bluuebunny/crossref_metadata_2025 huggingface dataset:
Pregnancy Decreases Myogenic Activity of Brain Parenchymal Arterioles: Role of Estrogen
Authors: Cipolla Marilyn J. , A Julie
Published: June 2021

This study investigated the influence of pregnancy and estrogen on myogenic activity of brain parenchymal arterioles (PA). PA were dissected from Sprague Dawley rats that were nonpregnant (NP, n=6), late‐pregnant (LP, n=6), ovariectomized and implanted with estriol (5.0 mg) + estradiol (0.5 mg) pellets for 2 weeks (OVX+E, n=8) to obtain levels of pregnancy or OVX implanted with a placebo pellet (OVX, n=6). PA were mounted on glass cannulas within an arteriograph chamber and lumen diameter recorded at intravascular pressures from 25–200 mmHg. Pregnancy significantly increased active diameters of PA, that also underwent forced dilatation at lower pressures vs. all groups. The active diameters of PA from LP vs. NP, OVX+E and OVX animals at 100 mmHg was (μm): 64±6 vs. 49±4, 42±3 and 42±3 (p<0.05). In addition, PA from LP animals had significantly diminished tone vs. all groups whereas the tone in OVX was increased vs. NP. The percent tone at 100 mmHg for LP vs. NP, OVX+E and OVX animals at 100 mmHg was: 34±5 vs. 44±3 (p<0.05 vs. LP), 52±1 (p<0.05 vs. LP) and 54±2 (p<0.05 vs. NP and LP). These results demonstrate that pregnancy diminishes myogenic activity of brain PA, an effect that was not related to elevated estrogen. The larger lumen of PA during pregnancy could decrease small vessel resistance and predispose the brain to hyperperfusion when blood pressure is elevated, as in eclampsia. Supported by RO1 NS045940 and AHA EI 0540081N.
Coronary arteriogenesis in the intraventricular septum of the post‐infarcted heart does not normalize maximal myocardial perfusion
Authors: Dedkov Eduard I , Zheng Wei , Christensen Lance P , Tomanek Robert J
Published: June 2021

The inadequate growth of coronary resistance vessels (arterioles) during compensatory hypertrophy has been commonly considered the prime cause of impaired maximal perfusion in the LV myocardium after MI. However, our previous studies have shown that maximal coronary conductance (flow/pressure) was also reduced in the remote region of post‐MI hearts that did not undergo compensatory hypertrophy. Accordingly, we investigated the adaptive arteriolar growth in the septum of 12‐mo‐old Sprague‐Dawley rats during 4 wks of post‐MI remodeling. We found that between day 7 and 14 after MI arterioles demonstrated a significant increase in DNA synthesis, mainly in vascular smooth muscle cells (VSMC), which was associated with up‐regulation of Ang‐2 and Tie‐2 protein expression. Although VSMC proliferation occurred throughout the entire arteriolar tree it was higher in larger arterioles (25–54 μm in diameter). As a result, 4 wks after MI we detected a significant increase in arteriolar length density in this segment of the arteriolar tree as compared to sham rats (P< 0.05). Mean arteriolar diameters and periarteriolar collagen content were similar in post‐MI and sham rats. Therefore, we suggest that despite significant arteriogenesis the perfusion deficit in the septum of post‐MI hearts of middle‐aged rats might be caused by geometric changes in the resistance vessels (length, tortuosity, etc.) or some functional deficit in their maximal vasodilation.
Right ventricular wall microvascular response to increased pulmonary artery pressure with particular reference to age of onset
Authors: Beighley Patricia E. , Dong Yue , Ouchi Hideo , Zamir Mair , Ritman Erik L.
Published: June 2021

Patients with congenital heart disease who have a morphological right ventricle (RV) serving as a systemic ventricle have an increased incidence of RV dysfunction. A different structural response of micro‐vessels to increased stress in the RV wall is a possible mechanism for this dysfunction. We examined this possibility by measuring the volume of myocardium perfused by arteries less than 200μm in lumen diameter (V) 7 weeks after a 5mg/kg IV injection of monocrotaline in three 2‐week old and three 18‐week old pigs. This information was obtained by use of 3D micro‐CT of transmural segments (~ 2cm3) of the RV wall. The table provides the average values of V (mm3/mm2 ± SD). Pulmonary artery pressures in the mature, treated pigs were 35±6 mmHg and in the juvenile were 25±2 mmHg. V did not differ from control in the treated 2.5‐month old pigs but trended down in the 6‐month old pigs.

We conclude that the volume of myocardium perfused by an arteriole in the RV wall may be an indicator of the perfusion reserve available to the microcirculation in response to increased wall stress. A more definitive answer would require delayed study of the early onset treatment group at the age of 6 months.

(Supported in part by NIH grant EB000305)
Cardiac reactive hyperemia is impaired in a rat model of hyperhomocysteinemia (HHcy)
Authors: Miklos Zsuzsanna , Kemecsei Peter , Koller Akos , Ivanics Tamas
Published: June 2021

The endothelium‐mediated vasomotor function of skeletal muscle arterioles has been shown to be impaired in HHcy. Thus we hypothesized that HHcy modulates the reactive hyperemic response of coronary circulation following occlusion. Rats received 1g/kg of body weight methionine daily, for 5 weeks in the drinking water to induce mild HHcy (~ 30 μmol vs. ~ 7 μmol). In Langendorff‐perfused hearts reactive hyperemia (RH) after 5 min of global ischemia was studied prior to and after the simultaneous administration of the nitric oxide synthase inhibitor L‐NAME (10−4M) and the cyclooxygenase inhibitor indomethacin (INDO 10−5M) into the perfusate. We have found that the early peak values of reactive hyperemia were not significantly different in HHcy and control (C) groups (at 2 min of RH: HHcy: 139±14 vs. C: 147±19% of basal flow). The later phase of reactive hyperemia however, was significantly diminished in HHcy hearts (at 8 min of RH: HHcy: 116±7 vs. C: 130±14% of basal flow). Presence of L‐NAME+INDO eliminated the difference between the reactive hyperemia of control and HHcy hearts. On the basis of these findings we propose that cardiac reactive hyperemia is impaired in HHcy, primarily due to reduced release of endothelium‐derived dilator factors. (Grants: Hungarian Sci. Res. Funds/OTKA T48376, 61694, 68502, T67984; and Am. Heart Assoc. NE Aff. 0555897T, USA).
Effects of carbon monoxide on sea lamprey and rainbow trout systemic arteries
Authors: Dombkowski Ryan Anthony , Motterlini Roberto , Olson Kenneth
Published: June 2021

Carbon monoxide (CO) is an endogenously produced gas involved in vascular, neural, and inflammatory responses in mammals. CO activity in non‐mammalian vertebrates, however, is unknown. To this extent, we utilized smooth muscle myography to investigate the effects of exogenously applied CO (via CORM‐3) on isolated lamprey (Petromyzon marinus) dorsal aortas and trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM dose‐dependently relaxed norepinephrine‐ (NE) and U46619‐, thromboxane‐A2 mimetic, ‐induced contractions of lamprey dorsal aortas. CORM relaxed both NE (10μM)‐and KCl (80mM)‐ prestimulated trout EBA and CMA dose dependently. EBA were 2‐fold more sensitive to CORM when prestimulated with NE than KCl and CORM relaxed 5‐fold more of the NE‐ than KCl‐induced tension. Glybenclamide (lOμM) inhibited a NE‐induced contraction, but did not effect a CORM induced relaxation. NS‐2028 (10μM), a solubule guanylyl cyclase inhibitor, had no effect on a NE‐contraction, but inhibited a subsequent CORM‐induced relaxation. Zinc protopophyrin‐IX (30μM), a heme‐oxygenase (HO) inhibitor, elicited a significant increase in baseline tension and eliminated the tri‐phasic EBA response to Na2S. These results implicate an active HO/CO pathway in trout vessels on resting vessel tonus and H2S‐induced vasoactivity that is partially mediated by soluble guanylyl cyclase.
Effects of Arginase I and II on local production of NO
Authors: Elms Shawn , Fulton David
Published: June 2021

Dysfunction of endothelial nitric oxide synthase (eNOS) has been implicated in cardiovascular diseases such as hypertension and diabetes. Arginase I and II (Arg I, II) degrade the substrate for eNOS, L‐arginine and decrease nitric oxide (NO) production. Altered expression and or activity of arginases have been proposed to account for reduced eNOS function in cardiovascular disease and also to provide a mechanism for the “arginine paradox”. Our hypothesis was that ArgI and ArgII inhibit eNOS activity by depleting arginine from specific intracellular pools. To test this hypothesis we transfected COS‐7 cells with arginase I (cytosolic) and arginase II (mitochondria) together with eNOS constructs targeted to various intracellular compartments; namely cytosol, plasma membrane, Golgi, mitochondria and nucleus. In preliminary studies the activity of subcellular eNOS targeting constructs were equally affected by Arg I and II, despite different locations within the subcellular environment. We submit that there are no spatially restricted pools of L‐arginine within COS‐7 cells but a homogenous and equally accessible amount of L‐arginine. Future studies will identify whether endothelial cells possess mechanisms for arginine compartmentalization.
Endogenous hydrogen peroxide is vasoactive in mesenteric arteries of spontaneously hypertensive rat
Authors: Kroetsch Jeffrey Thomas , Rush James W.E.
Published: June 2021

Hydrogen peroxide (H2O2) is an endogenous endothelium‐dependent dilator in resistance vessels; however, a role for H2O2 in the mesenteric vessels of the spontaneously hypertensive rat (SHR) has not been established. Mesenteric arteries were excised from SHR and normotensive (WKY) rats and myography was performed. Vessels were incubated with 3μM of the β‐blocker propranolol, pre‐contracted with norepinephrine, and cumulative dose‐response curves for acetylcholine (Ach) were conducted. Maximal dilation to Ach was impaired in SHR compared to WKY (76±4% vs 97±1%, p<0.05). Co‐incubation with the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine (L‐NAME, 0.3mM) and the cyclooxygenase inhibitor indomethacin (INDO, 5μM) reduced dilation in SHR (52±6%, p<0.05). Co‐incubation with L‐NAME, INDO, and the H2O2 scavenger catalase (CAT, 860U/mL) caused a further reduction in SHR dilation (32±3%, p<0.05). Incubation with CAT alone caused improved dilation (92±12%, p<0.05) while incubation with the superoxide dismutase mimetic 4‐hydroxy‐TEMPO (1mM) reduced dilation (59±7%, p=0.05) in SHR. WKY dilation was not altered by any treatment. Thus, in the presence of nitric oxide and prostaglandins, H2O2 acts as a constrictor; however, in the absence of nitric oxide and prostaglandins, H2O2 acts as a dilator in SHR mesenteric arteries.

Funding: NSERC/Heart and Stroke Foundation of Ontario
Effects of resveratrol on endothelial nitric oxide synthase (eNOS) phosphorylation and interaction with regulatory proteins
Authors: Araj Christina A , Slack Kristen N. , Harris M. Brennan
Published: June 2021

Previous studies have demonstrated that the polyphenolic compound, reseveratrol (RSV), from the skin of red grapes increases endothelial nitric oxide synthase (eNOS) activity. However, the mechanism by which RSV increases eNOS activity is unknown. Therefore, we have investigated changes in eNOS phosphorylation at Ser1179, Ser635, Thr495 as well as changes in eNOS association with Hsp90, Akt, and cdc37 in bovine aortic endothelial cells (BAECs) treated with varying doses of RSV for 0, 20, 60 min as well as 72 h. Acute RSV treatment (less than 1) resulted in no significant (P>0.05) changes in eNOS phosphorylation or changes in eNOS association with Hsp90, Akt or cdc37. In contrast, 72h treatment of BAECs at higher concentrations of RSV significantly (P<0.05) increased eNOS phosphorylation and protein association that was consistent with the increase in eNOS expression. In conclusion, these data suggest that RSV‐induced eNOS activity is primarily due to increased eNOS expression and that acute (less than 1 h) RSV treatment does not alter post‐translational eNOS regulatory mechanisms. Supported by AHA SDG 0430157N (MBH) and William & Mary HHMI Undergraduate Research Program Student Grant (KNS)
Regulation of VEGF mRNA expression in hypoxic pulmonary artery endothelial cells (PAECs) by histone deacetylases (HDACs)
Authors: Middleton Natavia S. , Ruchko Mykhaylo , Gorodnya Olena , Gillespie Mark N.
Published: June 2021

The mechanism by which HDACs regulate gene expression in hypoxia is not understood. Some evidence suggests that HDACs act directly on HIF‐1, a key transcription factor, to facilitate binding to the hypoxic response element (HRE). Other studies indicate that HDACs impact chromatin‐remodeling processes and modulate access of the HRE to transcriptional proteins. To explore these possibilities, we first used Western immunoblot and DNA affinity precipitation analyses to determine if the HDAC inhibitor, trichostatin A (TSA), altered abundances and DNA binding activities, respectively, of HIF‐1, p300, and HDAC1 in normoxic and hypoxic PAEC nuclear extracts. While HDAC inhibition caused minor changes in nuclear abundances of these transcriptional proteins, their association with a 65‐mer oligonucleotide sequence of the VEGF HRE was unaffected. Next, using chromatin immunoprecipitation assays to examine transcriptional complex formation in intact PAECs, we found that TSA suppressed HIF‐1, p300, and HDAC1 binding to the HRE of the VEGF gene. Nuclease digestion assays showed that TSA redistributed the VEGF HRE from transcriptionally active mononucleosomes to highly condensed chromatin structures. These findings suggest HDACs are recruited to the VEGF HRE in PAECs where they regulate hypoxia‐induced VEGF expression by remodeling chromatin and facilitating transcriptional complex formation.

Supported by NIH and AHA
Role of ErbB tyrosine kinase receptors in aging‐related cardiac dysfunction
Authors: RAJAGOPALAN VISWANATHAN , ZUCKER IRVING H , MA YING J
Published: June 2021

Background: Multiple factors are known to be involved in aging‐related cardiac dysfunction. We and others have showed that inhibition of cardiac ErbB tyrosine kinase receptor signaling lead to cardiac dysfunction. Also, ErbB expression is downregulated in aging‐related neural dysfunction. We hypothesized that compromised ErbB signaling is associated with aging‐related cardiac dysfunction.

Methods: Young adult (4–7 mo), middle aged (9–12 mo) and aged adult (18–22 mo) mice (n=4–5/group) were studied for cardiac function parameters using high frequency echocardiography and radiotelemetry (conscious). RT‐PCR and Western Blotting were performed to study ErbB expression.

Results: Compared to young adult mice, aged mice had significantly decreased ejection fraction (%; 55.3±1.5 vs. 66.6±3.1; p<0.01), fractional shortening and left ventricular (LV) dP/dtmax (mmHg/s; 3420±494.2 vs. 8423±486.2; p<0.001) but significantly increased LV systolic internal diameter (mm; 2.7±0.1 vs. 2.3±0.2; p<0.05), end‐diastolic pressure (mmHg; 22.8±0.8 vs. 6.2±0.5; p<0.001) and mass (mg/g b.w.; 4.3±0.2 vs. 3.3 ±0.2; p<0.05). Ligand‐binding ErbB‐1 and ErbB‐4 receptor expression of mRNA, protein and phosphorylated protein were significantly diminished in aged mice compared to young mice (OD; p<0.01).

Conclusion: Compromised ErbB signaling may be an important factor mediating aging‐related cardiac dysfunction.
Inflammatory Circulating Mononuclear Cell Phenotype in Healthy Older Adults with Low‐Grade Systemic Inflammation and Endothelial Dysfunction
Authors: Gano Lindsey B , Magerko Katie A. , Roeca Cassandra , Lawson Brooke R. , Jablonski Kristen L. , Seals Douglas R. , Donato Anthony J.
Published: June 2021

Chronic low‐grade inflammation may contribute to vascular endothelial dysfunction with aging. Changes in the inflammatory properties of circulating mononuclear cells (MC) may be involved, but data are limited. Compared with young controls (Y, 25±2 yr, n=9), healthy older adults (O, 65±2 yr, n=12) had elevated plasma concentrations of C‐reactive protein (0.85±0.15 vs. 0.42±0.10 mg/L, P<0.05) and interleukin‐6 (IL‐6, 1.32±0.22 vs. 0.64 ±0.05 pg/ml, P<0.05), and reduced maximal forearm blood flow responses to acetylcholine (12.8±1.3 vs. 17.9±2.2 mL tissue−1min−1, P<0.05). MC mRNA expression (real‐time RT PCR) of inflammatory cytokines monocyte chemoattractant protein‐1 (MCP‐1, 1.52±0.34 vs. 0.00±0.42 Fold change from Y[FC], P<0.05), tumor necrosis factor α (TNF‐α, 2.68±0.50 vs. 0.00±0.34 FC, P<0.05) and IL‐6 (1.98±0.91 vs. 0.00±0.83 FC, P=0.07) were greater, whereas the anti‐inflammatory nuclear transcription factor peroxisome proliferator activated receptor α (PPAR‐α, −1.28±0.50 vs. 0.00±0.51 FC, P<0.05) was lower in O. MC protein expression (Western blot analysis) of inflammatory molecules MCP‐1, TNF‐α and IL‐6 also was greater in a subgroup of O vs. Y. These preliminary results provide evidence that MC of healthy older adults exhibit a pro‐inflammatory phenotype that may contribute to chronic low‐grade systemic inflammation and vascular endothelial dysfunction with aging. NIH AG006537, AG013038, AG022241
Influence of CPZ and DMSO on the mechanism regulation of the plasma membrane Ca2+ ATPase of human erythrocyte
Authors: Domínguez‐Solís Carlos Augusto , Plenge‐Tellechea Fernando
Published: June 2021

The calcium pumping ATPase of the erythrocyte plasma membrane (hPMCA4b) is the principal active system that expulse intracellular calcium into extracellular space in eukaryotic cells. PMCA is regulated by calmodulin (CaM), incrementing its ATP affinity. The use of molecules like chlorpromazine (CPZ) and dimethylsulfoxide (DMSO), inhibitors to hPMCA4b activity (non specific), were used in this study as a molecular tools that could help to understand the regulating mechanism of biochemical activation by CaM activation and their relationship with them. These molecules inhibit the PMCA activity, affecting its calcium homeostasis. CPZ is a phenotiazine that interfere with CaM functionality and it is commonly used in the treatment of mental disease. Erythrocytes membranes were used as the model biological system in order to assess the PMCA due to the absence of other calcium ATPases.

PMCA activities in absence and presence of CaM and activated by trypsin exhibited inhibition by DMSO and CPZ. The ATPase activated with CaM in presence of CPZ does not interfere with the mechanism of regulation because there is no protection against the effect inhibitory of CPZ compared with the native PMCA. DMSO interacts with the regulation mechanism in the PMCA activated with CaM since there is the same profile of protection against the inhibition in comparison with the native enzyme. The recovery values of the activity in presence of CPZ and DMSO indicated the possible dilution of CPZ by the solvent.

Supported by the grants of Fondos Mixtos CONACYT‐Edo. De Chihuahua and PROMEP‐CAEC.
PGE2 inhibits the basolateral 50‐pS K channels in the thick ascending limb (TAL) of rat kidney by PKC and MAPK
Authors: Gu Ruimin , Jin Yan , Zhai Yuanyuan , Li Wennan , Yang Lei , Zhang Chengbiao , Wang Lijun , Kong Shumin , Yang Baofeng , Wang Wenhui
Published: June 2021

We used the patch‐clamp technique to study the effect of PGE2 on the basolateral 50‐pS K channel in the TAL. Application of 5 μM PGE2 inhibited basolateral 50‐pS K channels and decreased channel activity, defined as NPO, from 0.29±0.07 to 0.04±0.03. The inhibitory effect of PGE2 was abolished by inhibiting PKC with Calphostin C. To explore whether mitogen activated protein kinase (MAPK) is also involved in mediating the effect of PGE2 on the 50‐pS K channels, we examined the effect of PGE2 in the presence of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38). Although application of either PD98059 or SB203580 increased the channel activity, inhibiting p38 or ERK alone did not block the effect of PGE2 on the basolateral 50‐pS K channels. In contrast, simultaneously inhibiting both ERK and p38 completely abolished the effect of PGE2 on 50‐pS K channels. To further demonstrate that PGE2 stimulates PKC‐MAPK pathway, we examined the effect of PGE2 on the phosphorylation of p38 and ERK in the primary cultured mTAL cells. Application of PGE2 stimulates the phosphorylation of both p38 and ERK. The effect of PGE2 on MAPK phosphorylation was not affected by indomethacin but completely blocked by calphostin C. This suggested that PKC is involved in mediating the effect of PGE2 on MAPK phosphorylation. We conclude that PGE2 inhibits the basolateral 50‐pS K channels in the TAL of rat kidney through stimulating EP receptor and that PKC‐MAPK pathway is responsible for mediating the effect of PGE2 on the basolateral K channels.
Role of BK‐β4 in hypo‐osmotic activation of large conductance, Ca‐sensitive K (BK) channels expressed in HEK293 cells.
Authors: Bari Muhammad R , Holtzclaw J. David , Grimm P. Richard , Settles Deann C. , Foutz Ruth M. , Sansom Steven C.
Published: June 2021

BK channels, which are comprised of pore forming α and accessory β‐subunits, have been shown to eliminate intracellular K in response to hypo‐osmotic swelling. However, the role of the β subunits in this process has not been determined. We used HEK293 cells stably expressing BK‐α and examined a role for transiently expressed BK‐β4 (90% expression rate) to modulate the response of BK to cell swelling. Using patch clamp analysis, we determined NPo (cell attached, ‐Vp = −40 mV) and the % of observed channels per patch. The NPo and % channels per patch of BK‐α expressed alone in cells in isotonic (290 mOsm) solution was 0.71±0.05 (n=3) and 53%, respectively. This value was not significantly different from the NPo and % channels per patch for BK‐α in hypotonic (200 mOsm) solution (0.62±0.17, n=3 and 41%, respectively). The NPo and % channels per patch of BK‐α plus BK–β4 was significantly greater in hypotonic solution (1.32±0.15, n=3 and 76%, respectively) than in isotonic solution (0.70±0.15, n=8 and 41%, respectively). Compared to isotonic solution, when cells expressing BK‐α/β4 were in hypotonic solution, BK‐α exhibited more intense immuno‐staining at the plasma membrane and BK‐β4 was increased at the plasma membrane (western blot; OD = 0.20 in isotonic and 0.49 in hypotonic). These data suggest that the BK‐β4 subunit is necessary for trafficking of BK‐α and BK‐β4 to the plasma membrane in response to hypo‐osmotic stress.
Seasonal changes in thermoregulatory and hormonal changes in young and elder subjects
Authors: Sato Maki , Kanikowska Dominika , Inukai Yoko , Shimizu Yuuki , Nishimura Naoki , Sakurai Hiroki , Iwase Satoshi , Sugenoya Junichi
Published: June 2021

In Japan, there are four seasons, where environmental temperature changes very widely. With annual climate changes, body temperature regulation as well as hormonal regulation may be changed. The purpose of this study is to investigate the seasonal changes in temperature and hormonal regulation in elder subjects (69 ± 4.6 yrs; mean ± SD) and young subjects (21 ± 1.3 yrs; mean ± SD). The experiments were performed in the climatic chamber set at 26°C and 50% RH during spring, summer, autumn and winter. Each subject immersed his lower legs in hot water at 40 °C for 30 min in a sitting position. Core (tympanic) temperature, skin temperature and forearm sweat rates were recorded continuously. Blood was taken in pre‐immersion and in post‐immersion to measure the concentrations of antidiuretic hormone, plasma renin activity, angiotensin II, aldosterone, TSH, fT3 and fT4. The results obtained in spring and summer experiments showed that the rise of core body temperature and sweat rate during hot water immersion was lower in elder group than in young group. We will discuss the entire profile of the seasonal changes in thermoregulatory and hormonal function in young and elder subjects when the experiments in autumn and winter are completed.
Haematocrit and urine volume response to acute intravenous saline and oral water loading in normal volunteers
Authors: Chuen M. Jennyfer Ng Kam , Lip Gregory Y. H. , MacFadyen Robert J.
Published: June 2021

Introduction: Haematocrit (Hct) is widely recognized as a marker of haemodilution. Acute oral water (OW) and intravenous saline (IVS) have variable effects on blood volume. We aimed to test the utility of repeated measures of Hct in mapping this response.

Methods: 30 normal volunteers (16 male; age 48.7 ± 8.6, range 48–63; body mass index 24.7 ± 2.9 Kg/m2, range 19.1–31.9) were given in random order, no intervention (C), OW (1.5 L over 20 minutes) or IVS (1.5 L over 20 minutes) on 3 separate days. Hct was measured at 0, 0.5, 1, 2, 4, 6 and 8 h, and urine volume was measured at 4 and 8 h post intervention.

Results: Baseline Hct was similar before all treatments [C 40.8 (3.5); OW 41.5 (3.4); IVS 41.9 (4.3); p=0.083] and at 8h [C 39.4 (3.0); OW 39.5 (2.9); IVS 38.7 (3.3); p= 0.118]. On C, there was a significant decrease in Hct between 0 and 0.5 h [41.0 (3.4) versus 38.9 (2.8); p<0.0001]. The fall in Hct was significant and greater for IVS compared to OW, but not between C and either IVS or OW (Figure 1). Urine output rose significantly following both OW and IVS compared to C, with larger increase following OW (Figure 2).

Conclusion: The small relative rise in Hct following OW compared to C suggesting a transient reduction in blood volume is in keeping with previous studies. Urine volume response following OW is in contrast to that seen with Hct changes. The mediators of this differential response are unclear.

Funding: Haemostasis Thrombosis and Vascular Biology Unit charitable funds.
Aquaporins along the secretory pathway of the lactating bovine mammary gland
Authors: Mobasheri Ali , Kendall Bryony , Maxwell Judith , Sawran Ami , German Alexander , Marples David , Luck Martin , Royal Melissa
Published: June 2021

In this study we used custom‐designed rabbit polyclonal antisera to examine the expression and cellular localization of aquaporins along the secretory pathway of actively lactating bovine mammary glands by immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, connective and adipose tissues. AQP1 was found in capillary endothelia throughout the lactational mammary gland in addition to myoepithelial cells underlying teat duct epithelia. Expression of AQP2 and AQP6 was not detected and AQP9 was only found in leukocytes. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. AQP4 was observed in selected epithelial cells in the teat, cistern and secretory acini. AQP5 expression was confined to acini and glandular structures. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.
Hydrochlorothiazide causes sodium sensitization in rats
Authors: Grobe Connie L , Johnson Alan Kim
Published: June 2021

Sodium is an important ion for physiological functions, especially the maintenance of membrane potentials and osmotic gradients. The balance of sodium within the body is regulated both through sodium intake (primarily through food and drink), and output (in urine, saliva, and other bodily fluids). Previous studies have shown that repeated acute sodium depletion with the diuretic/natriuretic drug furosemide causes behavioral sensitization as indicated by the enhanced intake of sodium containing solutions (i.e., salt appetite) in response to the induced deficits. In humans, the diuretic compound hydrochlorothiazide (HCZ) is commonly used to treat hypertension. The present study was designed to examine if HCZ causes behavioral sensitization in the absence of a sodium deficiency in order to determine the mechanisms through which salt appetite is enhanced. Sprague‐Dawley rats maintained on standard‐sodium chow with access to 0.3 M NaCl and distilled water were injected with either vehicle or 2.5 mg/kg HCZ for 2 weeks. Ad lib intake of 0.3 M NaCl was significantly increased during HCZ treatment and importantly remained elevated beyond the washout period. In this paradigm, rats never experienced severe sodium deficiency, yet 0.3 M NaCl intake was increased even under ad lib conditions. These data have implications for humans treated with HCZ and directed to consume a low sodium diet. (Support: NIH HL14388 & HL007121).
Chronic treatment with a peroxynitrite scavenger attenuates blood pressure and improves renal hemodynamics in angiotensin II (ANG II) induced hypertensive rats
Authors: Matavelli Luis C , Wells Joel E , Castillo Alexander , Majid Dewan SA
Published: June 2021

ANG II induces the production of both nitric oxide (NO) and superoxide (O2−). NO reacts with O2− to form peroxynitrite (ONOO−). To determine the role of ONOO− in the pathogenesis of ANG II induced hypertension, we examined the responses to chronic administration of the ONOO− scavenger hesperitin (Hesp), an inhibitor of ONOO−‐mediated nitration of tyrosine, in ANG II induced hypertension. Four groups of Sprague‐Dawley rats were treated for 2‐wks: group 1 (G1) was given ANG II (osmotic minipump, 65ng/min; n=9); G2 was given ANG II + Hesp (oral gavage, 50 mg/kg/day; n=12); G3 was given Hesp alone (n=8); and, G4 was the non‐treated control (n=5). Systolic blood pressure (SBP) was measured every 3–4 days using tail‐cuff plethysmography. ANG II treatment in G1 increased SBP (121 ± 2 to 162 ± 4 mmHg) that was partially attenuated with Hesp treatment in G2 (149 ± 3 mmHg; p<0.05). SBP was similar in G3 and G4 (130 ± 1 and 127 ± 1 mmHg). After 2‐wks of chronic treatment, rats were anesthetized and subjected to renal hemodynamics and excretory function using standard clearance techniques. Renal blood flow was decreased in G1, but it was improved in G2 with Hesp treatment (G1, 7.0 ± 0.4; G2, 8.7 ± 0.2*; G3, 8.1 ± 0.5; G4, 9.1 ± 0.7* ml/min/g; *p<0.05 vs G1). Hesp treatment also increased glomerular filtration rate (G1, 1.11 ± 0.04; G2, 1.29 ± 0.03*; G3, 1.22 ± 0.05; G4, 1.24 ± 0.03 ml/min/g) and sodium excretion (G1, 0.6 ± 0.1; G2, 0.8 ± 0.1*; G3, 0.7 ± 0.1; G4, 0.8 ± 0.1 μmol/min/g) compared to G1. These data indicate that ONOO− plays a mechanistic role in the development of ANG II induced hypertension by influencing renal hemodynamics and excretory function. Supported by NHLBI grant RO1‐HL066432.
Kidney GFP expression through various techniques of Lentivirus (LV) delivery
Authors: Chen Gin‐Fu , Tain You‐Lin , Baylis Chris
Published: June 2021

The kidney is one of the most complicated organs to transfect due to heterogeneity of its structure, cellular makeup, and high rate of blood flow, reducing “dwell‐time”. We investigated methods of LV delivery to establish optimal routes allowing greatest and localized kidney expression. SD rats were injected with 1x107TU LV expressing GFP, delivered by: 1) Jugular vein injection (6ml in 10 sec; hydrodynamic delivery) and 2) carotid injection (0.5ml); 3) Intraparenchymal (IPC) injection (30–200ul) (border of inner and outer medulla) with normal renal blood flow (RBF) and 4) IPC injection with 7min cessation of RBF; 5) Retrograde ureter injection with the ureter obstructed for 15min (70ul). After on month the kidney, liver and lung was fixed for IHC to determine the GFP protein. Highest renal expression was by jugular vein injection but also widespread in other organs. The IPC injection with normal RBF gave highest level of transfection in the injected kidney and variable distribution to the contralateral kidney that increased with higher injected volume. Restricting RBF to increase dwell time actually decreased transfection in the injected kidney. Ureter delivery gave the most localized but weak transfection. Long lasting (>1 month) and stable transfection is possible in all regions of the kidney including the glomeruli. The expression and specificity can be varied by different delivery methods.
Flurbiprofen Inhibits Glioblastoma Proliferation and Migration by a COX‐Independent Mechanism
Authors: Kohler Missia , Qadri Yawar J , Fuller Catherine M , Benos Dale J
Published: June 2021

Glioblastoma multiformes (GBMs) are primary brain tumors that are difficult to treat. Non Steroidal Anti‐Inflammatory Drugs (NSAIDs) are a promising therapy for colon and prostate cancer. NSAIDs are classically described as non‐selective inhibitors of cyclooxygenase (COX) enzymes, which form prostaglandins that mediate processes such as inflammation and vasoconstriction. While there is evidence that upregulation of COX‐2 in GBMs coincides with a worse prognosis for patients, it is unclear whether NSAIDs affect GBMs through a COX‐mediated mechanism. This study examined whether a NSAID, flurbiprofen, affects GBMs through a COX–dependent mechanism. COX‐1 and COX‐2 were detected in all GBM cell lines using RT‐PCR. Using a tetrazolium‐based cell viability assay, flurbiprofen decreased the proliferation of a GBM cell line in a dose dependent manner. However, this effect was seen at similar doses (≥ 500 μM) with both COX‐active and COX‐inactive enantiomers. GBM cell migration was also measured using an electric cell‐substrate impedance sensing system, and migration was almost completely inhibited by 500 μM flurbiprofen in a non‐enatioselective manner. Ultimately, although COX‐1 and COX‐2 are present in glioblastomas, they do not appear to be involved in the effects of flurbiprofen on glioma migration and proliferation. This study was supported by NIH grant CA101952 and the AMA Foundation.
Azelnidipine inhibits the sympathetic nerve activity and improves baroreflex control of heart rate via anti‐oxidant effect in the rostral ventrolateral medulla
Authors: Konno Satomi , Hirooka Yoshitaka , Kishi Takuya , Koga Yasuaki , Araki Shuuichiro , Sunagawa Kenji
Published: June 2021

Azelnidipine (AZL), a long‐acting dihydropyridine calcium channel blocker, is known to inhibit sympathetic nerve activity (SNA). We previously demonstrated that oxidative stress in the rostral ventrolateral medulla (RVLM) in the brainstem causes the sympathetic hyperactivity. The aim of this study was to determine whether AZL inhibits the SNA and improves the baroreflex sensitivity (BRS) via anti‐oxidant effect in the RVLM. AZL (20 mg/kg/day, n=5) or hydralazine (HYD; 20 mg/kg/day, n=5) was orally administered for 30 days in stroke‐prone spontaneously hypertensive rats (SHRSP). Thiobarbituric acid‐reactive substances (TBARS) levels in the RVLM were measured as a marker of oxidative stress. Reductions in SBP were similar between the two groups (−32±11mmHg vs −28±9mmHg, ns). HR increased significantly in HYD but did not change in AZL. Urinary norepinephrine excretion (UNE), measured as a marker of SNA, decreased significantly in AZL (−0.25±0.06μg, P<0.01), whereas increased significantly in HYD. BRS, measured by sequence method, was significantly improved in AZL to a great extent than in HYD (8±2ms/mmHg vs 4±2ms/mmHg, P<0.05). TBARS levels in the RVLM decreased significantly in AZL (−0.21±0.09 μmol/g wet wt, P<0.01) but not in HYD. In conclusion, azelnidipine inhibits the SNA and improves the BRS possibly via its anti‐oxidant effect in the RVLM.
Activated Projection from Arcuate Nucleus to Rostral Ventrolateral Medulla by Electroacupuncture: relation to β‐endorphin
Authors: Guo Zhi‐Ling , Liu Yu , Moazammi Ali R. , Longhurst John C.
Published: June 2021

Electroacupuncture (EA) at the Neiguan‐Jianshi acupoints (P5–P6, overlying the median nerve) attenuates sympathoexcitatory responses through μ opioid receptors in rostral ventrolateral medulla (rVLM). Arcuate nucleus (ARC) in the hypothalamus is a critical brain area in which perikaryia containing β‐endorphin are distributed. However, direct neuronal projection from ARC to rVLM that could participate in central neural processing during EA has not been identified. The present study evaluated neuronal projection from the ARC to the rVLM, especially respect with neurons activated by EA. After unilateral microinjection of a rodamine‐conjugated microsphere retrograde tracer into the rVLM, rats were subjected to sham control or EA at P5–P6 acupoints for 30 min. Perikarya containing the microsphere tracer were found in the ARC. Some of these neurons (27%) co‐localized with β‐endorphin. Compared to controls (n=5), c‐Fos immunoreactivity and neurons double‐labeled with c‐Fos, an immediate early gene, and the tracer were increased significantly in the ARC of EA‐treated rats (n=7, all P<0.05). Moreover, some neurons were triple‐labeled with c‐Fos, the tracer and β‐endorphin in the ARC following EA. These results suggest that a β‐endorphin‐containing neural pathway projecting from the ARC to the rVLM may participate in EA‐modulation of premotor sympathetic activity (supported by NIH, HL‐72125 and HL‐63313).
Exercise training improves central NMDA‐induced erectile dysfunction in diabetes
Authors: Zheng Hong , Liu Xuefei , Patel Kaushik
Published: June 2021

Erectile dysfunction is a serious and common complication of diabetes mellitus. Apart from the peripheral actions, central mechanisms are also responsible for the penile erection. The goal of the present study was to determine the impact of exercise training (ExT) on the central NMDA‐induced erectile dysfunction in streptozotocin (STZ)‐induced diabetic rats (Type 1 diabetes‐T1D). In rats with T1D, ExT (treadmill run for 3 weeks) significantly improved the blunted erectile response to NMDA (50ng) microinjection within the paraventricular nucleus (PVN) (T1D+ExT 3.0±0.6 penile erection/rat; T1D 0.5±0.3 penile erection/rat within 20mins). Consistent with the erectile response, other behavior responses including yawning, stretching, chewing and grooming induced by central NMDA microinjection were significantly increased in T1D rats after ExT. Furthermore, ExT also improved erectile dysfunction induced by central administration of exogenous nitric oxide (NO) donor, sodium nitroprusside (SNP) in T1D rats. These results suggest that ExT may have beneficial effects on the erectile dysfunction in diabetes through improvement of central NO bioavailability within the PVN.
CVL‐projecting NTS neurons receive input from a distinct subset of vagal afferents
Authors: Aicher Sue A , Hermes Sam M
Published: June 2021

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). We previously showed that two tract tracers, cholera toxin B subunit (CTb) and isolectin B4 (IB4), label distinct populations of vagal afferents. In this study, we tested whether these fibers contact different populations of NTS neurons, including NTS neurons that project to the caudal ventrolateral medulla (CVL). To label CVL‐projecting NTS neurons, the retrograde tracer Fluorogold (2% in 0.9% saline; 50 nl, Fluorochrome Inc.) was injected into CVL; vagal afferents were labeled with injections of CTb and IB4 (2% solution, 1–4 ul) into the vagus nerve. Male Sprague‐Dawley rats (350–425 g) were used and surgeries were performed under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined using dual immunocytochemistry for Fluorogold (rabbit anti‐FG, Chemicon) and either CTb (goat anti‐CTb, List Biological Laboratories) or IB4 (goat anti‐IB4, Vector Laboratories). Primary antibodies were visualized using fluorescent secondary antibodies. By confocal microscopy, we found that CVL‐projecting NTS neurons were more than twice as likely to receive somatic input from CTb‐labeled afferents (79%) than IB4‐labeled afferents (34%). In somatic nerves, CTb and IB4 label myelinated and unmyelinated afferents, respectively. Therefore, our findings suggest that CVL‐projecting NTS neurons receive input mainly from myelinated vagal afferents that contain a distinct population of transporters, neurotransmitters, and receptors.

Support: NIH HL56301
Exploring the mechanisms for pH regulation in central respiratory chemoreceptors using a mathematical model
Authors: Cordovez Juan M , Clausen Chris , Moore Leon C , Solomon Irene C
Published: June 2021

Over the past few years, it has proven difficult to find a single marker that unequivocally identifies neurons as central respiratory chemoreceptors (CRCs). Previous studies in brainstem slices from neonatal rats suggested that CRC neurons show no pHi recovery during hypercapnic acidosis (HA) while non‐chemosensitive neurons show significant pHi recovery under the same conditions. This lack of pHi recovery, however, has been demonstrated in non‐chemosensitive neurons in adults, suggesting that is not a unique characteristic of CRCs, but rather a characteristic of neurons in brainstem respiratory regions. To identify potential mechanisms that may be responsible for this lack of pHi recovery during HA, we developed a mathematical model that allows us to investigate the role of different pHi regulatory mechanisms. Our previous work demonstrated that different Na+/H+ exchanger isoforms (i.e., NHE1, NHE3, NHE5) was not sufficient to explain this behavior during HA. Here, we examine a role for the HCO3−/Cl− exchanger (AE) in this pHi recovery response. We found that the model simulates neurons that show no pHi recovery during HA by increasing the activity of the AE while keeping the carbonic anhydrase activity low. This observation suggests that AE may be a possible cellular mechanism responsible for lack of pHi recovery, and therefore, it may represent a potential way to identify these neurons. Supported by NS04532.
Vitamin E Suppresses the Enhancement of Phagocytosis and Oxidative Burst of Neutrophil to E.coli by Systemic Hypoxia
Authors: Wang Jong‐Shyan , Liu Huang‐Chun
Published: June 2021

This study elucidates how various hypoxic interventions affect phagocytosis and oxidative burst of neutrophil to E.coli following treatment with/without vitamin E. Twenty‐four healthy sedentary men were randomly divided into vitamin E (n=12) and placebo (n=12) groups. These subjects were randomly exposed to 12% (severe hypoxia, SH), 15% (moderate hypoxia, MH), and 18% (light hypoxia, LH) O2 for 2 hours in a normobaric hypoxia chamber. Our results demonstrated that SH increased phagocyotic and oxidative burst activities as well as CD11b, CD16b and CD62L expressions of neutrophils, which correlated with an increase in urinary isoprostane level and decreases in plasma total antioxidant content. Following the treatment of the subjects with 1000 IU vitamin E, SH did not significantly alter isoprostane/total antioxidant levels, phagocyotic/oxidative burst activities and adhesion molecules expressions of neutrophils. Additionally, both neutrophil activity and redox status were constant in response to MH and LH in the two groups. Therefore, we conclude that SH promotes phagocytosis and oxidative burst of neutrophils, and this hypoxia effect was ameliorated by pretreatment with vitamin E.
Determination of MLSS: two tests are better than one
Authors: Yaeger Daniel B. , Stavrianeas Stasinos
Published: June 2021

The Maximum Lactate Steady State (MLSS) is often estimated using a hybrid VO2max/Lactate Threshold (LT) test even though the literature suggests that it yields a poor estimate of MLSS. We hypothesized that a second incremental test (SIT) improves estimation of MLSS. Trained cyclists (n=5) completed 2 incremental tests on a calibrated computer‐controlled cycle ergometer. After performing a VO2peak test the LTinitial was identified as visual deflection from baseline. In the SIT workload increased in 10 W increments from 30 W below to 30 W above the LTinitial. The LTSIT was then identified using the visual deflection method. To verify the LTSIT as the MLSS, participants performed three 30‐minute bouts at 95%, 100%, and 105% of LTSIT intensity. Although the three intensities differed in workload by only 10 ± 1 W, Heart Rate, VO2, and blood lactate levels were different between the three workloads at all times (p < 0.01). The LTSIT was 99 ± 4% of MLSS, whereas the LTinitial was 93 ± 10% of MLSS intensity. Furthermore, the SIT corresponded to 100% of MLSS for 2 of the cyclists and was within ± 5% of MLSS for all 5 cyclists. We conclude that the SIT greatly improves estimation of MLSS as compared to a single‐test protocol.

Supported by NSF‐DUE‐CCLI #0511219, the Mary Stuart Rogers Foundation, and an APS Undergraduate Summer Research Fellowship (DBY).
Superficial and deep aponeuroses movements in the human medial gastrocnemius during isometric plantarflexion contraction
Authors: Kinugasa Ryuta , Shin Dongsuk , Hodgson John A , Edgerton Reggie V , Sinha Shantanu
Published: June 2021

In pinnate muscle such as medial gastrocnemius (MG), the aponeuroses are located on both sides of the muscle. It has been reported that superficial (SA) and deep aponeuroses (DA) of the MG are stretched homogenously along its length by isometric contraction. However, it is impossible to elongate the entire aponeuroses, because the proximal edge of each aponeurosis attaches to the femur. We hypothesized that at certain locations each aponeurosis is shortened by isometric contraction. Velocity encoded‐phase contrast MRI was used to measure velocity and displacement of each aponeurosis in 4 subjects during repeated plantarflexion (20 and 40% loads). The velocities of each aponeurosis increased and was positive during the contraction phase, declined to zero at peak torque, and then became negative during the relaxation phase, suggesting both moved proximally during contraction and moved distally during relaxation. The displacement moved the least in middle region of the SA and proximal region of the DA along its length. These results indicate that the SA lengthened in the proximal region and shortened in the distal region, and the DA shortened in the proximal region. However, the displacement of the distal region may have been overestimated since the length of this region was of the same order as the fascicle length.

This work was supported by National Institute on Arthritis and Musculoskeletal and Skin Diseases Grant.
Physiological Responses of Elliptical and Treadmill Exercise in Collegiate Distance Runners
Authors: Garlatz T.J. , Brilla L.R. , Knutzen K.M. , Chalmers G.R.
Published: June 2021

Physiological responses of collegiate distance runners were compared in elliptical and treadmill exercise. Eighteen male subjects (age 19–23 yrs) performed four separate five‐minute exercise trials over two days, with a treadmill and an elliptical machine. One trial for each apparatus was performed at 75% of the subject's heart rate reserve (HRR), while the other trial was at 90% HRR. Oxygen consumption (VO2) and ratings of perceived exertion (RPE) were measured during all trials, and blood lactate concentrations were measured following exercise at 90% HRR. Significantly (P < 0.007) higher VO2 was measured during treadmill running (48.0 and 61.5 ml/kg/min at 75% and 90% HRR respectively) than during elliptical exercise (43.3 and 58.3 ml/kg/min). No significant differences existed in RPE between treadmill and elliptical exercise at 75% HRR, however, higher RPE values were observed in elliptical exercise (16.2) compared to treadmill running (14.6) during 90% HRR trials (P<0.05). Higher blood lactate concentrations were found in elliptical (7.97 mmol/L) than treadmill (4.77 mmol/L) in 90% HRR trials (P<0.05). Effect size analysis showed the effect of modality attributed 50.6% of the difference in blood lactates to differences between elliptical and treadmill exercise. Runners and coaches should consider these results when using the elliptical trainer as a cross training modality.