. Evaluation of αENaC mRNA levels over a 24 hour period revealed a circadian pattern of expression that was altered in mice lacking Per1. These data demonstrate a novel and functional role for the circadian clock protein Per1 in the regulation of the renal epithelial sodium channel. Supported by NIDDK, AHA.
A Small Molecule Screen Identifies Novel Inhibitors of the Neuronal K‐Cl cotransporter KCC2
Authors: Delpire Eric , Days Emily , Mi Dehui , Lewis Michelle , Lindsley Craig , Weaver David
Published: June 2021

KCC2, a neuronal‐specific K‐Cl cotransporter plays a major role in maintaining intracellular Cl− concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K‐Cl cotransporter is dominated by the loop diuretics furosemide and bumetanide. These molecules are used in clinical medicine as they inhibit the loop of Henle Na‐K‐2Cl cotransporter with much higher affinity. In order to identify novel molecules that affect KCC2 activity, we developed a fluorescent‐based assay suitable for high throughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. We characterized a small number of inhibitory compounds, some of which inhibit KCC2 activity in the micromolar or sub‐micromolar range, without substantially affecting NKCC1 activity. We synthesized a number of chemical variants, tested their effect on KCC2 function, and provide a first analysis of structure/activity relationship. We also used one of the compounds to demonstrate competitive inhibition in regard to external [K+], and non‐competitive inhibition in respect to external [Cl−].
Insertion of red fluorescent protein into the amino terminus of Na,K‐ATPase catalytic subunit does not disrupt post‐translational processing
Authors: Pressley Thomas A
Published: June 2021

The first five amino acids of the catalytic α1 isoform from the Na,K‐ATPase undergo proteolytic cleavage during or after translation. Previous work suggests that amino acids downstream of the first nine are not required for proteolysis. To explore this inference, I tested whether increasing the distance between the amino terminus and the main body of the protein would influence cleavage. A chimeric cDNA was prepared in which sequence encoding a variant of DS‐red, mCherry, was inserted into a highly‐conserved leucine‐lysine pair just downstream from the amino terminus of the nascent polypeptide. The resulting mutant was introduced into opossum kidney cells (OK) by DNA‐mediated gene transfer, and stable recipients were examined by fluorescence microscopy and immunoblotting. Cells exhibited both surface and intracellular fluorescence, indicating that the chimeric catalytic subunit was targeted successfully to the plasmalemma. A site‐directed antibody raised against the first nine residues of the α1 nascent chain (anti‐VGR) did not bind to membranes from the transfected cells. However, a band with a mobility appropriate for the chimeric α was detected with an antibody that is specific for mCherry and a second site‐directed antibody specific for rat α1, further confirming expression of the chimeric protein. Because anti‐VGR does not recognize cleaved α1, the absence of binding suggests that the α1‐mCherry construct underwent normal processing, despite the insertion of irrelevant sequence in the amino terminus. Consistent with earlier work, these results suggest strongly that the determinants of amino terminal processing in the catalytic subunit of the Na,K‐ATPase reside within the first few residues of the nascent polypeptide. [Supported by NIH RR‐19799.]
Altered Transport In Na/K pumps With α‐Subunits Truncated At Its 5 C‐Terminal Residues
Authors: Yaragatupalli Siddhartha , Gatto Craig , Artigas Pablo
Published: June 2021

Truncation of the α‐subunit's last 5 residues renders Na/K pumps with a reduced affinity for intracellular Na (Nai), without effects on affinity for external K (Ko) (Morth et al, 2007). We used two electrode voltage clamp, and cut‐open oocyte to investigate the effect of the ΔKESYY truncation on electrogenic transport by Xenopus Na/K pumps (Q120RN131D‐α1+β3). Pump currents (IP) were measured 2‐3 days after cRNA injection in Nai‐loaded oocytes. The K0.5 for IP activation by Ko in Na‐free NMDG external solutions was ≈0.2 mM, both in eggs expressing truncated (TP) or normal (NP) pumps; TP expressing eggs showed larger IP (IPmax≈300 nA) than NP expressing eggs (IPmax≈150 nA). Western blots suggest better expression of TP at the plasma membrane. Surprisingly, in Ko‐free solutions, TP but not NP, presented a voltage‐ and Nao‐dependent inward current of ≈ ‐250 nA at ‐160 mV with 135 mM Nao, that is reduced by ≈50% at 67 mM Nao. Consistent with a low Nao affinity in TP, the voltage‐dependent distribution of transient charge movement (which describes the E1‐E2 occupancy in the Na branch of transport) is shifted by ≈‐100 mV compared with NP. The effect of combining ΔKESYY with other mutations that modify external Na affinity will also be presented.