bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis of astrocyte mediated by toll‐like receptor 4 and apoptosis signal‐regulating 1 (ASK1) in cardiovascular center causes excessive sympathoexcitation in hypertensive rats
Authors: Kishi Takuya , Hirooka Yoshitaka , Sunagawa Kenji
Published: June 2021

Background

Long considered merely a mechanical support to neurons, astrocyte has been shown to protect and mediate neural function. We previously demonstrated that oxidative stress and apoptosis in the rostral ventrolateral medulla (RVLM) of brainstem increase sympathetic nerve activity (SNA). ASK1 plays a key role in the stress‐induced apoptosis. TLR4, known to regulate inflammatory reactions, activates ASK1. We hypothesized that apoptosis of astrocyte in the RVLM mediated by ASK1/TLR4 results in sympathoexcitation in hypertension.

Methods and Results

We used 16–18 weeks old stroke‐prone spontaneously hypertensive rats (SHRSP) as a hypertensive model with increased SNA and Wister‐Kyoto rats (WKY) as control. Immunohistochemical staining revealed that the number of astrocyte in the RVLM was significantly lower in SHRSP than in WKY. The expression of TLR4 and ASK1 in the RVLM was significantly higher in SHRSP than in WKY (TLR4; 23±2%, ASK1; 44±5%, n=5 for each, p<0.01). Infusions of TLR4 antagonist (intracerebroventricularly, for 14 weeks) significantly reduced SNA only in SHRSP (−38±2%, n=5, p<0.01), not in WKY. The number of astrocyte and the expression of ASK1 in the RVLM were significantly higher in TLR4 antagonist‐treated SHRSP than in vehicle‐treated SHRSP.

Conclusion

Apoptosis of astrocyte in the RVLM mediated by TLR4/ASK1 plays a key role for sympathoexcitation in hypertensive rats.
Increased Group I Metabotropic Glutamate Receptor Activity in Paraventricular Nucleus Contributes to Elevated Sympathetic Vasomotor Tone in Hypertension
Authors: Li De‐Pei , Pan Hui‐Lin
Published: June 2021

Enhanced glutamatergic input in the paraventricular nucleus (PVN) contributes to elevated sympathetic outflow in hypertension. We determined the role of group I metabotropic glutamate receptors (mGluRs) in the PVN in the control of sympathetic outflow in spontaneously hypertensive rats (SHR). Lumbar sympathetic nerve activity (LSNA), arterial blood pressure (Bp), and heart rate (HR) were recorded from anesthetized SHR and WKY rats. Bilateral microinjection of MPEP (1–10 nmol), a mGluR5 antagonist, or LY367385 (10–100 nmol), a mGluR1 antagonist, into the PVN dose‐dependently decreased LSNA and Bp in SHR but not in WKY. MPEP‐induced decreases in LSNA and Bp were greater than that produced by LY367385 in SHR. Furthermore, microinjection of group I mGluR agonist DHPG (10–100 nmol) caused dose‐dependent increase in LSNA and Bp in both WKY and SHR. DHPG‐induced responses were significantly attenuated by MPEP or LY367385 and abolished by a combination of MPEP and LY367385 in both WKY and SHR. In addition, prior injection of NMDA receptor antagonist AP5 (1.4 nmol) into the PVN decreased LSNA and Bp in SHR and attenuated DHPG‐induced sympathoexcitation in both WKY and SHR. Thus, our study suggests that elevated sympathetic outflow is maintained by activation of group I mGluR receptors in the PVN in SHR. Increased group I mGluR receptor activity contributes to increased glutamatergic input via NMDA receptor in hypertension.
Altered regulation of sympathetic nerve activity by the caudal ventrolateral medulla and nucleus tractus solitarius in obese Zucker rats
Authors: Huber Domitila A. , Schreihofer Ann M.
Published: June 2021

Obese Zucker rats (OZR) have higher sympathetic nerve activity (SNA) and arterial pressure (AP) compared to lean Zucker rats (LZR). The higher SNA in OZR is due to reduced GABAergic inhibition of rostral ventrolateral medulla (RVLM), the source of drive for SNA. Caudal ventrolateral medulla (CVLM) provides this inhibitory input to RVLM, and CVLM is driven by a glutamatergic input from nucleus tractus solitarius (NTS). We hypothesized the CVLM provides reduced tonic inhibition to RVLM in OZR due to a reduced glutamatergic drive from NTS. Adult male rats were anesthetized with urethane (1.5 g/kg, iv), ventilated, and paralyzed to record splanchnic SNA and AP. Compared to LZR (n=13), OZR (n=14) had higher SNA (2.7 ± 0.4 vs. 1.6 ± 0.2 μV, P<0.05) and mean AP (130 ± 2 vs. 123 ± 2 mmHg, P < 0.05). Inhibition of glutamatergic inputs to CVLM (kynurenate; 5.4 nmol/100 nl) evoked smaller rises in SNA in OZR (n=7) vs. LZR (n=7; 169 ± 23 vs. 289 ± 17 %, P < 0.05). This was not due to a reduced ability of GABA to inhibit RVLM because injections of GABA into RVLM (0.3, 1.0, 3.0 nmol) evoked comparable decreases in SNA in OZR (n=7) and LZR (n=7). Inhibition of NTS (muscimol; 100 pmol) evoked smaller rises in SNA in OZR (n=7) vs. LZR (n=6; 62 ± 11 vs. 112 ± 14 %, P < 0.05). These results suggest higher SNA in OZR may be due to reduced tonic GABA from CVLM. Further, reduced glutamatergic inputs from NTS to CVLM may contribute to diminished inhibition from CVLM to RVLM in OZR.

Grant Support: NIH R01HL086759
Chronic exposure to estradiol‐17 β (E2) causes hypertension in female rats: Role of IL‐1β and norepinephrine
Authors: Subramanian Madhan , Hamman D W , Mohankumar S MJ , Fink G D , Mohankumar PS
Published: June 2021

Cardiovascular disease is the most prevalent cause for morbidity and mortality in women. Previous findings from our lab suggest that chronic exposure to low levels of E2 causes hypertension. The mechanisms underlying this effect are unknown. We believe that E2 affects central sites such as the Paraventricular nucleus (PVN) of the hypothalamus to cause hypertension, since the PVN plays a key role in the regulation of sympathetic activity. We hypothesized that chronic E2 exposure most probably increases cytokine levels in the PVN to affect norepinephrine (NE) levels leading to hypertension. To test this, 3–4 month old female Sprague Dawley rats were implanted s.c. with slow‐release E2 pellets. Animals were exposed to 20ng of E2/day for 30, 60 or 90 days (n=6). At the end of the treatment, animals were sacrificed and the PVN was microdissected and analyzed for NE and the cytokine, IL‐1β. Exposure to E2 produced a duration‐dependent increase in IL‐1β and NE levels. IL‐1β (pg/μg protein; mean±S.E.) was 19.4±1.2 after 30 days and increased significantly after 60 & 90 day exposure (34.1±5.9 & 24.9±3.0 respectively) compared to the control group 9.0±2.4 (p<0.05). Similarly NE values (pg/μg protein; mean±S.E.) were 5.01±1.04 after 30 days and increased significantly after 60 & 90 day exposure (53.8±12.4 & 78.9±14.5 respectively) compared to the control group (8.99± 3.23; p<0.05). The increase in IL‐1 β after chronic E2 exposure could stimulate NE release to increase sympathetic outflow contributing to hypertension. Supported by NIH AG027697.
A phylogeny driven genomic encyclopedia of bacteria and archaea
Authors: Eisen Jonathan A
Published: June 2021

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms, including those related to human health. Unfortunately, the available genomes are from a highly biased subset of the known phylogenetic diversity of microbes. In order to explore the value added by choosing microbial genomes for sequencing based on their evolutionary relationships, the Joint Genome Institute has sequenced and analyzed the genomes of 56 culturable species of bacteria and archaea selected to maximize phylogenetic coverage. I will present results from analysis of these genomes that demonstrate that there are pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and novel biological properties, and the prediction of functions for known genes from other organisms. The results strongly support the need for systematic ‘phylogenomic’ efforts to compile a phylogeny‐driven ‘Genomic Encyclopedia of Bacteria and Archaea’ in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.
Focal synaptic recruitment to second order solitary tract nucleus neurons with minimal electrical shocks
Authors: McDougall Stuart J. , Andresen Michael C.
Published: June 2021

Second order neurons in the solitary tract (ST) nucleus (NTS) receive vagal primary afferent as well as input from other central neurons. Less is known about non‐ST inputs. In horizontal brainstem slices, ST activation evoked low jitter EPSCs (σ of latency; 101±6 μs) identifying 2nd order NTS neurons (n=38), ST‐EPSCs (amplitude 213±25 pA) never failed. Using a fine glass focal electrode, minimal electrical shocks activated local inputs that did not interact with ST‐EPSCs (failed collisions). Probing the surface of the slice 10–400 μm from voltage clamped cells, focal shocks at minimal intensities evoked mostly EPSCs (30/38 cells). Focal‐EPSCs were unitary, all‐or‐none (amplitude 176±22 pA; jitter 102±5 μs; 0% failure rate) and blocked by the non‐NMDA antagonist NBQX, indicating that minimal focal shocks selectively recruit monosynaptic glutamatergic inputs. High focal intensities evoked complex PSCs consistent with recruitment of multiple inputs, many exhibiting high jitter (>200 μs), indicating polysynaptic pathways. In NBQX, focal stimulation (5/38 cells) recruited unitary, low‐jitter (138±22 μs) IPSCs with highly variable amplitudes (184±49 pA) and frequent failures (47±16%). GABAA receptor antagonist SR95531 blocked IPSCs. This approach allows for selective recruitment of converging inputs to NTS neurons and indicates that GABA release has lower release probability than afferent glutamate.
PLASTICITY OF SYNAPTIC INPUT TO LIVER‐RELATED HYPOTHALAMIC NEURONS IN A MODEL OF TYPE 1 DIABETES
Authors: Zsombok Andrea
Published: June 2021

Glucose homeostasis is controlled by the central nervous system, via regulation of the activity of the autonomic nervous system, including preautonomic neurons of the paraventricular nucleus (PVN). The PVN regulates the sympathetic and parasympathetic output controlling visceral functions such as hepatic gluconeogenesis. Endovanilloids, the endogenous ligands for transient receptor potential vanilloid type 1 (TRPV1), affect autonomic function via excitation of synaptic inputs to most neurons. We used whole‐cell patch‐clamp recordings in hypothalamic slices from control and streptozotocin‐treated (STZ), hyperglycemic mice to identify synaptic characteristics in liver‐related PVN neurons. Application of the TRPV1 agonist capsaicin (CP) increased mEPSCs frequency by 55±2% in control mice. However, CP had no effect on mEPSC frequency in liver‐related neurons from STZ‐treated mice. Pre‐incubation with insulin restored TRPV1 activity in a PKC/PI3K‐dependent manner. This effect of insulin was prevented by brefeldin A, suggesting that insulin stimulates TRPV1 receptor trafficking to the plasma membrane. Therefore, insulin deficiency and hyperglycemia appear to affect TRPV1 signaling; which contributes to autonomic dysfunction and dysregulation of glucose metabolism. These findings support the hypothesis that the autonomic output from liver‐related PVN neurons is altered in type 1 diabetes.
Maternal Separation affects neurocircuitry of ether and restraint stress in adulthood
Authors: Foscolo Daniela Rocha Costa , Foscolo Rodrigo Bastos , Coimbra Candido Celso
Published: June 2021

We investigated the effect of maternal separation on neural circuitry activation in response to stress in adulthood. Rats were submitted to maternal separation (180 min/day) during 2 first weeks of life. Two stress protocols were randomly performed at 10–12 weeks of age. 1) Restraint animals were placed into plastic tubes for 20 min or 2) exposed to ether for 10 min. After 90 min of the onset of stress situation rats were euthanized and brains were removed and processed for cFos immunohistochemistry in paraventricular (PVN), amygdaloid medial (AM), supraoptic nucleus (SO) and preoptic area (POA). Cathecolaminergic nucleus of brainstem tyrosine hydroxilase was also co‐localized. Maternal separation increased Fos activated neurons in both stress situations in most of nuclei evaluated, especially after ether stress. The number of neurons Fos positive was 180% higher on Locus Coeruleus (LC), 136% PVN and 170% APO after ether stress when compared to control group. On the other hand on restrain stress was 86% in LC, 125% in PVN, and 56% in APO higher. Maternal separation only affect activation in SO after restraint stress (73%). AM showed a similar response on both stress protocols (about 135% higher). Our data showed that maternal separation induces a heterogenic response on neural circuitry activation and interferes with the neuroendocrine programming early in life.

Financial support CNPq, Fapemig
Effect of 17β‐estradiol on temporomandibular joint nociception in intact and castrated male rats
Authors: Bellinger Larry L , Kramer Phillip R
Published: June 2021

Meal duration (MD) is a biological marker of complete Freund's adjuvant (CFA) induced temporomandibular joint (TMJ) nociception in rats and mice. In female rats the proestrus rise in 17β‐estradiol (E2) attenuates CFA induced TMJ nociception (Endocrin. 150:3680, 2009). We determined if a proestrus increase in E2 would also attenuate CFA TMJ nociception in intact and castrated (CAST) rats (~240g). A artificial E2 cycle had low E2 (~10 pg/ml via Alzet pumps) during diestrus‐1, ‐2; estrus and metestrus and ~70 pg/ml E2 boost during proestrus. The TMJ CFA (15μg/30μl) groups included: intact or CAST with or without E2 and their respective control groups receiving TMJ saline (SAL) The rats (n=8 per group) in the computerized feeders received three artificial E2 cycles and then were TMJ injected with SAL or CFA. Meal size was similar in all groups prior to and following TMJ injections. CFA increased MD (P<0.01) in all groups compared to SAL controls. MD was (P<0.01) longer in the CAST + no E2 + TMJ CFA compared to all other CFA injected groups. The data demonstrate the lack of gonadal steroids increase CFA induced nociception. Importantly, the data show that giving CAST male rats proestrus levels of E2 decreased (P<0.01) nociception when compared to the CAST + no E2 + CFA TMJ group. Proestrus levels of E2 can attenuate TMJ nociception in both female and male rats. NIDCR DE12657‐01 (LLB) and DE07188 (PRK).
BPP‐10c from Bothrops jararaca venom changes behavioral and cardiovascular responses to acute stress exposure
Authors: Ianzer Danielle Alves , Bernardes Isabella , Lima Augusto M , Santos Robson A S , Xavier Carlos H , Fontes Marco A P
Published: June 2021

Bradykinin‐potentiating peptides (BPPs) from Bothrops jararaca venom display strong antihypertensive and bradycardic effects in spontaneously hypertensive rats (SHR). We assessed the contribution of BPP‐10c to locomotor/exploratory activity and cardiovascular reactivity to acute stress exposure. BPP‐10c (71 nmol/Kg) or vehicle (NaCl 0.9%) were i.v. injected in male SHR prior to evaluation of locomotor activity. The behavior was recorded during 210min. Another group was submitted to air jet stress (10L/min‐10 min) at 120min following injections of BPP‐10c or vehicle. Renal sympathetic activity was also measured in anesthetized SHRs. The entire exploratory episodes sampled after BPP‐10c administration were significantly reduced, when compared to vehicle (running: 54±9 vs. 93±12; rearing: 38±7 vs. 79±11; grooming: 2.7±0.4 vs. 5.8±1.4; P<0.05). Despite absence of effects on cardiovascular reactivity to stress, BPP‐10c greatly reduced HR in post stress recovery period (P<0.05 for all). In anesthetized animals, BPP‐10c did not change sympathetic tone sampled from renal nerve. We conclude that BPP‐10c has central effects, modulating behavior and improves recovery of homeostasis after acute stress exposure. Support: CNPq, FAPEMIG and FAPESP.
Reduced Distal Nephron Sodium Reabsorption in Cyp1a1‐Ren2 Transgenic Rats with Inducible ANG II‐Dependent Malignant Hypertension
Authors: Alam Zubia , Mitchell Kenneth D
Published: June 2021

The present study was performed to determine the ANG II‐dependence of distal nephron sodium reabsorption in Cyp1a1‐Ren2 transgenic rats [strain name: TGR(Cyp1a1‐Ren2)] with inducible ANG II‐dependent malignant hypertension. To assess distal nephron sodium reabsorptive function, we compared sodium excretion before and after blockade of the two main distal nephron sodium transporters by intravenous administration of amiloride (AMIL; 25 μg) plus bendroflumethiazide (BFTZ; 25 μg) in pentobarbital sodium‐anesthetized male Cyp1a1‐Ren2 transgenic rats (n=5). Cyp1a1‐Ren2 transgenic rats induced with 0.3% I3C for 8–10 days exhibited a markedly elevated mean arterial pressure (MAP; 185±5 vs. 131±3 mmHg, P<0.001) compared with non‐induced rats (n=5). Administration of AMIL+BFTZ did not alter MAP in either group, but elicited a greater increase in urinary sodium excretion in the non‐induced control rats than in the hypertensive rats (6.36±1.08 vs. 3.13±0.42 μEq/min, P<0.05). Distal nephron sodium delivery was not different between the two groups (7.43±1.41 vs. 5.24±0.86 μEq/min). However, absolute distal nephron sodium reabsorption and fractional reabsorption of distal nephron sodium delivery were markedly lower in the hypertensive rats than in the normotensive rats (3.13±0.42 vs. 6.36±1.08 μEq/min and 63.37±7.86 vs. 87.97±4.58 %, respectively, P<0.05 in both cases). These findings demonstrate that distal nephron sodium reabsorptive function is markedly decreased in Cyp1a1‐Ren2 rats with ANG II‐dependent malignant hypertension. Such impaired distal nephron sodium reabsorptive function suggests that ANG II‐dependent stimulation of distal nephron sodium reabsorption does not contribute substantively to the hypertension in these rats.
ATP mediates flow‐induced nitric oxide production in thick ascending limbs
Authors: Cabral Pablo Daniel , Garvin Jeffrey Lawrence
Published: June 2021

Mechanical stimulation caused by increasing flow induces the release of ATP and other nucleotides. Both luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in the thick ascending limb (TAL). We hypothesized that luminal flow‐induced NO production in TALs is mediated by ATP. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. The rate of increase in fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 17±15 to 130±34 arbitrary units (AU)/min (p < 0.04; n=7). When an ATP scavenging system (hexokinase; 10 U/ml plus 5.5 mM glucose) was added to the luminal perfusate and basolateral bath, luminal flow did not increase NO production (control: 32±5; after ATP scavenging: 19±4 AU/min; n=6). When luminal flow was increased with the ATPase apyrase (5 U/ml) in the luminal perfusate and basolateral bath, NO levels did not change significantly (control: 41±12; after apyrase: 59±11; n=6). The P2 receptor antagonist suramin (300 umol/L) in the lumen and bath reduced flow‐induced NO production by 73% (control: 56±11; with suramin: 13±12; n=5). We concluded that ATP mediates flow‐induced NO production in thick ascending limbs via P2 receptors.

Support: PO1 HL 090550, RO1 HL 070985
Cadmium (Cd)‐induced DNA damage triggers G2/M arrest via chk1/2 and cdc2 in p53‐deficient kidney proximal tubule cells
Authors: Lee Wing‐Kee , Bork Ulrich , Kuchler Anna , Dittmar Thomas , Thévenod Frank
Published: June 2021

Carcinogenesis is a multi‐step process that is frequently associated with p53 inactivation. The class 1 carcinogen Cd causes renal cancer and is known to inactivate p53. G2/mitosis(M) arrest contributes to stabilization of p53‐deficient mutated cells, but its role and regulation in Cd exposed p53‐deficient renal cells is unknown. Using the Comet assay, DNA damage was induced by Cd (50–100 μM) within 1–6 h in p53‐inactivated kidney proximal tubule (PT) cells. This was associated with peak formation of reactive oxygen species (ROS) at 1–3 h, measured with dihydrorhodamine 123, and G2/M cell cycle arrest at 6 h, which were both abolished by the antioxidant α‐tocopherol (100μM). G2/M arrest by Cd was enhanced ~2‐fold upon release from cell synchronization (double thymidine block or nocodazole) and resulted in ~2‐fold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN‐01 (0.3μM) abolished Cd‐induced G2/M arrest, increased apoptosis, and prevented Cd‐induced cyclin‐dependent kinase cdc2 phosphorylation at tyrosine15, shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53‐inactivated PT cells Cd‐induced ROS formation and DNA damage trigger signaling of check‐point activating kinases ATM/ATR to cause G2/M arrest. This may promote survival of pre‐malignant PT cells and Cd carcinogenesis. Funded by DFG TH 345/10–1.
Pregnant Diabetic Mice have Aberrant Blood Pressure Control and Renal Dysfunction Associated with Fetal Growth Restriction and Death
Authors: Burke Suzanne Diana , Barrette Valerie F , Adams Michael A , Croy B Anne
Published: June 2021

Cardiovascular adaptations of pregnancy involve structural and functional changes in the heart and kidneys. Pregnant women with Type 1 Diabetes (T1D) are at high‐risk for developing pregnancy complications. Using radiotelemetry, we sought to investigate the blood pressure regulation of the pregnant T1D non‐obese diabetic (NOD) mouse.

Diabetic NOD mice had a normal drop in blood pressure from gestation day 5–9, but became hypotensive and severely bradycardic during late gestation in comparison to pregnant, normoglycemic NOD mice. These acute hemodynamic changes were accompanied by biochemical and histopathological signs renal failure. Pups born to diabetic dams were growth‐restricted, suggesting placental insufficiency. The paradox of rapid onset renal dysfunction, combined with unexpected hypotension and bradycardia suggests that, in diabetes, pregnancy‐induced renal adaptations accelerate renal disease progression, compromising maternal and fetal circulatory health.

Funded by CIHR, Canadian Foundation for Women's Health and the Canada Research Chairs Program

Grant Funding Source: CIHR
Toad skin sensory mechanisms: correlation between behavioral, neural and electrophysiological responses associated with water absorption.
Authors: Hillyard Stanley D , Willumsen Niels J
Published: June 2021

Dehydrated toads respond to ionic and osmotic properties of water in potential rehydration sources. Thus, when exposed to high NaCl concentrations (>125 mM) the time showing hydration behavior is significantly reduced relative to distilled water (DI). Similarly, hydration behavior time is strongly reduced when the animals are presented with hyperosmotic (500 mM) mannitol. Previous studies with intracellular microelectrodes indicated that exposure of the isolated skin epithelium to Ringer made hyperosmotic with 207 mM mannitol caused a depolarization of the basolateral membrane potential (Vb) of skin keratinocytes similar to that of 250 mM NaCl. Thus, Vb appears sensitive to osmotic as well as ionic stimuli. We investigated the afferent nerve activity of spinal nerve #6 when the ventral skin was exposed to DI water and hyperosmotic solutions known to reduce water absorption behavior. The data indicated that Ringer elicits a neural response significantly greater than DI but comparable to 250 mM NaCl while 500 mM NaCl elicited a substantially larger neural response. Interestingly, the neural responses to the same solutions were often amplified by pre‐treatment with 500 mM mannitol, significantly so with 500 mM NaCl. Our data indicate that the neural response associated with water absorption behavior is determined partly by ionic composition and partly by osmotic strength.
Inhibition of strong midgut alkalinization in larval yellow fever mosquitoes (Aedes aegypti) with HEPES buffer
Authors: Onken Horst , Brandwein Lynsey , Maniscalco Julianna , Mashkulli Medije , Moffett Stacia B , Moffett David F
Published: June 2021

Like some other larval insects, larval mosquitoes generate an extremely alkaline (pH 10–12) compartment in their anterior midgut. The mechanisms of midgut alkalinization are not completely understood. However, digestive enzymes are adapted to this high pH and it is believed that the alkaline environment increases nutrient assimilation. Because larval mosquitoes drink the medium, it could be anticipated that buffering the medium could prevent the animals from alkalinizing the anterior midgut. Alkalinization of the midgut can be studied by addition of the pH indicator m‐cresol purple to the medium and observing the color of the midgut. In larvae raised under control conditions the midguts showed a purple color, indicating midgut alkalinization. When maintained in water containing 25 mmol/l HEPES buffer midgut alkalinization was hardly detectable. When raising larvae in water containing 50 mmol/l HEPES, no alkalinization was observed. In buffered water, the development of larvae through the four instar stages was slower and many larvae did not reach the pupal stage. Financial support by the NIH (1R01AI063463‐01A2) and by a Wagner College Faculty Research Grant is gratefully acknowledged.
Regulation of Cl−/HCO3− Exchange in the Lower Villous Epithelium of Murine Duodenum
Authors: Clarke Lane L. , Walker Nancy M. , Soleimani Manoocher , Liu Jinghua , Brazill Jennifer M. , Hoover Erin E.
Published: June 2021

Previous studies of duodenal lower villous epithelium revealed that Slc26a3 (Dra) was solely responsible for apical membrane Cl−/HCO3− exchange. These studies were performed using luminal glucose to lower intracellular pH for measurements with the pH‐sensitive dye BCECF. However, Na+‐coupled glucose transport also depolarizes the apical membrane and may minimize activity of Slc26a6 (Pat‐1) which is also expressed and reportedly has an electrogenic 1Cl−:2HCO3− stoichiometry. To investigate regulation of Cl−/HCO3− exchangers, the effects of luminal glucose and cAMP stimulation (to activate CFTR) on the rates of Cl−in/HCO3−out exchange in Pat‐1 knockout (KO), DraKO and wild‐type (WT) littermate mice were measured. In the absence of luminal glucose (replaced with mannose), Cl−/HCO3− exchange was reduced ~50% in both the Pat‐1KO and DraKO, as compared to WT. With luminal glucose, Cl−/HCO3− exchange was unaffected in the Pat‐1KO and abolished in the DraKO. cAMP stimulation reduced Cl−/HCO3− exchange by 50% in WT and DraKO villi, but had no effect on Pat‐1KO villi. We conclude that Dra and Pat‐1 contribute to basal Cl−/HCO3− exchange, whereas regulation by luminal glucose or cAMP stimulation reduces activity of Pat‐1 but not Dra in duodenal lower villous epithelium. These findings are consistent with electroneutral Dra and electrogenic Pat‐1 Cl−/HCO3− activities. Supported by NIH.
Regulation of V‐ATPase traffic in submandibular gland through Rab GTPases
Authors: Oehlke Oliver , Martin Henno , Osterberg Nadja , Roussa Eleni
Published: June 2021

Redistribution of acid‐base transporters is a crucial regulatory mechanism for many cells to cope with extracellular pH changes. Moreover, in epithelial cells, translocation of acid‐base transporters ultimately leads to changes in vectorial transport of H+ and HCO3−. However, the specific proteins involved in regulated vesicular traffic of acid‐base transporters in epithelial cells are largely unknown. In the present study we have investigated the impact of members of the Rab family of small GTPases on the acidosis‐induced trafficking of the vacuolar‐type H+‐ATPase (V‐ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. We demonstrate distinct subpopulations of intracellular V‐ATPase pools in HSG cells. We also show differential co‐localization of V‐ATPase with Rab family members. Extracellular acidosis causes translocation of the V‐ATPase from the Golgi apparatus toward the plasma membrane, a process mediated by PKA and Src kinases. Loss‐of‐function experiments using specific siRNA against distinct Rab family members prevent acidosis‐induced V‐ATPase translocation. The data introduce members of the Rab family as crucial regulators of acidosis‐induced V‐ATPase traffic in duct cells of submandibular gland. Funded by the Deutsche Forschungsgemeinschaft.
Mitochondrial fragmentation causes cellular acidification in the nematode C. elegans
Authors: Johnson David , Nehrke Keith
Published: June 2021

Mitochondrial structural dynamics (MSD) occur through the opposing processes of membrane fission and fusion. Diseases such as autosomal dominant optic atrophy and Charcot‐Marie‐Tooth syndrome Type 2A result from haploinsufficiency at genetic loci whose gene products regulate MSD and null mutations in these genes are lethal in mice. However, the nematode C. elegans tolerates a complete loss of fission and fusion proteins, and the physiologic consequences of MSD loss‐of‐function (lf) can be studied in a whole animal genetic model. Dynamic fluorescent imaging of genetically‐encoded biosensors in live transgenic worms indicates that mitochondrial fragmentation caused by mutation of the profusion gene eat‐3 (Opa1 in man) induces cellular acidification in multiple tissues. Epistasis analysis further suggests that fragmentation itself rather than an unrelated protein function is causative. The results of dichloroacetate supplementation experiments demonstrate that it is unlikely that the acidosis is caused by lactate accumulation, while AMP/ATP ratios suggest that basal energy balance is conserved in MSD mutants. However, using a redox‐sensitive fluorescent probe we have found that the mitochondrial matrix in eat‐3lf mutants is significantly oxidized compared to that of wildtype worms. We are currently exploring the hypothesis that reactive oxygen species play a role in the acidification. Support: USPHS F31GM084506 (DWJ).
The role of hyperglycemia on cardiac fibroblast function: proliferation and ERK activation
Authors: Shamhart Patricia E. , Luther Daniel J. , Raman Priya , Meszaros J Gary
Published: June 2021

Cardiac fibroblasts (CFs) are critical mediators of remodeling and upon activation secrete extracellular matrix, proliferate, migrate, and differentiate to the hypersecretory myofibroblast phenotype. Myofibroblasts are significant regulators of wound healing, a process that is compromised in diabetic patients. The MAPK pathway is a classical proliferation pathway in many cell types including CFs as previously reported by our laboratory. The goal of this study was to determine the influence of hyperglycemia on CFs with a particular focus on proliferation and ERK activation. Our previously published study reported that fibroblasts isolated from 6 week streptozotocin‐induced type 1 diabetic hearts had a 15.6% higher proliferation rate versus those from control hearts. In this study we utilized hyperglycemic media (25 mmol/l) to mimic diabetes in vitro. Surprising, short term hyperglycemia significantly attenuated basal ERK activation: ERK phosphorylation decreased to 14.8 ± 4.8% control in CFs treated for 20 minutes with high glucose and 6.4 ± 2.7% control at 1 hour. We also observed that short term high glucose incubation slightly attenuated angiotensin II induced ERK phosphorylation. These data suggest high glucose attenuates basal ERK activation and hyperglycemia‐induced CF proliferation may be independent of the classical MAPK pathway.
Luteotropic Hormone (LH) effects on T‐type calcium currents and intracellular calcium transients are mediated by PKA in mice Leydig cells
Authors: Costa Roberta Ribeiro , Aguiar Jose Fernando , Varanda Wamberto Antonio
Published: June 2021

Previous results from our laboratory have shown that LH is able to increase the intracellular Ca2+ concentration ([Ca2+]i) in Leydig cells, a process triggered by calcium entry through a T‐type Ca2+ channel. Here we describe the effects of modulators of PKC and PKA on the Ca2+ currents, measured with the patch clamp technique, and on the overall [Ca2+]i transients with fluorescence detection. Treatment of the cells with LH (1 μg/ml) increased the peak current at −20 mV from −2.4 ± 0.7 to −3.8 ± 0.8 pA/pF (n = 22, p = 0.0381). PMA (1 μM) increased the peak current to −3.6 ± 0.4 pA/pF (n = 6, p = 0.00033). Only minor changes were observed in the voltage dependence of activation, inactivation or deactivation of the currents. Experiments with the calcium sensitive dye Fluo‐3, showed that inhibition of both PKC and PKA with 400 nM staurosporine blocks the [Ca2+]i changes induced by LH (n = 37 cells). The specific inhibitor of PKA, H89 (10 μM) abolished the LH induced rise in [Ca2+]i (n = 48 cells). PMA slowly increases the fluorescence, but the subsequent addition of 1 μg/ml LH still triggers the typical [Ca2+]i transient showing that block of PKC alone does not inhibit the effect of LH. Chelerythrine, a PKC inhibitor, also does not avoid the Ca2+ transients. Taken together, our results support the conclusion that PKA plays a leading role in mediating the LH induced intracellular Ca2+ changes.

Financial Support: FAPESP, CNPq
Hypertonic stress disrupts cellular protein homeostasis and causes rapid protein damage in vivo
Authors: Burkewitz Kristopher , Choe Keith P. , Strange Kevin
Published: June 2021

It has long been assumed from in vitro studies that hypertonic stress (HS) disrupts protein structure and function. However, there is little in vivo evidence to support this hypothesis. We recently reported that protein degradation genes are essential for survival of C. elegans in hypertonic environments and that HS induces aggregation of the fluorescent protein Q35::YFP. We characterized the kinetics and cell biology of Q35::YFP aggregation by time‐lapse confocal microscopy. Q35::YFP aggregates accumulate in the cytosol and between myofilaments of muscle cells. Aggregates form within 1 h after HS, develop at a rate of 10 aggregates/worm/h, and double in volume every 15 to 30 min. Protein folding per se is also disrupted by HS in vivo. Temperature‐sensitive (ts) point mutations give rise to misfolded proteins and mutant phenotypes at high but not low or “permissive” temperatures. We characterized the phenotypes of worms harboring ts mutations in paramyosin and ras. Exposure to high temperature alone or HS at permissive temperatures caused identical mutant phenotypes indicating that cellular water loss disrupts in vivo protein folding. These and our previous findings demonstrate that HS causes rapid protein damage that must be ameliorated to ensure survival. Our results have important implications for understanding how cells respond to HS and how these response mechanisms may be impacted by disease and aging.

This work was supported by NIH grant DK61168.
Nanoparticle Internalization and Transport by Caveolae
Authors: Wang Zhenjia , Tiruppathi Chinnaswamy , Minshall Richard D , Malik Asrar B
Published: June 2021

Caveolae are flask‐shaped membrane invaginations in endothelial cells involved in uptake of macromolecules such as albumin and viral particles. It is unknown whether caveolae can internalize nanoparticles. Here we investigated uptake of nanoparticles of different sizes ranging from 20 nm to 100 nm in endothelial cell monolayers. We observed that BSA (bovine serum albumin) coating of nanoparticles was required for the internalization of nanoparticles and the uptake is dependent on particle size. Co‐localization of caveolae staining and BSA‐coated nanoparticles indicated nanoparticles were internalized in endothelial cells via caveolae. Using a nanoruler made of a dual‐color nanoparticle pair to measure caveolae size and assembly dynamics in live endothelial cells, we found that only nanoparticles less than caveolae size were internalized. Uptake of nanoparticle cargo in caveolae was followed by its transport across the endothelial monolayer via caveolae. Transport was dependent on size of nanoparticles; e.g., transport of 40 nm particles was 2‐fold greater than 100 nm particles. The present results suggest that nanoparticles of a particular size (~40nm diam) coated with albumin provide an efficient means of delivering therapeutic proteins across the vascular endothelial barrier.
Serotonin‐induced vascular contractions are regulated by rhoA localization within caveolar microdomains
Authors: Lamping Kathryn G , England Sarah K , Nuno Daniel W
Published: June 2021

RhoA is compartmentalized within caveolae in vascular tissue to regulate activation. We hypothesized that in the absence of caveolin‐1 (cav‐1), a requirement for caveolar formation, localization of rhoA is disrupted increasing activation of rhoA and its downstream effector, rho kinase (ROCK) and enhancing ROCK‐dependent contractions. These studies compared targeting of rhoA and ROCK to caveolar microdomains in aorta from wild‐type (WT) and cav‐1 deficient mice (cav‐1 KO) before and after contraction to serotonin (1 uM). In WT, rhoA and ROCK were primarily localized in high‐density sucrose gradient fractions. Following serotonin, rhoA and ROCK expression increased in low‐density cav‐1 containing fractions. In the absence of cav‐1, rhoA and ROCK no longer compartmentalized in aortic membranes in the presence or absence of serotonin. In agreement with these findings, contractions to serotonin were increased in aortic rings from cav‐1 KO mice compared to WT (serotonin 1uM WT 0.70±0.11g; cav‐1 KO 1.02±0.06g, p<0.05). The increased contractions were inhibited by H1152 (1 uM), a specific ROCK inhibitor. We conclude that localization of rhoA in caveolar microdomains impedes its activation and attenuates contractions to rho‐dependent agonists. Disruption of rhoA localization within microdomains may lead to increased activation in cardiovascular disease. (Support: Dept Veterans Affairs and HD37831)
Leptin Signaling Through the PI3K‐PDE3B Pathway is Impaired During Chronic Hypoxic Exposure
Authors: Litvin David G , Tsipis Constantinos P , Puchowicz Michelle C , LaManna Joseph C
Published: June 2021

Background

Leptin signaling in the brain plays a crucial role in energy homeostasis. Recent evidence suggests that in addition to JAK2‐STAT3, leptin activity is also mediated through an insulin‐like signaling pathway that relies on PI3K and PDE3B. Recent work indicates this pathway becomes deregulated during leptin resistance. We hypothesized that exposure to chronic hypoxia, a trigger of leptin release, may alter leptin signaling, thereby producing leptin resistance.

Objective

To investigate whether chronic hypoxic exposure alters central leptin signaling.

Methods

male C57BL/6 mice were exposed to either hypobaric hypoxia at a pressure of ~300 torr or normoxic conditions for 7 or 21 days. PDE3B and pSTAT3 positive cells were identified immunohistochemically. Central leptin levels were measured by western blot.

Results

Robust expression of pSTAT3 & PDE3B was observed at day 7 of chronic hypoxic exposure compared to normoxic controls. At 21 days exposure, pSTAT3 staining appeared indistinguishable from 7‐day samples. Conversely, PDE3B staining appeared markedly decreased when compared with 7‐day PDE3B positive samples, but still above normoxic controls.

Conclusions

These findings suggest chronic hypoxia may impair PI3K‐PDE3B signaling. PDE3B may mediate the mechanism leading to leptin resistance induced by sustained leptin release during chronic hypoxic exposure.

Grant Funding Source: NIH Grant NS 38632
An Amphipathic Alpha‐Helical Domain Identified in Nucleoporins Plays a Role at The Nuclear Pore Complex of Saccharomyces Cerevisiae
Authors: Lazarus Michelle Denise , Natarajan Paramasivam , Wente Susan
Published: June 2021

All macromolecular transport between the nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) embedded in the nuclear envelope. NPCs are composed of multiple copies of proteins, termed nucleoporin (Nups) which assemble sequentially into nascent nuclear pores generated following fusion of the inner and outer nuclear membranes. While much is known about NPC structure, the mechanisms governing NPC biogenesis remain largely undefined. Recently, an amphipathic α‐helix (ALPS for ArfGAP1 lipid packing sensor) motif, which mediates protein association with highly curved liposomes, has been defined. This ALPS motif was identified in protein components of two early associating S. Cerevisiae (sc) NPC subcomplexes; the scNup84 subcomplex (specifically Nup85 and Nup120) and the scNup170 subcomplex (specifically Nup170 and Nup188) and may help proteins associate with newly developing NPCs. Utilizing microscopy techniques and yeast genetics we have identified an important role for the ALPS domain in vivo and our evidence suggests that ALPS domains of different NUPs have redundant functions. Utilizing in vitro liposome binding assays we find that the ALPS domain is sufficient for liposome binding and that this binding is dependent on liposome size. Together, this data provides the first evidence for an in vivo function of the ALPS motif with an impact in the broad membrane biology field, and concomitantly expands our understanding of NPC assembly.

Grant Funding Source: IRACDA & RO1
Distribution Patterns of Soft and Hard Keratin Proteins in Finger Nails, Cat Claws, and Horse Hooves
Authors: Bragulla Hermann H. , Foster Antoine L. , Stokes Ashley M. , Lopez Mandy J. , Homberger Dominique G.
Published: June 2021

Nails, claws, and hooves are the three basic forms of digital end organs that cover the tip of a digit and serve various primary functions, e.g., palpation, catching prey or weight bearing. Digital end organs are complex structures with varying growth rates and mechanical properties to meet locally defined functional demands. Different segments of digital end organs vary in their type of keratinization, but the specific keratin proteins have not yet been determined. In the present study, the keratin proteins were studied in digital end organs using a fractionated extraction method. The extracted keratin proteins were separated on SDS‐PAGE gels, transferred to nitrocellulose or PVDF paper and blotted using an array of monoclonal and polyclonal antibodies. The distribution of the keratin proteins in the digital end organs and their possible influence on the mechanical properties of digital end organs are discussed to establish the base‐line information needed to be able to identify pathologies afflicting digital end organs. Understanding the segment‐specific synthesis of keratin proteins in digital end organs in situ is a prerequisite for studying their keratinization process in cell culture.
The involvement of CD2AP and endocytic proteins during enteropathogenic E. coli (EPEC) pedestal formation
Authors: Guttman Julian Andrew , Lin Ann E. , Veiga Esteban , Cossart Pascale , Finlay B Brett
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that generate actin‐rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections and their efficient formation in vitro routinely requires the phosphorylation of the EPEC effector protein Tir at Tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474 that is utilized for actin nucleation through a downstream N‐WASP/Arp2/3‐based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis‐associated and cortactin binding protein, is a novel and important component for EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck then clathrin prior to actin polymerization at pedestals during the Nck‐dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.

Grant Funding Source: CIHR, NSERC
Spectrin is a crucial component of EPEC pedestals
Authors: Ruetz Tyson Joel , Guttman Julian
Published: June 2021

Enteropathogenic E.coli (EPEC) is one of the main pathogens contributing to roughly 2 million diarrhea related child deaths worldwide each year. Upon ingestion of contaminated food or water, EPEC colonize the intestinal tracts of their hosts, attach to the surface of enterocytes and generate characteristic attaching and effacing (A/E) lesions. These lesions involve the collapse of host microvilli and the generation of pedestal‐like structures atop infected cells. Pedestal formation and disease progression is induced by bacterial proteins (effectors) that are translocated into the host cell cytoplasm by a syringe‐like type‐three secretion system (T3SS). The three major components of the host cell cytoskeleton; actin, microtubules and intermediate filaments, are all altered during EPEC pathogenesis. We have identified another major host cytoskeletal network that is involved in EPEC disease, the spectrin cytoskeleton. Here we demonstrate that spectrin is recruited to the base of EPEC pedestals and through the use of EPEC effector mutants, we show that this localization is mediated by the translocated intimin receptor (Tir). Spectrin knockdowns using siRNA have revealed that spectrin is necessary for pedestal formation to occur. Our findings provide the first evidence of spectrin's involvement in EPEC pathogenesis and identify it as a crucial protein involved in pedestal formation and disease progression.

Grant Funding Source: CIHR, NSERC
Inflammation inhibits patterning but not dedifferentiation in amphibian limb regeneration
Authors: Mescher Anthony Louis , Neff Anton W. , King Michael W.
Published: June 2021

The ability of larval anuran limbs to regenerate is lost as larvae develop, grow and approach metamorphosis. Our previous microarray analyses of gene expression and proteomic analysis of protein levels in developing Xenopus hindlimbs after amputation indicate that events of tissue inflammation and its resolution are more strongly up‐regulated and persist longer at the later, regeneration‐incompetent stages. This finding raises the possibility that such inflammation interferes with important aspects of limb regeneration, leading to the ontogenic loss of regenerative capacity. We have tested this hypothesis using a classic inhibitor of amphibian limb regeneration, beryllium. Exposure of freshly amputated, regeneration‐competent Xenopus limbs to aqueous solutions of beryllium exacerbates the local inflammatory response, as well as inhibiting regeneration. PCR shows that local expression of genes related to both inflammation and resolution of inflammation are more strongly and more persistently up‐regulated after beryllium treatment. Treated limb stumps exhibit normal expression levels of markers for cellular dedifferentiation and reprogramming, but fail to express genes important for patterning of the regeneration blastema. Our data support the hypothesis and show that although inflammation has little effect on dedifferentiation, it can inhibit certain aspects of patterning that are required for normal limb regeneration. This work was supported by National Science Foundation Grant IOS‐0814399.