bluuebunny/crossref_metadata_2025 huggingface dataset:
Teaching Renal Physiology in the Laboratory with Case Studies
Authors: Goodman Barbara Eason
Published: June 2021

Students from middle school through undergraduates appreciate learning about diseases with the Kidney Under Pressure unit. The unit was written for Frontiers in Physiology of the American Physiological Society by Dr. Jeffrey Osborn and colleagues at the Medical College of Wisconsin. It is being modified and field‐tested for web publication by the Education Office of the APS. I have repeatedly used parts of the unit in my own teaching and outreach activities. The unit includes patient note cards for 8 patients with different kidney diseases (renal failure following long‐term hypertension, diabetic nephropathy, severe dehydration, kidney stones, kidney infection, renal failure caused by congestive heart failure, trauma‐induced acute renal failure, and polycystic kidney disease). Simulated urine is prepared with glucose and/or protein and can be easily analyzed with urine test strips. In addition, cytology slides are available of a smear of the patient's urine. Students are in small groups of 3‐4 and fill out a patient health chart. They may request various laboratory tests to evaluate the symptoms. After obtaining additional information, their task is to diagnose the disease. Their written report includes the evidence that they obtained to lead them to choose the particular disease and their diagnosis. Another section asks them to describe in detail the disease that their patient had and the physiological problems caused by the disease. They are asked to reflect on what they learned in the laboratory. Each group presents their patient to the rest of the class in a mini grand rounds format and there is discussion about what they found and what it means. Students of all ages enjoy the "playing doctor" aspects of this activity. If resources about kidney diseases are provided, they justify their diagnoses carefully. Students comment on how much fun the activity is and how it helped them learn about the causes of kidney problems.
Neuropeptide Y concentration within sympathetic nerve endings of skeletal muscle arterioles
Authors: Evanson Kirk Wendell , Stone Audrey J , Kluess Heidi A
Published: June 2021

The purpose of the study was to determine neuropeptide Y (NPY) concentration within sympathetic nerve endings of gastrocnemius 1A arterioles. Arteriole segments were dissected from the gastrocnemius muscle of male and female F‐344 rats. The 2mm segments were then placed in 400 μl of Krebs‐Ringer buffer and pulverized with a pestle for three minutes. The samples were centrifuged for 15 minutes to remove vascular debris. A competitive peptide enzyme immunoassay (EIA) was used for NPY quantification. Analysis revealed .56±.04 ng/ml and .49 ng/ml in male and female gastrocnemius 1A arterioles, respectively. The intra‐assay coefficient of variation was 5%. These data suggest that NPY levels within gastrocnemius 1A arterioles are substantive in concentration to quantify using this competitive peptide EIA. This research was supported by the Arkansas Biosciences Institute and the National Institute on Aging.
Global analysis of miRNA expression in endothelial cells under different flow patterns
Authors: Wang Kuei‐Chun , Li Yi‐Shuan Julie , Nguyen Phu , Chun Jennifer , Chien Shu
Published: June 2021

Fluid shear stress plays an important role in regulating functions of vascular endothelial cells (ECs). Focal nature of atherosclerotic plaques reveals that pulsatile flow (PS) along the straight part of arterial tree has athero‐protective effects, whereas oscillatory flow (OS) at bifurcations induces pro‐atherosclerotic responses. MicroRNAs (miRNAs) affect post‐transcriptional modulation of stability of target mRNAs; there is evidence that miRNAs may modulate EC functions. Using microfluidic microarray (µParafloTM), we profiled miRNA expression levels in human ECs under 24‐hr PS and OS flows, as well as static control. 24‐hr PS flow caused significant changes in expression of 42 miRNAs (12 up‐regulated, 30 down‐regulated) in ECs in comparison with static cells. We also found that 24‐hr OS flow caused differential expressions of 68 miRNAs (42 up‐regulated, 26 down‐regulated) in comparison to ECs under 24‐hr PS flow. Many of these miRNAs have been shown to regulate cell cycle and inflammatory proteins. Our results demonstrated that miRNA expression level can be differentially regulated by different flow patterns, consequently modulating EC functions related to proliferation and inflammation.
Human internal mammary and radial arteries express different level of endothelial nitric oxide synthase (eNOS)
Authors: FAN Li , GROVE Kevin L , WEI Wei , FURNARY Anthony , YANG Qin , HE Guo‐Wei
Published: June 2021

The internal mammary artery (IMA) has better long‐term patency rate than the radial artery (RA) after coronary artery bypass grafting surgery with unknown mechanisms. We investigated endothelial nitric oxide synthase (eNOS) in these arteries taken from redundant parts during surgery. Cumulative concentration‐relaxation curves for acetylcholine (ACh, ‐9 ~ ‐4.5 log M) against U46619 were established in IMA and RA rings pretreated with/without eNOS inhibitor Nω‐nitro‐L‐arginine (L‐NNA, 300 μM). Expression levels of eNOS in the whole tissue, as well as in the endothelial cells (EC) and smooth muscle cells (SMC) of human IMA and RA were quantified with quantitative real‐time PCR (qRT‐PCR). Immunohistochemistry (IHC) technique was also adopted to determine the localization of eNOS. Without L‐NNA, ACh‐induced relaxation was similar between IMA and RA rings (Rmax: 48.5 ± 5.2% vs 55.9 ± 6.0%, n = 8, 9). L‐NNA pretreatment significantly inhibited Rmax in IMA (14.7 ± 3.0%, p < 0.001) and in RA (15.2 ± 3.2%, p < 0.01). eNOS mRNA expression level in integrated tissues was similar in IMA and RA, but significantly higher in EC of IMA than in RA (1.03 ± 0.19 vs 0.53 ± 0.09, p < 0.05, n = 7). In addition, IHC showed that eNOS immunoreactivity was higher in EC of IMA than that in RA. Thus, the EC of IMA has higher eNOS expression than that in RA. This may be the cause of better long‐term patency of IMA.

Supported by St. Vincent Medical Foundation.
ATP‐mediated vasodilation is not reduced in aging humans
Authors: Kirby Brett Sean , Voyles Wyatt F , Crecelius Anne R , Dinenno Frank A
Published: June 2021

Endothelium‐dependent vasodilation is reduced with age, as evidenced by blunted vasodilator responsiveness to acetylcholine (ACH) infusions. Circulating adenosine triphosphate (ATP) has been implicated in the control of skeletal muscle vascular tone during mismatches in oxygen delivery and demand (e.g., exercise) via binding to purinergic receptors on the endothelium, and aging is typically associated with reductions in muscle blood flow under such conditions. Therefore, we tested the hypothesis that ATP‐mediated vasodilation is impaired in aging humans and that ascorbic acid (AA) infusion would acutely restore this impairment. We measured forearm blood flow (FBF; venous occlusion plethysmography) responses to local intra‐arterial infusions of ACH, ATP, and sodium nitroprusside (SNP) with and without AA infusion in 13 young and 13 older adults. The peak increase in FBF to ACH was significantly impaired in older compared with younger adults (260±70% vs 620±99%; P<0.05), and this difference was abolished during AA infusion (520±80% vs 560±70%). Peak FBF responses were not different between older and young adults to either ATP (675±105% vs 735±125%) or SNP (1120±110% vs 1140±150%) and AA infusion did not alter these responses (both NS). Thus, in contrast to the observed impairments in ACH responses, the vasodilatory response to exogenous ATP is not reduced with age in healthy humans.
Extracellular Superoxide Dismutase Activity is Reduced with Aging in Humans: Relation to Impaired Vascular Endothelial Function and Exercise Capacity
Authors: LaRocca Thomas J. , Seals Douglas R. , Walker Ashley E. , Eskurza Iratxe , Pierce Gary L.
Published: June 2021

Aging is associated with impaired exercise capacity and vascular endothelium‐dependent dilation (EDD), the latter mediated by oxidative stress and reduced nitric oxide (NO ?) bioavailability. The antioxidant enzyme extracellular superoxide dismutase (EC‐SOD) binds to the endothelial wall via a heparin binding domain and protects NO ? from inactivation by superoxide (O2 ?−). However, the effects of aging on EC‐SOD and its relation to EDD and exercise capacity are unknown. Two groups of healthy non‐obese adults were studied: young sedentary (YS: n = 7; age 22 ± 2 yr; 2M) and older sedentary (OS: n = 12; 62 ± 2 yr; 6M). EDD (brachial artery flow‐mediated dilation) was lower (P < 0.05) in OS (5.4 ± 0.7 Δ%) vs. YS (7.4 ± 0.9 Δ%). EC‐SOD activity, measured by the release of EC‐SOD into plasma following intravenous heparin bolus (5000U), also was lower (P < 0.01) in OS vs. YS (AUC15min: 5.4 ± 1.4 vs. 14.0 ± 1.7 U/mL/min). EC‐SOD activity was inversely related to age (r = ‐0.61, P < 0.01) and positively related to EDD (r = 0.64, P < 0.01) and exercise capacity (maximal oxygen consumption, r = 0.50, P < 0.05). EC‐SOD activity was inversely related to plasma oxidized low‐density lipoprotein (r = ‐0.44, P < 0.05), a circulating marker of oxidative stress, but not to any other anthropometric or metabolic risk factor. These results provide the first evidence that EC‐SOD activity is reduced with aging in healthy humans and may contribute to oxidative stress‐associated vascular endothelial dysfunction and impaired exercise capacity.

NIH AG000279, AG013038, AG022241, AG006537, RR00051
Role of delta‐opioid and adenosine receptors in preventing NMDA receptor dependent excitotoxicity in anoxic turlte cortex
Authors: Buck Leslie Thomas , Pamenter Matthew Edward
Published: June 2021

Delta‐opioid (DOR) and adenosine A1 (A1R) receptor activation are neuroprotective against short‐term anoxic insults in mammalian brain. Protection may be conferred by inhibition of N‐methyl‐D‐aspartate receptors (NMDARs); activation of which leads to excitotoxic cell death (ECD). In anoxic turtle cortex NMDAR activity decreases 50% and ECD is avoided. DORs and A1Rs are expressed in turtle brain but their roles in anoxic turtle brain NMDAR regulation have not been investigated. DOR blockade with naltrindole potentiated normoxic NMDAR currents by 78%, and increased [Ca2+]c 13%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated 70%, and [Ca2+]c increased 5‐fold above anoxic controls. The naltrindole‐mediated depolarization and increased [Ca2+]c were prevented by NMDAR antagonism or by perfusion of the Gi protein agonist mastoparan, which also reversed the naltrindole‐mediated potentiation. Normoxic agonism of A1Rs with CPA decreased NMDAR currents by 58% but antagonism during anoxia with DPCPX did not prevent the anoxia‐mediated decrease. The Gi protein inhibitor pertusis toxin (PTX) prevented both the CPA and anoxia‐mediated decreases in NMDAR currents. Our results suggest that the long‐term anoxic decrease in NMDAR activity is activated by a PTX‐sensitive mechanism that maybe be related by DORs but is independent of A1Rs.
Chemosensory properties of the amphibian skin epithelium
Authors: Hillyard Stanley D. , Willumsen Niels J.
Published: June 2021

Toad skin has chemosensory properties analogous to mammalian taste buds enabling assessment of the chemical composition of potential hydration sources in the environment. Thus, dehydrated toads rejected 250 mM NaCl given as a rehydration medium which correlates with a burst of activity in spinal nerve 6. We hypothesized that the neural activity is initiated by depolarization of the basolateral membrane potential (Vb) of skin keratinocytes. We addressed this by combining behavioral studies on dehydrated toads with Ussing chamber studies of isolated skin epithelia using intracellular microelectrodes. As predicted, increasing the external NaCl concentration from 112 to 250 mM depolarized Vb by 16.5±10.2 mV. Furthermore, the depolarization of Vb was reduced and hydration time prolonged by amiloride. In contrast, toads given 250 mM Na‐gluconate showed longer periods of hydration behavior relative to 250 mM NaCl which was associated with transient hyperpolarization of Vb by 6.5±5.6 mV followed by a sustained depolarization of 11.6±8.9 mV. Neither the behavioral nor the above responses of Vb were affected by amiloride. Exposure to Ringer made hyperosmotic with mannitol caused a transient hyperpolarization of Vb followed by depolarization that was similarly not affected by amiloride, suggesting the amiloride‐insensitive components of Vb and behavior result from an osmotic response of the epithelial cells.
Survival of a Choanoflagellate Following Acute Exposure to Abiotic Stressors
Authors: Rodriguez Sara Scarlet , Julian David
Published: June 2021

Choanoflagellates are the unicellular antecedents of animals. Among them, the Acanthoecidae are a diverse group found in aquatic habitats that can contain steep clines of temperature, salinity, oxygen, and hydrogen sulfide (H2S). It is unclear whether acanthoecids in general are tolerant of many stressors, or if they exhibit endemism, with individual species being adapted to some stressors.We tested stress tolerance of the acanthoecid Diaphoneca grandis after acute 24h changes in temperature, pH, salinity, O2, and H2S under normoxia and anoxia. Survival was assessed using morphology, propidium iodide labeling to indicate loss of plasma membrane integrity, and phosphatidylserine (PS) externalization. Compared to control conditions (0 H2S, 10 °C, 33 ppt salinity, pH 8.0, 21% O2), viability was significantly decreased at 0.11‐3.3 mM H2S, =20 °C, =20 or =50 ppt salinity, pH =7.7 or =8.3 and 0.21‐2.1% O2 but not anoxia. When H2S exposure was coupled with anoxia, cell viability was higher than under normoxia. PS externalization was significantly increased following exposure to high temperature, hypersalinity, alkaline pH, and H2S, which may indicate multiple cell death pathways. The tolerance range of D. grandis to each abiotic stressor was substantially less than that predicted by the maximum ranges of acanthoecids as a group, suggesting that the high stress tolerance of acanthoecids results from endemism.
HIF signaling during molting in Crustacea
Authors: Miller Wyatt A. , Terwilliger Nora B. , Towle David W.
Published: June 2021

Increasing evidence has demonstrated HIF regulation of gene activity in response to both hypoxia and non‐hypoxic stimuli. Normoxic upregulation of vertebrate HIF alpha has been reported during tissue reconstruction, vascularization, and the immune response, events also integral to the crustacean molt cycle. This study investigated the potential role of HIF alpha in mobilizing genes during molting in normoxic crabs. In premolt, the epidermis underlying the exoskeleton hypertrophies, hemocytes and reserve cells proliferate, cryptocyanin increases, and selected muscle proteins atrophy. New exoskeleton secretion occurs from premolt through postmolt. Tissue growth, vascularization and sclerotization begin in postmolt. A cohort of juvenile Dungeness crab Cancer magister was sampled daily for an entire molt cycle. Analysis of HIF alpha, arthrodial membrane protein 6, anti‐lipopolysaccharide factor, cryptocyanin 2 and B‐actin mRNA expression was carried out via real‐time quantitative PCR using SYBR GreenER qPCR SuperMix, ABI Prism 7700 instrumentation (Applied Biosystems) and non‐degenerate primers. Each gene showed a specific pattern of translational upregulation consistent with its putative role in molting. The changes in HIF alpha transcript support the hypothesis that HIF alpha is upregulated in normoxic crabs to coordinate gene expression during molting. Supported by MDIBL NIA (NBT).
Activation of AMP‐activated protein kinase (AMPK) increases phenylephrine mediated contraction in murine corpus cavernosum
Authors: Labazi Hicham , Wynne Brandi M. , Webb R. Clinton
Published: June 2021

AMP‐activated protein kinase (AMPK) is a serine/threonine kinase that was shown to phosphorylate eNOS at ser1177 and also at thr495. Although the role of AMPK in the vascular system has been the recent subject of much investigation, its role in erectile tissue has yet to be defined. Using a pharmacological approach, our objective was to elucidate its role in the corpus cavernosum. A concentration response curve (CRC) to 5‐aminoimidazole‐4‐carboxamide‐1‐b‐riboside (AICAR), a known AMPK activator, in phenylephrine (Phe) contracted corpus cavernosal strips did not cause relaxation, even at high concentrations (3mM). However, with AICAR (1mM) pre‐incubation, an increase in phenylephrine‐mediated contraction was observed compared to control (1.22±0.06 vs. 0.71± 0.03). The CRC to U‐46619 (a thromboxane receptor agonist) and endothelin‐1 did not show any significant difference in contraction between the two groups. However, with AICAR pre‐incubation, there was a significant attenuation of acetylcholine‐mediated relaxation (57.17± 2.87 % in Aicar vs. 71.66± 2.53% in control) and also relaxation mediated by sodium nitroprusside (43.24± 2.59 % in Aicar vs. 60.01± 2.47% in control). We speculate that activation of AMPK in penile tissue leads to phosphorylation of eNOS at Thr 495 producing inactivation of eNOS. Thus, NO bioavailability is reduced resulting in decreasing relaxation and increasing contraction.
Activation of Vinculin Induced by Cholinergic Stimulation Regulates Tension Development in Tracheal Smooth Muscle (TSM)
Authors: Huang Youliang , Craig Susan W. , Gunst Susan J.
Published: June 2021

The adhesion protein vinculin functions in focal adhesions to link actin filaments to membrane adhesion junctions by binding to the integrin‐binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). In the inactive (closed) state, Vh and Vt bind to each other, masking the binding sites for actin and talin; in the active (open) state, vinculin binding sites for talin and actin are exposed. We hypothesized that the contractile activation of TSM might regulate conformation of vinculin and thereby contribute to the regulation of tension development. Stimulation of TSM tissues with acetylcholine (ACh) increased the co‐immunoprecipitation of vinculin and talin from tissue extracts, and increased vinculin phosphorylation at tyr 1065, an indicator of vinculin activation. Recombinant Vh (EGFP‐Vh) expressed in TSM tissues competed with endogenous vinculin for binding to talin and inhibited vinculin tyr 1065 phosphorylation. Expression of EGFP‐Vh depressed TSM contraction to ACh by 60% and inhibited ACh‐induced actin polymerization. EGFP‐Vh did not affect myosin light chain phosphorylation. We conclude that vinculin is activated in TSM by ACh, and that vinculin regulates tension by promoting connections between actin filaments and talin‐integrin adhesion complexes and by facilitating the initiation of actin polymerization.

Support: AHA 0820074Z, HL 29289, HL 074099
Change in Calcium and Contractile Responses of Middle Cerebral Artery from Stroke‐prone Spontaneously Hypertensive Rats
Authors: Hashimoto Terumasa , Nobe Koji , Ohata Hisayuki , Honda Kazuo
Published: June 2021

In the present study, we have compared the calcium and contractile responses of middle cerebral artery from stroke‐prone spontaneously hypertensive rats (SHR‐SP) and Wistar‐Kyoto rats (WKY) utilizing confocal laser microscopy. 5‐hydroxytryptamine (5‐HT) ‐induced [Ca2+]i and contractile responses in SHR‐SP at 3 months of age were significantly greater than those in age‐matched WKY. Furthermore, [Ca2+]i oscillation and vasomotion were observed. Treatment of ketanserin, a 5‐HT2A receptor antagonist dose‐dependently lowered the 5‐HT‐induced [Ca2+]i and contractile responses. In the highest concentration of ketanserin, 5‐HT‐induced [Ca2+]i oscillation and vasomotion in SHR‐SP disappeared. Additionally, both responses were also abolished in the absence of extracellular calcium. On the other hand, dysfunction of endothelial cell induced by air treatment in WKY failed to induce the [Ca2+]i oscillation, although [Ca2+]i and contractile responses were enhanced in comparison with intact. These observations suggest that influx of extracellular calcium via stimulation of 5‐HT2A receptor is responsible for the 5HT‐induced [Ca2+]i oscillation and thus vasomotion, but endothelial cell may not play a key role.
Voltage‐gated K+ (Kv) currents sensitive to stromatoxin‐1 in Guinea pig urinary bladder smooth muscle
Authors: Hristov Kiril L. , Chen Muyan , Petkov Georgi V.
Published: June 2021

Members of the voltage‐gated K+ (Kv) channel superfamily have been suggested to mediate the resting membrane potential and the repolarization phase of the urinary bladder smooth muscle (UBSM) action potential. Here, we tested the hypothesis that stromatoxin‐1 (ScTx1)‐sensitive Kv channels are expressed and control the membrane excitability in Guinea pig UBSM cells. Freshly isolated UBSM cells were studied using RT‐PCR, immunocytochemistry and the perforated mode of the whole cell patch‐clamp technique. In all patch‐clamp experiments 100 nM paxilline, a specific BK channel inhibitor, was present in the recording solutions to separated the KV currents from the BK currents. Whole‐cell current was elicited by voltage‐step depolarizations to potentials between ‐70 to +30 mV. Application of 100 nM ScTx1 inhibited about 60% of the remaining whole‐cell current (n=5; P<0.05) and the effect was more pronounced at positive voltages. We have further identified the molecular fingerprints of the ScTx1‐sensitive Kv currents. RT‐PCR experiments on isolated cells revealed mRNA expression for Kv2.1 and Kv9.3 family members (n=3). The immunocytochemical experiments on freshly isolated single UBSM cells confirmed the expression of the Kv2.1 channel protein (n=3). Our data revealed that ScTx1‐sensitive Kv2.1 channels are expressed and serve to control cell excitability in Guinea pig UBSM cells. Supported by DK070909 to GVP
Hyperthyroid rats display increased adrenergic ‐mediated contraction in cavernosal tissue
Authors: Carneiro Zidonia Nunes Noetzold , Carrillo ‐Sepulveda Maria Alicia , Carneiro Fernando Silva , Giachini Fernanda Regina , Lima Victor Vitorino , Webb R Clinton , Tostes Rita C
Published: June 2021

Introduction

Disturbances in thyroid function lead to sexual dysfunction in humans. However, the mechanisms by which hyperthyroidism causes erectile dysfunction (ED) are unknown. Whereas nitric oxide (NO) is the mediator of erection, sympathetic activation causes detumescence.

Objective

We hypothesized that hyperthyroidism increases sympathetic‐mediated contractility in cavernosal tissue.

Methods and Results

Wistar male rats were submitted to experimental hyperthyroidism (70ug/Kg T3 for 14 days). Thyroid hormone T3 plasmatic levels were increased in hyperthyroid animals (Hyper) (Control: 0.43±0.08 vs Hyper: 1.45±0.64, ng/mL). After euthanasia, penises were excised and dissected. Cavernosal strips were mounted in a myograph and stretched to a resting force of 3.0 mN. Cavernosal contractility to phenylephrine (PE) (Control: 1.72±0.2 vs Hyper: 3.06±0.2, mN) and sympathetic‐nerve stimulation (Control: 0.9±0.4 vs Hyper: 2.03±0.3, mN) were increased in Hyper rats. Cavernosal responses to nonadrenergic‐noncholinergic‐mediated relaxation and endothelium‐independent relaxation were not altered.

Conclusion

Our results suggest that increased sympathetic‐ mediated responses contribute to ED in hyperthyroidism.
Chloroquine and resveratrol stimulate ATM‐independent phosphorylation of AMPK and AKT
Authors: Spears Larry D. , Hobbs Supriya B. , Zhang Zhihong , Ralston Lyle , Liang Cheryl A. , Royer Nathaniel K. , Fisher Jonathan S.
Published: June 2021

It was hypothesized that ataxia telangiectasia mutated (ATM) activators would increase phosphorylation of the AMP‐activated kinase (AMPK) and AKT. To test this hypothesis, C2C12 cells were lentivirally transduced with short hairpin RNA (shRNA) against ATM, which decreased expression of ATM by ~80%. A control C212 cell line was produced by lentiviral transduction with green fluorescent protein (GFP), and non‐transduced C2C12 cells (wild‐type) were also used controls. Myotubes were incubated with or without 500 μM chloroquine, an ATM activator, for one hour. AMPK phosphorylation and AKT‐T308 phosphorylation were stimulated by chloroquine in all cell lines (i.e. wild‐type, cells expressing GFP, and cells expressing shRNA against ATM). In wildtype C2C12 cells, one hour of incubation with 150 μM resveratrol, another ATM activator, increased phosphorylation of AMPK even in the presence of KU55933, a specific inhibitor of ATM. Thus, chloroquine and ATM both cause AMPK and AKT phosphorylation in an ATM‐independent fashion.

This work was supported by NIH DK080437 and by NICHD/NCMRR through a Pilot Project subcontract from the National Skeletal Muscle Research Center.
Serotonergic cell transplant and cervical spinal cord injury
Authors: Dougherty Brendan J. , White Todd E. , O'Steen Barbara E. , Reier Paul J. , Fuller David D.
Published: June 2021

Phrenic motor recovery following cervical hemisection (HS) positively correlates with recovery of serotonergic input to ipsilateral phrenic motoneurons (PhrMNs). In preliminary studies, we are testing the hypothesis that respiratory motor recovery following HS can be enhanced by intraspinal grafting of embryonic (E14) brainstem tissue containing serotonergic (5‐HT) neurons or their precursors. E14 dorsal raphe nucleus tissue was placed into an acute C4HS cavity in adult male rats. At 8 weeks post‐surgery histological analysis showed that donor tissue filled the cavity and robust 5‐HT immunostaining was seen in the ipsilateral spinal cord rostral and caudal to the graft. Ventilation was assessed in conscious rats with plethysmography. Normoxic breathing patterns were similar between control (C4HS only) and transplant animals. However, during hypoxic challenge (10% O2) graft rats had increased frequency and minute ventilation (both p=0.01) compared to controls. Although additional control experiments are needed to determine if graft effects are specific to DRN tissue (vs. a non‐specific effect of embryonic tissue), these preliminary findings suggest that neurotransplantation may be a viable strategy for promoting respiratory motor recovery by enhancing serotonergic innervation of PhMNs.

Funding: NIH 1R01HD052682‐01A1 (DDF), NIH RO1 NS054025 (PJR), NIH T32 HD043730 (BJD)
Respiratory neural output during hypercapnia in a rat model of central dysmyelination
Authors: Baker‐Herman Tracy Lee , Mitchell Gordon S. , Duncan Ian D. , Johnson Rebecca A.
Published: June 2021

Respiratory complications contribute to morbidity/mortality in advanced myelin disorders, but little is known concerning mechanisms whereby dysmyelination impairs breathing. Preliminary studies using a genetic model of CNS dysmyelination, the Long Evans shaker rat (les), suggest that ventilation is impaired with advancing disease. We tested the hypothesis that respiratory impairment is associated with abnormal inspiratory neural output in les rats. Surprisingly, les rats (P185, n = 3) had higher phrenic burst frequencies than wild‐type controls in baseline (45 vs 33 bursts/min, respectively) and hypercapnia (52 vs 41 bursts/min, respectively; both p<0.05). Although percent increase in phrenic burst amplitude from baseline during hypercapnia was increased in les rats, this apparent difference was due to a tendency for decreased baseline amplitudes (0.6 vs 1.8 volts, respectively; p>0.05). Since peak voltages during hypercapnia (2.7 vs 3.6 volts, respectively; p>0.05) were similar in les and wild‐type rats, it appears that the capacity to increase phrenic burst amplitude is preserved. However, abnormalities were observed in the pattern of phrenic activity during a burst, including exaggerated synchronized activity and a decrementing discharge pattern, which may suggest a degree of dyscoordination in sculpting the phrenic burst. Support: NIH HL69064, NMSS TR3761, UW ICTR ‐ NIH CTSA 1UL1RR025011
Multi‐Resolution Wavelet Analysis of Time‐dependent Physiological Responses in Syncopal Youths
Authors: Ocon Anthony James , Nowak Jennifer A. , Taneja Indu , Medow Marvin S. , Stewart Julian M.
Published: June 2021

Prior studies indicate that fainting relates to thoracic hypovolemia and increased vascular resistance until late during head‐up tilt (HUT). Splanchnic resistance, cardiac output and BP decreased throughout HUT. We investigated alterations of baroreflex activity that occur with time dependent changes in HR, BP and TPR, using BP Mayer waves to estimate sympathetic baroreflex. Discrete wavelet transform with multiresolution analyses (MRA) measured time dependence. We studied 22 patients 13 to 21 yo, 14 who fainted within 10 minutes of HUT (fainters), and 8 controls. MRA of BP, HR and respirations during 70o upright tilt was obtained at different scales corresponding to frequency bands. Power concentrated in scales at 0.125 Hz and 0.25 Hz. A major difference from control was observed at the 0.125 Hz BP scale which progressively decreased from early HUT. The alpha index at 0.125 Hz was increased fainters. RR‐interval 0.25 Hz power decreased in fainters and controls but was markedly increased in fainters with syncope corresponding to increased vagal tone. The data imply a rapid reduction in time‐dependent sympathetic baroreflex activity in fainters but not control subjects during HUT.
Preferential baroreflex control of left ventricular diastolic function at rest and during mild dynamic exercise.
Authors: Sala‐Mercado Javier , Ichinose Masashi , Coutsos Matthew , Li ZhenHua , O'Leary Donal S
Published: June 2021

Carotid sinus hypotension elicits large baroreflex‐induced increases in peripheral resistance but has relatively little effect on left ventricular systolic function. However, baroreflex control over left ventricular relaxation is unknown and diastolic function can contribute importantly to overall cardiac performance, especially during periods of stress such as dynamic exercise. Therefore, we investigated the relative effects of bilateral carotid occlusion (BCO) on left ventricular systolic and diastolic function (±dP/dtmax/min) in conscious dogs at rest and during exercise (3.2 km/h). As expected, exercise caused a significant increase in heart rate (HR), +dP/dtmax, stroke volume (SV), and therefore cardiac output (CO) but also in total vascular conductance (TVC) and as a result arterial pressure (AP) remained unchanged. No significant change occurred in ‐dP/dtmin, thus ±dP/dtmax‐min ratio substantially increased. At rest and during mild dynamic exercise, BCO caused no significant change in CO and therefore the rise in AP was due to a significant decrease in TVC. BCO increased ‐dP/dtmin, with no change in +dP/dtmax, therefore ±dP/dtmax‐min ratio significantly decreased. We conclude that the increase in sympathetic activity to the heart elicited by carotid baroreceptor unloading preferentially improves left ventricular diastolic function. Supported by HL‐55473.
Skin‐surface cooling increases left‐ventricular preload but not contractility in older humans
Authors: Gao Zhaohui , Wilson Thad E. , Hess Kari L. , Monahan Kevin D.
Published: June 2021

Skin‐surface cooling acutely increases arterial blood pressure, left‐ventricular (LV) afterload, and myocardial oxygen demand. To determine whether skin surface cooling increases LV preload and contractility more in older than in young adults, we measured indices of LV preload and myocardial contractility using standard and tissue‐Doppler imaging (TDI) echocardiography in 6 young (23 ?1 yrs; mean ?SE) and 6 older (65 ?3 yrs) adults during normothermia (35° C water perfused through a tube‐lined suit) and skin‐surface cooling (15‐18° C water perfused for 20 min). Cooling increased LV end‐diastolic volume (EDV, from 98±1 to 108±1 ml, P<0.001) and LV internal diameter in diastole (IDd, from 4.61±0.03 to 4.77±0.03 cm, P<0.01) in older, but not young adults (EDV=101±1 and 99±1 ml, IDd=4.67±0.03 and 4.62±0.03 cm, for normothermia and cooling, respectively). In contrast LV ejection fraction and TDI myocardial functional indices (myocardial acceleration during contraction and peak velocities during systole and diastole) were unchanged by cooling in both young and older adults. These data indicate that skin surface cooling acutely increases LV preload, without influencing LV contractility in older, but not young adults. Increases in LV preload and afterload likely contribute to greater increases in myocardial oxygen demand during cold exposures in older than in young adults.

Support: NIH AG24420 and M01 RR10732
Administration of 17‐ β estradiol in the medial preoptic area prevents reduction of body temperature during cold exposure in ovariectomized rats
Authors: Uchida Yuki , Tohi Madoka , Tokizawa Ken , Nagashima Kei
Published: June 2021

BACKGROUND

We reported that ovariectomized rats could not maintain body temperature (Tcore) well in the cold; however, local administration of 17‐β estradiol (E2) in the medial preoptic area (MPO) restored the cold intolerance. The E2 administration had no effect on UCP1 mRNA level in the interscapular brown adipose tissues, suggesting less influence on metabolic heat production.

PURPOSE

To test the hypothesis that the local E2 administration in the MPO affects heat dissipation, the tail surface temperature was assessed by thermography.

METHODS

After ovariectomy for 9‐wk old rats, a stainless steel canula was placed in the MPO for local administration of E2 or cholesterol. A rat was placed at 10 or 25°C for 2h, 48 h after the administration E2 or cholesterol. Tcore and tail temperature (Ttail) were measured by telemetry and thermography.

RESULTS

Both the groups showed decrease in Ttail at 10°C; however the reduction was greater in the estrogen group than in the cholesterol group.

CONCLUTION

Without estrogen in the MPO, heat dissipation from the tail in ovariectomized rats may not be fully suppressed in the cold, resulting in reduction of Tcore.
Structures of RNA polymerase II ‐ TFIIF and Mediator complexes
Authors: Kornberg Roger , Calero Guillermo , Takagi Yuichiro , Spahr Henrik , Imasaki Tsuyoshi
Published: June 2021

Structures of an RNA polymerase II transcribing complex in association with general transcription factor TFIIF and of the Mediator head module will be presented.
Effects of ganoderma lucidum spores on the change of hyaluronic acid content and glutamine synthetase activity in epilepsy rats
Authors: Wang Shu‐Qiu , Kang Yu‐Ming , Zhang Shi‐Ling , Wang Xian‐Gang , Qin Wen‐Bo , Zhao Xiao‐Lian , Liu Lei , Wang Fang‐Fang , Zhao Lu , Zhang Ting
Published: June 2021

To investigate the effects of ganoderma lucidum spores on the mossy fiber sprouting (MFS), the hyaluronic acid (HA) content, the expression of glial fibrillary acidic protein (GFAP) and the activity of glutamine synthetase (GS) in hippocampus of rats with epilepsy induced by pentylenetetrazol (PTZ), and to further study the pathogenesis of epilepsy and the anti‐epilepsy mechanism of ganoderma lucidum spores. Thirty‐six male SD rats were randomly divided into control group, PTZ epilepsy group and ganoderma lucidum spores (GLS) group. The results showed that MFS, HA content and GFAP‐positive cells in rats of PTZ model group were significantly higher than those in control group and GLS group. The Timm scores were significantly higher in PTZ model group than those of control group and GLS group at the CA3 region, also Timm scores in GLS group were higher than control group. In PTZ model group, the astrocytes activation phenomenon in hippocampus was more obvious than GLS group, while the activity of GS was much lower than control group and GLS group.

Conclusion

ganoderma lucidum spores can reduce MFS and the content of HA in hippocampus, and enhance the function of astrocytes, and then reduce the seizures.
Increased CaM kinase II‐mediated phosphorylation of ampa receptors precedes status epilpeticus
Authors: Schafer Johanna Marlies , Churn Severn , Kurz Johnathon , Low Brian
Published: June 2021

This study was conducted to analyze the change in calcium/calmodulin‐dependent kinase II (CaMK II)‐mediated phosphorylation of AMPA receptors at times points throughout the duration of stats epileptics (SE). The GluR1 subunit of the AMPA receptor is phosphorylated at ser831 by CaMK II; the progression of seizure activity into the continuous seizures of status epilepticus may be due to potentiation of excitatory postsynaptic neurotransmission by AMPA‐receptor‐mediated currents.

Methods

SE was induced by a pilocarpine injection. At three time‐points of discrete seizures preceding SE, 20 and 40 minutes after the onset of SE, and 24 hours post‐SE, cortical and hippocampal brain regions were isolated into homogenization buffer and used for biochemical studies. Proteins were resolved by SDS‐PAGE and subjected to western blot analysis. GluR1 phosphorylation was assessed using phosphoGluR1‐specific antibodies and computer‐assisted densitometry.

Results

All time point proceeding and during SE had over a 150% increase in phosphorylation at ser831compared to the controls. The increase in pGluR1 was observed well into overt SE. Although, the last time point, 24 hours post‐SE, did not demonstrate a statistically significant change in phosphorylation levels.

Conclusions

This data suggests that elevated AMPA‐directed CaMK II activity may increase the functionality of excitatory AMPA receptors prior to and during status epilepticus. At the 24 hour time‐point, GluR1 phosphorylation appeared to return to control levels, suggesting a return of CaMK II activity to normal levels.
SMALL CONDUCTANCE CALCIUM‐ACTIVATED POTASSIUM CHANNELS (SK) LIMIT THE EXCITABILITY OF PVN NEURONS PROJECTING TO RVLM (PVN‐RVLM)
Authors: Chen Qing‐Hui , Toney Glenn M.
Published: June 2021

In vivo studies indicate that under normal condition pre‐autonomic PVN neurons are often silent or have low spontaneous activity. In spite of their importance in regulating sympathetic outflow, mechanisms that control the excitability of pre‐autonomic PVN neurons remain largely unexplored. Here, we determined the role of SK channels in limiting excitability of PVN‐RVLM neurons. In voltage‐clamp recordings, an outward current (IK) (amplitude: 72 ± 10 pA; decay constant: 234 ± 55 ms, n=8) was nearly abolished by the SK channel blocker apamin or UCL1684. Activation of IK was Ca2+ dependent and reversed at ‐97±3 mV (n=3) which was close to the EK value (‐103 mV). In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 3 Hz (n=8) and was significantly greater (P<0.05) in the presence of apamin (100 nM) (47 ± 8 Hz, n=8) or UCL1684 (100 nM) (39 ± 5, n=5). Apamin and UCL1684 each blocked a prominent medium after hyperpolarization potential (mAHP: ‐11 ± 2 mV) that was evident in response to current injection. With blockade of the mAHP, a prominent after depolarization potential (ADP) was revealed (amplitude: apamin +9 ± 1; UCL1684 +7 ± 2 mV). We conclude that SK channel activation limits the excitability of PVN‐RVLM neurons. An underlying mechanism may be the ability of the mAHP to oppose the ADP. Support: AHA0865107F (QHC) & HL076312 (GMT)
Changes in expression and localization of glial cell line‐derived neurotrophic factor in skeletal muscle from rats at 3, 5 and 8 weeks of age
Authors: Ousley Dominique D. , Deering Scott J. , Spitsbergen John M.
Published: June 2021

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to play a role in motor neuron survival and plasticity. As GDNF plays a role in neural development we were interested in determining where and when GDNF was expressed in skeletal muscle during the final stages of neuromuscular junction development. We hypothesized that GDNF protein would be expressed at high levels at the end‐plate in 3 week old rats and that levels of expression would decline with increased age (5 and 8 weeks). Soleus, extensor digitorum longus and extensor hallucis longus muscles were removed and GDNF protein localization was examined using immunocytochemical methods. Muscles taken from 3 week old rats contained end‐plate regions at various stages of development, while muscles taken from older rats contained no immature end‐plates. The results showed little or no staining for GDNF protein at end‐plate regions in muscles from 3 week old rats. Muscles taken from older rats showed clear staining for GDNF protein at the end‐plate region. The results suggest that GDNF is not expressed at higher levels at developing end‐plates. These observations may suggest that GDNF plays a role at the neuromuscular junction beyond initial development, possibly being necessary for maintenance and growth of junctions as muscles continue to enlarge with animal growth. Supported by NIH Grant 1R15AG022908‐01A2, MSU‐KCMS, and Western Michigan University.
Differential effect of glutamate receptor blockade on dendritic outgrowth in developing chicken lumbar motoneurons
Authors: Ni Xianglian , Martin‐Caraballo Miguel
Published: June 2021

Glutamate receptor‐mediated changes in intracellular Ca2+ may have important implications for activity‐dependent regulation of early embryonic development. Although NMDA receptors were originally considered to be the sole source of Ca2+ influx through glutamate receptors, AMPA receptors also allow a significant influx of Ca2+ ions. Previously, we have demonstrated that chicken lumbar motoneurons express Ca2+ permeable AMPA receptors at embryonic day (E) 6. By E11, the Ca2+ permeability of AMPA receptors decreases three‐fold. In this study we have explored the role of transiently expressed Ca2+ permeable AMPA receptors in regulating the dendritic outgrowth of developing motoneurons. The AMPA receptor blocker CNQX (1 mg/day), when applied between E5 and E8, causes a significant increase in dendritic outgrowth as compared with vehicle‐treated embryos. CNQX causes both an increase in dendritic length and branching. Inhibition of NMDA receptor function with MK‐801 (0.2 mg/day) during this period, however, does not have any effect on dendritic or cell body morphology. Treatment of chicken embryos with CNQX between E8 and E11 (when most receptors become Ca2+ impermeable) did not have any effect on dendritic outgrowth. However, MK‐801 application to chicken embryos between E8 and E11 causes a significant reduction in dendritic length and branching. These findings indicate AMPA receptor activation limits dendritic outgrowth at early stages of development, whereas NMDA receptor activation is involved in dendritic remodeling after the establishment of synaptic contacts with sensory afferents.
Autonomic, cardiovascular and thermoregulatory responses to central neropeptide Y or neuromedin U in rats
Authors: Tanida Mamoru , Nagai Katsuya
Published: June 2021

Previous studies have demonstrated that neuropeptide Y (NPY) or neuromedin U (NMU) affects cardiovascular functions such as blood pressure (BP) or heart rate (HR) in rats. Here, we examined the effects of the central injection of various doses of NPY or NMU on renal sympathetic nerve activity (RSNA) and BP in urethane‐anesthetized rats. Central injection of NMU elevated RSNA, BP and HR in a dose‐dependent manner, while NPY injection reduced RSNA, BP and HR RSNA, BP and HR.

In addition, central injection of NMU suppressed gastric vagal nerve activity (GVNA) and activated brown adipose tissue sympathetic nerve activity (BAT‐SNA) of anesthetized rats, and elevated brown adipose tissue temperature (BAT‐T) of conscious rats. Oppositely, NPY affected autonomic nerves and elevated the GVNA and suppressed BAT‐SNA and BAT‐T.

Moreover, neither pretreatment of thioperamide, an antagonist of histaminergic H3‐receptor, or of diphenhydramine, an antagonist of histaminergic H1‐receptor, abolished increasing effects of NMU on RSNA, BP and HR However, pretreatment of thioperamide eliminated suppressive responses of RSNA, BP and HR to NPY injection. Thus, these lines of evidence suggest that NMU or NPY affects neural activities of autonomic nerves containing RSNA, GVNA or BAT‐SNA, and BP by mediating central mechanism, and possible role of histaminergic neurotransmittion in autonomic controls by NPY.
Comparative analysis of cardioprotective and antiarrhythmic properties of opioid receptor agonists in a rat model of myocardial infarction
Authors: Brown Stephen A. , Maslov Leonid N. , Oeltgen Peter R.
Published: June 2021

We examined the effect of opioid receptor (OR) agonists/antagonists and inhibitors on infarct size (IS) and arrhythmia in rats following coronary artery occlusion and reperfusion. Pretreatment with the specific δ2‐OR agonist Deltorphin II reduced the IS and was prevented by treatment with naltrexone, naloxone and the specific δ2‐OR antagonist naltriben (NTB). The infarct‐limiting effect of Deltorphin II was also abolished by inhibition of Protein Kinase C (PKC) and potassium ATP‐sensitive K (KATP) channels but not by the specific δ1‐OR antagonist, 7‐Benzylidennaltrexone (BNTX). All deltorphins tested exhibited antiarrhythmic properties during phase 1b of ischemia, but only Deltorphin II prevented ventricular arrhythmias during phase 1a and phase 1b. The antiarrhythmic effect of deltorphin II was abolished by treatment with naltrexone, naloxone methiodide and the selective δ2‐OR antagonist NTB but not by the selective δ1‐OR antagonist BNTX. PKC inhibition, but not KATP channel inhibition, eliminated the antiarrhythmic effect of Deltorphin II. Pretreatment with other opioids D‐Ala2, N‐Me‐Phe4, Gly5‐ol‐Enkephalin, Dermorphin H, D‐Pen2,5 Enkephalin, Deltorphin‐D, Deltorphin‐E, and U‐50,488, nociception did not reduce the IS or arrhythmias. We conclude that peripheral δ2‐OR activation induces infarct size reduction and prevents ischemia induced ventricular arrhythmias.
Bufalin Prevents Rrestoration of Blood Volume after Hemorrhage.
Authors: Darlington Daniel N. , Gann Donald S.
Published: June 2021

Shock is associated with cellular depolarization, a rise in extracellular K and intracellular Na, and a movement of water from the extracellular to the intracellular space. These phenomena can be explained by an inhibition of Na/K ATPase. We have found that plasma (extracts) from shocked rats inhibit Na/KATPase and increases water movement into cells. Recently, bufodienolides have been identified in mammalian plasma and are strong inhibitors of Na/K ATPase. To determine if Bufalin or Marinobufogenin (MBG) can prevent normal restoration of blood volume to mild hemorrhage, we treated awake Sprague‐Dawley rats, with either 1) 20ml/kg hemorrhage, 2) an infusion of Bufalin or MBG, or 3) both (n=>8 all groups). Hemorrhage led to an immediate fall in plasma volume followed by recovery to normal by 60min. However, rats given bufalin and non‐lethal hemorrhage failed to restore blood volume and died within 60min. Bufalin alone led to no significant change in blood volume. Marinobufogenin, given at this dose and higher did not prevent restoration of blood volume. These data suggest that Bufalin but not MBG, prevents normal restoration of blood volume after hemorrhage.