bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial restoration of TG secretion via bile salt supplementation is sufficient to fully restore GIP secretion in bile‐diverted rats
Authors: Li Luke , Yang Qing , Tso Patrick , Yoder Stephanie
Published: June 2021

The incretin hormone glucose‐dependent insulinotropic polypeptide (GIP) is secreted by enteroendocrine K cells in response to ingested nutrients. Previously, it has been shown that fat‐stimulated GIP secretion is suppressed in dogs with chronic bile diversion but is restored upon oral feeding of bile in a dose‐dependent manner, suggesting that an aspect of intraluminal bile is required for GIP secretion. Our aim was to explore the effect of sodium taurocholate (NaTC) supplementation on the secretion of GIP in the bile‐diverted lymph fistula rat. To test this aim, we inserted a duodenal feeding tube and cannulated the intestinal lymphatic duct of male Sprague‐Dawley rats. A subset of these animals also underwent a bile duct cannulation. After an overnight recovery, bile‐diverted and bile‐intact animals were provided a 3 ml Ensure bolus. A second group of bile‐diverted animals were infused with 19 mM NaTC at 3 ml/h and provided a 3 ml Ensure or saline bolus. Lymph was collected for 3 h and analyzed for TG and GIP. In the bile‐diverted animals, postprandial TG and GIP secretion was suppressed. However, upon the addition of NaTC to these animals, TG secretion was partially restored, while GIP secretion was fully restored. The data suggest that partial restoration of TG via the addition of NaTC is sufficient to fully restore GIP, indicating a potential link between TG absorption and GIP secretion.

Funded by DK059360 NIH/NIDDK.
Sigma1 receptors and neuroblastoma differentiation
Authors: Schrock Joel Michael , Kim Felix J , Pasternak Gavril W
Published: June 2021

The sigma1 receptor is an integral membrane protein of ~26kD which shares no structural homology with any traditional receptor family. Sigma1 binding sites are highly conserved across mammalian species and are present in a broad range of tissue, with particularly high expression in distinct regions of the CNS. Given the CNS expression of sigma1 receptors and the neuro‐active properties of many sigma1 receptor ligands, we hypothesize that sigma1 receptors may play a role in neuronal differentiation. Differentiating neuroblastoma cells upregulates sigma1 receptors, with high levels in distinct vesicular structures throughout neurite outgrowths in contrast to an endoplasmic reticulum distribution in undifferentiated cells. These data suggest an association of sigma1 receptors with neuronal differentiation.

Funded by grants to GWP (DA0641, DA02615 and DA00220) from the National Institute of Drug Abuse
Intestinal Microbiome in Transporter Null Mouse Models
Authors: Engevik Melinda A , Worrell Roger T
Published: June 2021

A growing interest exists in the interrelation between host organism and bacteria colonization as exemplified by the NIH Human Microbiome Project. This study sought to determine the effect of altered intestinal ion transport on the luminal micobiome in mice. Ammonia is both used and produced by gastrointestinal bacteria. Changes in ammonia have been associated with shifts in bacteria species in vitro. Thus changes in transport function likely influence bacterial location and quantity. NKCC1 and cH/K‐ATPase are both implicated in epithelial cell ammonia transport. Universal, family specific, and species specific bacterial primers based on 16S ribosomal DNA were used with real time PCR to quantify bacterial populations from luminal flushes of mouse terminal ileum, ceacum, proximal and distal colon. Flushes from NKCC1−/− and cH/K‐ATPase−/− mice showed no change in total bacteria present when compared to wild type littermates. Using family specific primers, Bacteroides and Firmicutes family members were also unchanged. Interestingly however, Bifidobacterium, a probiotic bacteria, was decreased in both ileum and ceacum of null mice, yet unchanged in colon. These data suggests that long term differences in intestinal ion transport can result in a decrease of “good” bacterial species which may increase the susceptibility of the host to intestinal dysfunction. Supported by NIH DK079979.
Trans effects of Na + and glucose on Na+/glucose co‐transport
Authors: Hummel Charles S. , Hirayama Bruce A. , Loo Donald D.F. , Wright Ernest M.
Published: June 2021

The human intestinal Na+/glucose co‐transporter 1 (SGLT1) is responsible for glucose absorption on the brush border of the small intestine. Previously, it has been shown that two Na+ ions bind to the extracellular surface of SGLT1 prior to sugar binding and translocation. However, the order of release on the cytoplasmic surface is not well understood. Molecular dynamics simulations based on the X‐ray crystal structure of a homolog of SGLT1 suggest that Na+ is the first substrate to dissociate from the cytoplasmic (i.e. trans) side of the transporter during a reaction cycle. To test this, we obtained whole cell patch clamp recordings in human SGLT1‐expressing HEK293T cells with varying trans substrate concentrations. Compared with control experiments under zero trans conditions, 85 mM trans glucose reduced the Vmax for transport by greater than two‐fold. In contrast, trans Na+ alone had no effect on Vmax. These results are consistent with kinetic modeling that predicts sugar is first to dissociate on the cytoplasmic surface of SGLT1. This work was supported by NIH grant #DK19567 and pre‐doctoral fellowship #DK821532 to CSH.
Time‐course expression of PGC‐1 in acute inflammation
Authors: Llimona Flávia , Lima‐Salgado Thais Martins , de Souza Heraldo Possolo
Published: June 2021

Peroxisomal proliferator‐activated receptor coactivator 1 (PGC1) controls expression of several genes involved in cell energy homeostasis. PGC1 isoforms (α and β) are present in tissues with high oxidative metabolism and enhance mitochondrial biogenesis, β‐oxidation of fatty acids and gluconeogenesis in response to exposure to cold, fasting and exercise. We hypothesized that PGC1 expression may also be modulated during inflammatory response. Systemic inflammation was induced in male Wistar rats by acute pancreatitis (AP), caused by retrograde perfusion of sodium taurocholate into biliopancreatic duct. Animals were sacrificed after 2, 24 and 48h and PGC1 α and β expression were measured by quantitative PCR.

Systemic inflammation was confirmed by increased white blood cell count and circulating cytokines. PGC1α expression was increased in liver and spleen (2.0±0.1 and 4.3±0.5 fold vs sham, respectively), 48h after AP. At same time, lungs and leukocytes presented enhanced PGC1β expression (2.2±0.3 and 6.2±1.3 fold vs sham, respectively). On the other hand, PGC1β expression was decreased in lymph nodes 2h after AP induction (0.5±0,1 vs sham). Thus, we conclude that during systemic inflammation there is increased expression of PGC1α and β especially in the later stages of the disease, which may be related to the increasing demand of energy in different tissues. Financial Support: CAPES, FAPESP# 09/1141‐1
Calcium interactions with divalent metal–ion transporter–1 (DMT1)
Authors: Shawki Ali , Mackenzie Bryan
Published: June 2021

Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interaction is not understood. DMT1 is the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border and, since the scallop DMT1 homolog has been reported to operate as a calcium transporter [FEBS Lett 579, 2727 (2005)], we tested the hypothesis that human DMT1 also transports calcium. We expressed human DMT1 in RNA‐injected Xenopus oocytes and examined DMT1 activity using radiotracer assays and the voltage clamp. At pH 5.5, DMT1 did not mediate significant uptake of 2 μM or 100 μM 45Ca2+ (P > 0.25). Instead, we found that 20 mM Ca2+ inhibited the uptake of 2 μM 55Fe2+ (P < 0.001) and blocked the Fe2+‐evoked currents at all membrane potentials from −150 to +50 mV. Ca2+ decreased the Vmax for 55Fe2+ uptake without effect on K0.5 suggesting that Ca2+ is a noncompetitive inhibitor of DMT1 (Ki ≈ 10 mM). The mechanism of calcium inhibition did not involve intracellular Ca2+ signaling since the effect persisted even after microinjection of EGTA. The alkaline earth metal ions Ba2+, Sr2+ and Mg2+ also inhibited DMT1‐mediated iron‐transport activity. We conclude that Ca2+ is a low‐affinity noncompetitive inhibitor—but not a transported substrate—of DMT1, explaining in part the effect of dietary calcium on iron bioavailability.
Expression of Soluble Epoxide Hydrolase (SEH) in Brains of Female Offspring of Rats Fed High Salt During Pregnancy
Authors: Porter James Paul , Johnson Clinton L , Rogers Kristina
Published: June 2021

SEH inactivates epoxyeicosatrienoic acids (EETs) which may function in the brain to regulate blood pressure. We have previously shown that adult female rats born to mothers fed a high‐salt (HS) diet during pregnancy exhibit a hyperresponsive pressor and tachycardic response to acute stress. Microarray analysis of mRNA from brains of these HS rats suggested that SEH expression was significantly decreased. In the present investigation we used Western blot to compare the expression of SEH protein in whole brains of HS and normal salt (NS) female rats. Relative expression of SEH protein was significantly increased in the HS rats (0.97 ± 0.06 NS vs 1.72 ± 0.32 HS, n=6). Immunohistochemistry using the ABC reaction showed that SEH staining was most prominent in the medial basal region of the hypothalamus in the area of the arcuate nucleus and in regions surrounding the subfornical organ and area postrema. Morphologically the stained cells appeared to be astrocytes. These brain areas are known to be involved in the stress response and/or control of the cardiovascular system. This raises the possibility that long‐term alteration in EETs metabolism in these areas could contribute to the altered stress responsiveness programmed by prenatal high salt. Supported by a BYU Mentored Environment Grant
Enhanced activation of the median pre‐optic nucleus contributes to the activation of the paraventricualr nucleus in heart failure
Authors: Zheng Hong , Liu Xuefei , Patel Kaushik P
Published: June 2021

The purpose of the study was to determine if altered median pre‐optic nucleus (MnPO) mediated input to the paraventricular nucleus (PVN) results in increased renal sympathetic nerve activity (RSNA) in rats with chronic heart failure (HF). The left coronary ligation‐induced HF model in the rat was used. In the MnPO, FosB immunoreactivity (a marker for neuronal activation) increased significantly in HF rats compared to sham‐operated rats. In alpha‐chloralose and urethane anesthetized Sprague Dawley rats, microinjection of N‐methyl‐D‐aspartic acid (NMDA, 50–200pmol) into the MnPO produced dose‐dependent increases in RSNA, blood pressure (BP) and heart rate (HR) (n=6/group) in both sham‐operated and HF rats. The response to NMDA was significantly potentiated (50% at highest dose) in HF rats compared with sham‐operated rats. On the other hand, microinjection of the NMDA receptor antagonist AP5 (4–16nmol) into the MnPO caused decreases in RSNA, BP and HR only in rats with HF but not in sham‐operated rats. Furthermore, prior microinjection of AP5 (4nmol, bilaterally) into the PVN was able to significantly blunt the responses to NMDA in the MnPO. These data indicates that a higher forebrain site (MnPO) contributes to the sympatho‐excitation observed during late stages of HF via increased neuronal activity within the PVN.
Involvement of the cholinergic system of the posterior hypothalamic nucleus (PHN) during the reflex compensatory response to hemorrhage
Authors: Webb Zachary T. , Martin John R.
Published: June 2021

Stimulation of muscarinic receptors within the PHN of the rat evokes a pressor response. It is not known whether this cholinergic system is activated during a depressor event in an effort to overcome the depressed blood pressure. The goal of the present study was to evaluate whether the PHN cholinergic system is activated during the compensatory reflex response to moderate hemorrhage. Since cannabinoids (CBs) appear to modulate acetylcholine release, the effect of the presence of a CB agonist in the PHN during recovery to hemorrhage was also studied. Carbachol (CCh), a cholinergic agonist, methyl‐atropine (MeATR), a muscarinic receptor antagonist, or CP 55,940, a CB1 receptor agonist, were administered into the PHN of urethane‐anesthetized rats following moderate hemorrhage (20% blood loss). None of the treatments affected the percent MAP recovery or the change in heart rate post‐hemorrhage. While CCh did not affect the rate of recovery of the mean arterial pressure (MAP), both MeATR and CP 55,940 decreased the recovery rate of MAP. The decreased rate of MAP recovery in the presence of CP 55,940 was reversed by pretreatment with AM 251, a CB1 receptor antagonist/inverse agonist. Furthermore, CP 55,940 decreased survival compared to saline‐treated rats. These results suggest that cholinergic activity within the PHN increases during the reflex compensatory response to moderate hemorrhage, and that CBs can decrease this activity through the CB1 cannabinoid receptor. (Supported by a grant from the KCOM Graduate Program Committee.)
The involvement of guanylyl cyclase‐G in intestinal ischemia and reperfusion‐induced jejunal apoptosis in mice
Authors: Lo Hui‐Chen , Yang Ruey‐Bing , Huang Yu‐Chan , Lai Chun‐Hong
Published: June 2021

Our previous study indicated that guanylyl cyclase (GC)‐G gene plays a certain role in maintaining jejunal morphology and regulating systemic and local inflammatory responses. In this study, we further investigated the molecular mechanism of GC‐G gene in modulating intestinal apoptosis. Sham operation and 45 min intestinal ischemia with 3h, 6h, 12h, or 24h reperfusion were performed in both male WT and KO mice. Our results showed that KO mice had significantly increased protein expressions of active caspase‐3 (17kDa), cytochrome c, and Bax in the jejunum compared to WT mice before intestinal I/R injury. After suffering from intestinal I/R, active caspase‐3, Bax, and Bcl‐2 were significantly increased at 3h and reversed at 12h in WT mice, whereas those were significantly decreased at 12h in KO mice. There was no significant difference in cytosol IκBα between WT and KO mice before and after intestinal I/R; however, nuclear phosphorylated NFκB was significantly decreased at 24h in KO mice compared to WT mice. In addition, intestinal I/R‐induced increases in phosphorylated JNK1 and Erk1 were occurred at 3h in WT mice and 6h in KO mice. Taken together, our results demonstrated that KO mice have attenuated changes in intestinal morphology and apoptosis signaling pathways when suffered from intestinal I/R. These results suggest that mGC‐G plays important roles in maintaining jejunal morphology and regulating apoptosis.
High sodium intake alters the hemodynamic response to mental stress in normotensive subjects after systemic beta adrenergic blockade
Authors: Silva Bruno Moreira , Elvebak Rachel L. , Knutson Jean N. , Warner Nathaniel D. , Joyner Michael J. , Eisenach John H.
Published: June 2021

We tested the hypothesis that dietary Na+ intake alters the hemodynamic response to mental stress (MS) in normotensive subjects, which could be mediated by β‐adrenergic receptors (ADRB). As part of an ongoing ADRB2 phenotyping protocol, 12 subjects (age 23±5 year) completed 5 days of low (10 mmol day−1), normal (150) and high (400) dietary Na+ intake. We measured heart rate (ECG), mean arterial pressure (MAP, Finometer), and calculated stroke volume (SV), cardiac output (CO) and systemic vascular resistance (SVR) responses to arithmetic MS, before and after administration of propranolol. The high Na+ diet decreased CO (6.3±0.3 vs. 5.7±0.3 l min−1, p<0.01) and increased SVR (1187±47 vs. 1311±68 dynes s−1 cm−1, p<0.01) at rest, but MAP was unchanged (92±2 vs. 93±3 mmHg, p=0.37). During MS, the increase in HR, CO, and MAP was not affected by diet, nor was the decrease in SVR. However, on the high Na+ diet β‐blockade increased resting MAP (93±3 vs. 97±2 mmHg, p<0.01), while the absolute MAP response to MS was unchanged, the ΔMAP was blunted (7.3 vs. 4.3 %, p=0.03) and ΔSV was decreased (1.7±3.5 vs. −5.3±2.0 %, p=0.03). In conclusion, reactivity to MS was not influenced by dietary Na+, but systemic β‐blockade after high Na+ blunted the SV response during MS. This may be suggestive of augmented ADRB function or blunted non‐ADRB mechanisms of ventricular function during MS after high Na+. Support: R‐01 HL‐089331 and CTSA RR‐024150.
Impact of increased muscle sympathetic nerve activity on conduit artery shear rate patterns
Authors: Padilla Jaume , Young Colin N. , Simmons Grant H. , Deo Shekhar H. , Newcomer Sean C. , Sullivan John P. , Laughlin M. Harold , Fadel Paul J.
Published: June 2021

Disturbed flow patterns induce a proatherogenic endothelial cell phenotype; however, the factors accountable for the oscillatory shear profiles in peripheral conduit arteries are not fully understood. We tested the hypothesis that acute elevations in muscle sympathetic nerve activity (MSNA) are accompanied by increases in conduit artery retrograde shear and oscillatory shear index (OSI). Fourteen healthy men (25±1 yrs) performed three sympatho‐excitatory maneuvers: progressive lower body negative pressure (LBNP) from −10 to −40 Torr, cold pressor test (CPT), and 35% maximal voluntary contraction handgrip followed by postexercise ischemia (PEI). MSNA, arterial blood pressure (BP), and brachial artery velocity and diameter were recorded continuously. All maneuvers elicited significant increases in MSNA total activity from baseline; whereas mean BP was only increased during CPT and PEI (p<0.05). Interestingly, retrograde shear (−3.96±1.2 baseline vs. −8.15±1.8 s−1 −40 LBNP, p<0.05) and OSI (0.09±0.02 baseline vs. 0.20±0.02 a.u. −40 LBNP, p<0.05) were primarily increased during LBNP in which elevated MSNA was not accompanied by increases in BP. Together, these data suggest that elevated MSNA may promote an increase in retrograde and oscillatory shear; however, these effects may be influenced by concomitant changes in BP. Support: NIH RO1HL093167, HL52490
Influence of progesterone and estradiol on cardiovagal baroreflex sensitivity in young healthy women
Authors: Martini Emily R , Miner Jennifer A , Brunt Vienna E , Smith Michael M , Kaplan Paul F , Halliwill John R , Minson Christopher T
Published: June 2021

Objective

Our goal was to measure cardiovagal baroreflex (CVBR) sensitivity in young healthy women during gonadotropin‐releasing hormone antagonist (GnRHa), progesterone only (GnRHa+P4), estrogen only (GnRha+E2), and combined progesterone plus estrogen (GnRHa+P4+E2) conditions.

Methods

We suppressed endogenous estrogens and P4 in 31 premenopausal women with GnRHa for 10 days. Subjects were randomized into 2 groups and studied as indicated in figure below. CVBR sensitivity was assessed by lowering and raising blood pressure with intravenous administration of sodium nitroprusside and phenylephrine. CVBR sensitivity was determined as the slope relating R‐R interval and systolic blood pressure.

Results

No differences in slopes were seen on day 4 (trial 1) and day 10 (trial 3) in Groups 1 or 2. Slopes were significantly higher after P4 only (17.18 ± 1.28) compared to GnRHa only (14.07 ± 1.17), P4+E2 (14.12 ± 1.23), and E2 only (12.33 ± 1.21). Estradiol CVBR slopes were significantly lower than GnRHa only and P4+E2. Thus, P4 only > GnRha only = P4 + E2 > E2 only; all p<0.0001.

Conclusions

Exogenous sex hormones in young healthy women significantly altered the CVBR slope. P4 increases and E2 decreases this slope, and appear to antagonize each other.

Supported by NIH Grant HL081671.
Alterations in carotid baroreflex control of arterial blood pressure during the menstrual cycle in young women
Authors: Kim Areum , Deo Shekhar H , Fisher James P , Fadel Paul J
Published: June 2021

Limited studies have suggested that menstrual cycle variations in sex hormones may influence arterial baroreflex control of heart rate (HR) and sympathetic nerve activity; however, results are equivocal. In addition, the baroreflex control of blood pressure (BP) has not been directly examined as pharmacological perturbations were mainly used to assess baroreflex function. Therefore, 5s pulses of neck suction (NS, −60 Torr) and neck pressure (NP, +40 Torr) were applied to load and unload the carotid baroreflex (CBR), respectively in young women during the early follicular (EF, day 1–5, low estrogen, low progesterone), late follicular (LF, day 10–12, high estrogen, low progesterone), and mid luteal (ML, day 20–24, high estrogen, high progesterone) menstrual cycle phases. Resting HR and BP were not different between the 3 phases. Similarly, HR responses to NP and NS were not significantly different between phases. In contrast, mean BP responses to NP were significantly greater in the ML phase (Δ17±1 vs. Δ13±1 EF; Δ13±2 LF mmHg; P<0.05); however, responses to NS were not significantly different between phases (EF Δ−10±2, LF Δ−10±1, and ML Δ−10±1 mmHg). These preliminary findings suggest a maintained CBR control of HR throughout the menstrual cycle whereas concomitant elevations in estrogen and progesterone selectively augment CBR‐mediated BP responses to hypotension without effect on CBR responses to hypertension.

Supported by R01HL093167.
Augmented skeletal muscle metaboreflex function in hypertensive adults
Authors: Delaney Erin Paul , Edwards David G. , Rose William C. , Fadel Paul J. , Farquhar William B.
Published: June 2021

Recent animal studies have reported that exercise pressor reflex mediated increases in blood pressure are exaggerated in hypertensive (HTN) rodents. Whether these findings can be extended to human hypertension remains unclear.

PURPOSE

We sought to investigate the metabolic component of the exercise pressor reflex (muscle metaboreflex) in HTN humans.

METHODS

Mean arterial pressure (MAP), muscle sympathetic nerve activity (MSNA), and metabolites were measured at baseline, during static handgrip (HG) exercise at 30% and 40% maximal voluntary contraction, and during post‐exercise ischemia (PEI) to isolate the muscle metaboreflex in normotensive (NTN; n=23; 60±1 years) and HTN (n=15; 63±1 years) subjects. MAP and MSNA responses were also quantified during a non‐exercise sympatho‐excitatory stimulus (cold pressor test).

RESULTS

The changes in MAP and MSNA from baseline to PEI were greater in HTN subjects after the 30% HG trial (MAP: NTN, Δ 13±1 vs. HTN, Δ 16±2 mmHg, p=0.05; MSNA: NTN, Δ 54±10 vs. HTN, Δ 101±11 AIU/min, p=0.009) and after the 40% HG trial (MAP: NTN, Δ 15±1 vs. HTN, Δ 22±2 mmHg, p=0.007; MSNA: NTN, Δ 57±10 vs. HTN, Δ 136±11 AIU/min, p≤0.001). There were no group differences in metabolite concentration. MAP and MSNA responses to the cold pressor test were not different between groups.

CONCLUSION

These data suggest that muscle metaboreflex function is augmented in HTN humans.

Supported by HL07485
Anti‐hypertensive action of intermittent hypoxia in young spontaneously hypertensive rats
Authors: Jasti Dinesh , Manukhina Eugenia , Willams Arthur , Howard Linda , Downey Fred
Published: June 2021

Hypobaric intermittent hypoxia (IH) has been shown to have anti‐hypertensive action, and IH therapy is used clinically to treat hypertensive patients in Eastern European countries. The present study evaluated the effects of normobaric IH on the progression of hypertension in spontaneously hypertensive rats (SHR). Four‐five week old SHR were subjected to either IH (n=15) or to sham IH (n=10). IH rats were exposed for 20 consecutive days to intermittent hypoxia (5 to 8 cycles of 9.5–10 % O2, each 5 to 10 min, separated by 4 min normoxia).Sham rats were subjected to the same regimen but with no change in inspired O2. Systolic blood pressure (SBP) was measured in all the animals using the tail cuff method (IITC Life Science). Pre‐treatment SBP (mean ± SEM) of IH rats (131 ± 2 mmHg) and sham rats (132 ± 3 mmHg) were similar. After 20 days of IH, SBP of IH rats (162 ± 2 mmHg) was significantly lower (P < 0.001) than that of sham rats (202 ± 3 mmHg). IH associated with sleep apnea is known to cause hypertension. However, the IH regimen of this study, which differs from the IH of sleep apnea, clearly blunted the development of hypertension in young SHR. The anti‐hypertensive mechanism of this regimen of IH merits further investigation.
Early Intervention is important for inhibiting pulmonary hypertension progression
Authors: Ou Jing‐song , Ou Zhi‐jun , Wei Wei , Huang Da‐da , Wang Zhi‐ping , Zhang Xi
Published: June 2021

L‐arginine(L‐Arg) has been shown to attenuate pulmonary hypertension (PH). To investigate how L‐Arg affects the nitric oxide (NO) to inhibit PH, SD rats were treated with monocrotaline (MCT) once ± L‐Arg for 3 or 5 weeks (wk). The right ventricular systolic pressure (RVSP), NO level in plasma, superoxide anion (O2·−) generation in lung and pulmonary arteries wall thickness were measured. The proteins expressions and the association in lung were determined by western blot and immunoprecipitation. MCT increased RVSP, pulmonary arteries wall thickness and decreased endothial NO synthase (eNOS) and heat shock protein 90 (HSP90) expression and association, phosphorylation of eNOS at S1177 and T495. O2·− was increased at 3 wk post‐MCT but not at 5 wk post‐MCT. However, NO production was decreased at 3 wk post‐MCT and restored to control level at 5 wk post‐MCT despite progressive PH. L‐Arg can decrease RVSP, O2·− generation, pulmonary arteries wall thickness and increase NO production. L‐Arg can increase eNOS expression, phosphorylation at S1177, the association of eNOS and HSP90 at 3 wk post‐MCT but not at 5 wk post‐MCT without altering the HSP90 expression and phosphorylation at T495. Our findings indicate that L‐arg can inhibit the further development of PH despite of losing the preventive effect on protein expression and association to generate NO in late stage PH and emphasize the importance of early intervention of PH.
Synergism between Ca2+ and Rho‐kinase signaling during pulmonary arterial vasoconstriction in chronic hypoxic newborn lamb
Authors: Papamatheakis Demosthenes G , Vemulakonda Srilakshmi , Merritt Travis , Longo Lawrence D. , Blood Arlin B. , Wilson Sean M.
Published: June 2021

Pulmonary artery (PA) contractility is mediated in large part by increases in cytosolic Ca2+, which activates myosin light chain (MLC) kinase and in turn phosphorylates myosin light chain20 (MLC20). Rho‐kinase further enhances phosphorylation of MLC20 and contractility through inhibition of MLC phosphatase. We hypothesized that Ca2+ and Rho‐kinase signaling synergistically regulate pulmonary vascular tone in chronically hypoxic (CH) newborn lambs. This hypothesis was tested by performing wire myography on serotonin (5‐HT) stimulated PA rings isolated from 12 to 17 day old lambs maintained at high altitude (3801 m) for the final 100 days of gestation and after birth (CH). 5‐HT elicited dose‐dependent contractions were attenuated with various Ca2+ entry or Rho‐kinase blockers. Voltage dependent and independent Ca2+ entry was inhibited with 30 μM KB‐R7943 (KBR), 100 μM flufenamate (FFA), 30 μM SN‐6, or 10 μM nifedipine (NIF) in the absence or presence of 50 μM SKF‐96365 (SKF). Rho‐kinase was inhibited with 10 μM fasudil or Y27632. The potency order for the Ca2+ entry antagonists was KBR ≥ NIF+SKF ≫ NIF > FFA ≫ SN‐6, whileY27632 > fasudil. Fasudil substantially augmented the inhibitory action of the Ca2+ entry blockers. These findings support the rationale for treating pulmonary hypertension with medications that target both Ca2+ and Rho‐kinase pathways. NIH P01HD031226, R01HD003807 (LDL), R01HL95973 (AB)
Regulation of vascular smooth muscle contraction by Ca2+ signaling and Ca2+ sensitivity in intrapulmonary arteries
Authors: Perez‐Zoghbi Jose F , Mukherjee Seema , Sanderson Michael J.
Published: June 2021

The contraction of vascular smooth muscle cells (SMCs) of small pulmonary arteries is critical for the regulation of pulmonary vascular resistance and plays an important role in the development of pulmonary hypertension. We have studied the regulation of SMC contraction by Ca2+ and Ca2+ sensitivity in intrapulmonary arteries by measuring artery contraction and Ca2+ signaling in mouse lung slices with phase‐contrast and confocal microscopy. The agonists serotonin (5‐HT) and endothelin (ET) induced artery contraction and stimulated Ca2+ oscillations in their SMCs. Conversely, nitric oxide (NO) caused relaxation and decreased the frequency of Ca2+ oscillations in arteries pre‐stimulated with agonists. Ca2+ permeabilization with caffeine and ryanodine caused artery contraction but agonists stimulate further contraction. Activation of PKC with phorbol 12‐myristate 13‐acetate (PMA, 1–10 μM) induced strong and sustained artery contraction whereas the PKC inhibitor GF‐109203X (1 μM) inhibited the agonists‐induced contraction. In conclusion, our results suggest that the contraction of intrapulmonary arteries is regulated by the frequency of Ca2+ oscillations and a PKC‐dependent increase in Ca2+ sensitivity.
Aberrant eNOS phosphorylation in Idiopathic Pulmonary Arterial Hypertension
Authors: Ghosh Sudakshina , Mavrakis Lori , Janocha Allison J. , Comhair Suzy A.A. , Erzurum Serpil C.
Published: June 2021

Based upon its critical role in mediating vasodilation, nitric oxide (NO) deficiency has been implicated in the pathogenesis of Idiopathic Pulmonary Arterial Hypertension (IPAH). We have shown that NO production by pulmonary artery endothelial cells (PAEC) derived from IPAH lungs is lower than control lungs. We hypothesized that the low NO production by IPAH PAEC is due to reduced activity of the endothelial nitric oxide synthase (eNOS). Agonist‐induced (Bradykinin, BK, 1 mM) NO production nearly doubled in control PAEC (n = 4) [nitrite pmol/ml‐min, nonstimulated: 0.14 ± 0.01; stimulated: 0.24±0.01, p < 0.0001] whereas NO production increased only minimally in IPAH PAECs (n=6) [pmol/ml‐min, nonstimulated: 0.15 ± 0.01, stimulated: 0.18 ± 0.01, p = 0.01]. Dimerization of eNOS, which is required for active enzyme, was similar among control and IPAH PAECs. Activity of dimeric eNOS is determined by phosphorylation; Serine 1177 (S1177) phosphorylation is required for enzyme synthesis of NO and Threonine 495 (T495) phosphorylation inhibits activity. BK stimulation caused phosphorylation of S1177 in IPAH PAEC, but less than control cells (pS1177/total eNOS, control: 1.25 ± 0.01, IPAH: 0.92 ± 0.03, p = 0.01). BK also rapidly induced dephosphorylation of T495 in control PAEC, but not in IPAH PAEC (pT495/total eNOS, control: 0.11 ± 0.01, IPAH: 2.1 ± 0.06, p = 0.01). These findings point to abnormalities in eNOS phosphorylation in the decreased NO synthesis of IPAH cells.
The Rab‐GAP, AS160, participates in the regulation of apical membrane epithelial sodium channel (ENaC) density and recycling
Authors: Liang Xiubin , Butterworth Michael B. , Peters Kathryn W. , Frizzell Raymond A.
Published: June 2021

The Rab‐GAP, AS160 is a recently discovered Akt kinase substrate. Since phosphorylation sites for Akt are similar to those for the aldosterone (Aldo)‐induced kinase, SGK1, we postulated that phosphorylation of AS160 may promote ENaC trafficking during aldo stimulation. AS160 expression was detected in mCCD cells, in which its expression was increased by its transfection or reduced by shRNA‐mediated knockdown, followed by assays of ENaC expression and function. Aldo induced a time‐dependent increase in AS160 expression, and its phosphorylation at five sites: T568, S588, T642, S318 and S751, detected by phospho‐specific antibodies. Under non‐stimulated (basal) conditions, AS160 knockdown increased apical ENaC without changing total ENaC expression and increased Na transport. After AS160 knockdown, aldo did not further augment apical ENaC density or Na transport. Under basal conditions, AS160 over‐expression increased total ENaC expression 2.5‐fold but did not increase surface ENaC expression. However, aldo increased surface ENaC expression and Na current 2.5‐fold in epithelia expressing AS160. These findings suggest that AS160 normally retains ENaC within intracellular compartments, and that SGK1‐mediated AS160 phosphorylation blocks its Rab‐GAP activity, permitting ENaC trafficking to the apical membrane in response to aldosterone. [Supported by AHA fellowship 0725416U and NIH DK54814]
PSEUDOHYPERKALAEMIA INDUCED BY BLOOD COLLECTION AND CORRECTIONS FOR HEMOLYSIS IN BLOOD SAMPLES FROM EXPERIMENTAL MICE
Authors: Yan Qingshang , Du Zhaopeng , Gotoh Nanami , Giebisch Gerhard , Wang Tong
Published: June 2021

Pseudohyperkalaemia is commonly caused by improper blood collection and hemolysis in blood samples. The reported blood potassium concentrations range from 4mM to 7mM in wild‐type mice of different genetic backgrounds. It is not clear whether such large differences are due to genetic background or technical problems during blood collection. In this study, we measured and compared the plasma K+ concentrations in mice with similar genetic background by using different blood collection methods and also assessed K+ concentrations in blood samples with hemolysis. Five commonly used blood collection methods were performed (via carotid artery cannulation; cardiac puncture; retro‐orbital bleeding; abdominal aorta puncture; and trunk artery by decapitation) and K+ concentrations were measured from these samples. The K+ concentrations, in increasing order, were (in mM): 4.06±0.10; 4.48±0.12; 4.84±0.13; 6.00±0.18 and 6.94±0.14 respectively from blood samples collected by the above methods. We also measured the hemoglobin in these samples and found that hemoglobin concentrations are proportional to the K+ concentration and the increase in plasma potassium and changes in hemoglobin show a linear relationship with a formula of Y=0.08609 + 5.355* (X−0.08266), R2=0.9911 (where Y= change in hemoglobin; X= change in K+). These results indicate that carotid artery cannulation, cardiac puncture, and retro‐orbital bleeding methods produce relatively accurate K+ values but pseudohyperkalaemia results from abdominal aorta puncture and trunk artery collections, which can be corrected by our new equation.
Sex and Racial Background Influence Angiotensin Peptide Metabolism in Young Adults
Authors: Gwathmey TanYa M. , Shaltout Hossam A. , Nixon Patricia A. , Rose James C. , Washburn Lisa K. , Chappell Mark C.
Published: June 2021

It is established that men have higher blood pressures than women and that African Americans (AA) have an increased likelihood of developing hypertension and renal disease compared to their Caucasian (CN) counterparts. However, the mechanism(s) accounting for these differences remain to be established. In the present study, we assessed mean arterial pressures (MAP), as well as ACE and ACE2 that form and metabolize Angiotensin (Ang) II, the primary effector peptide of the renin angiotensin system (RAS), in urine from AA male (n=8), AA female (n=9) and CN female (n=4) adolescents at 14 years of age. MAP did not differ significantly between groups [AA males: 74±2 mmHg; AA females: 76±1mmHg; CN females: 73±1mmHg]. ACE activity was lower in AA females [0.7±0.2 fmol/min/g creatinine] than AA males [1.3±0.2 fmol/min/g creatinine; p<0.05], but did not differ from CN females [0.8±0.3 fmol/min/g creatinine]. Conversely, ACE2 activity was lower in AA females [0.9±0.2 fmol/min/g creatinine] compared to CN females [1.7±0.4 fmol/min/g creatinine; p<0.05] but not in AA males [1.0±0.3 fmol/min/g creatinine]. These findings suggest that sex and racial differences in urinary ACE and ACE2 activities are evident in young adults in the absence of changes in blood pressure. We speculate that lower ACE2 activity may contribute to the development of cardiovascular disease particularly in AA females. HD047584‐S3; HL‐56973; M01‐RR07122
Inhibition of estradiol synthesis attenuates diabetic renal disease in male streptozotocin (STZ)‐induced diabetic rats
Authors: Manigrasso Michaele B. , Marbury David C. , Sawyer R. Taylor , Flynn Elizabeth R. , Maric Christine
Published: June 2021

Previous data showed that STZ‐diabetic males have increased renal estradiol (E) levels correlating with renal injury. The aim of this study was to examine whether inhibition of E synthesis, by blocking the aromatization of testosterone to E using anastrozole, is renoprotective. The study was performed in male STZ‐diabetic (D) and STZ‐diabetic rats treated with 0.15mg/day of anastrozole (Da) for 12 weeks. Urine albumin excretion (UAE) was determined by ELISA and calculated by measuring urine albumin concentration and daily urine output. Glomerulosclerosis (GSI) and tubulointerstitial fibrosis (TIFI) indices were graded using a semiquantitative scoring method. Activated macrophages (CD68) and podocyte integrity (nestin) were assessed by immunohistochemistry, followed by image analysis. Treatment reduced diabetes‐associated increases in UAE (D 20.4±4.7; Da 10.8±3.2 mg/day; NS), GSI (D 1.94±0.13; Da 1.35±0.19 AU; p<0.05), TIFI (D 0.76±0.07; Da 0.52±0.08 AU; p<0.05), decreased the number of renal cortical CD68‐positive cells (D 6.04±0.42; Da 3.90±0.30; p<0.05) and increased the density of nestin–positive podocytes (D 15.8±1.2; Da 18.8±1.2; NS) and podocin protein expression (D 0.31±0.08; Da 0.47±0.16 ROD; NS). Our results suggest that blocking aromatase activity in males, thereby reducing E synthesis, protects against diabetes‐induced UAE, inflammation, renal pathology and podocyte injury.
Phospholipase A2 group VIA regulates Na‐K‐2Cl cotransporter activity in the thick ascending limb
Authors: Roeschel Tom , Seidel Saskia , Kahl Thomas , Mutig Kerim , Mrowka Ralf , Shan Qixian , Bleich Markus , Ferreri Nick R , Bachmann Sebastian , Paliege Alexander
Published: June 2021

In the present study we attempted to characterize the renal expression pattern of phospholipase A2 group VI isoforms in response to vasopressin (AVP) treatment. To this end we used microarray analysis, PCR, immunohistochemistry, and Western blot in control rats and in Brattleboro rats (DI rats) treated with the selective V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or vehicle via osmotic minipump. Cultured rat medullary TAL cells (mTAL cells) and the R‐ and S‐enantiomere of the iPLA inhibitor, bromoenol lactone (BEL), were employed to study the effect of iPLA on NKCC2 phosphorylation. Microarray analysis and quantitative real time PCR showed a significant downregulation of iPLA2 group VIA mRNA in the outer medulla of treated animals whereas other iPLA2 isoforms were not affected. Immunohistochemistry demonstrated iPLA2 expression in the entire distal nephron. Western blotting revealed a dominant band at 70kDa which was reduced to 50±7% by dDAVP (p<0.05). Treatment of mTAL cells with 5μM S‐BEL for 1h markedly increased phospho‐NKCC2 abundance and membrane localization without effect on total NKCC2. R‐BEL was less effective. In summary, we have shown that the group VIA‐2 isoform of iPLA2 is expressed in the distal nephron of rat kidney and has an inhibitory effect on NKCC2 phosphorylation. Downregulation of iPLA may therefore be an important mechanism of AVP mediated urine concentration.
Bioimpedance Spectroscopy for the Estimation of Body Fluid Volumes in Mice
Authors: Chapman Mark Edward , Kohan Donald , Plato Craig , Hu Lufei
Published: June 2021

Conventional indicator dilution techniques for measuring body fluid volume (BFV) are time‐consuming, expensive and require bulky equipment. Bioimpedance spectroscopy (BIS) may be a useful alternative for BFV determination due to being rapid, minimally invasive and allowing repeated measurements. BIS has not been reported in mice; such analysis would be helpful particularly in rare transgenic or knockout mouse lines. Hence, we examined how well BIS estimates BFV in mice. Using C57/Bl6 wildtype mice, the BIS system demonstrated <5% inter‐mouse variation in total body water (TBW), extracellular (ECV) and intracellular fluid volume (ICV) between animals of similar body weight (BW). TBW, ECV and ICV differed between heavier male and lighter female mice, however, the ratio of TBW, ECV and ICV to BW did not differ within mice and corresponded closely to values in literature. Default resistance coefficients used by the BIS system defined for rats produced body composition values for TBW that exceeded BW in mice. Therefore, body composition was measured in mice using a range of resistance coefficients. Resistance values at 10% of those defined for rats provided TBW, ECV and ICV ratios to BW that were similar to those published in literature. Further evaluation of the sensitivity of the BIS system was determined by its ability to detect volume changes after saline infusion. One mL saline provided the predicted increases in compartmental fluid volumes in mice. In summary, BIS is a sufficiently sensitive and accurate method for the estimation of body composition in mice. The ability to perform serial measurements will be a useful tool for future studies.
Expected hypotension in the Af17 knockout mouse
Authors: Zhang Wenzheng
Published: June 2021

Our previous studies suggest that Dot1a‐Af9 complex represses the transcription of the epithelial Na+ channel α subunit (α‐ENaC) gene by hypermethylating histone H3 K79 at the α‐ENaC promoter, that aldosterone treatment or Af17 overexpression relieves this repression by different mechanisms, and these transcriptional mechanisms are important in regulating ENaC‐mediated Na+ transport. However, the significance of Af17 in regulating Na+homeostasis and thus blood pressure in vivo in mouse has not been tested. Accordingly, we hypothesize that deletion of Af17 leads to downregulation of ENaC transcription, ENaC activity and thus hypotension. To test this hypothesis, we generated Af17−/− mice using gene trap ES cells in which Af17 was specifically disrupted. Af17−/− mice are viable and have no obvious morphological or growth defects till 6 months old. However, compared to controls, Af17−/− mice have a larger volume of urine and a lower blood pressure, regardless of the diet containing very low, low, normal or high amount of Na+. While control animals can tolerate high Na+ (1%) in drinking water, Af17−/− mice die from severe dehydration within 4 days of the treatment. Detailed characterization of the renal physiological phenotype and identification of the underlying molecular mechanisms are underway. Our data confirm and extend our previous findings regarding Dot1a‐Af9‐Af17 mediated regulation of ENaC transcription, ENaC activity, Na+ transport and thus blood pressure, and highlight the importance of Af17 in these biological processes.
NK‐1 receptors expressing neurons in the Locus coeruleus (LC) play a role in cardiorespiratory response to CO2
Authors: Gargaglioni Luciane H. , de Carvalho Debora , Bicego Kenia C , Ferreira da Silva Glauber S , Castro Olagide W , Garcia‐Cairasco Norberto
Published: June 2021

Male Wistar rats were injected in the LC with substance P‐saporin conjugate (SP‐SAP; 2μM), to kill NK‐1 receptors (NK1R) expressing neurons, or IgG‐SAP as a control. Pulmonary ventilation (VE), mean arterial pressure (MAP) and heart rate (HR) were determined after 60 min of hypercapnic exposure (7% CO2 in air). To verify the effectiveness of the lesions, immunohistochemistry for NK1Rs was performed. Immunohystochemistries for tyrosine hydroxylase (TH‐ir) and Glutamic Acid Decarboxylase (GAD‐ir) were also performed to verify if catecholaminergic and GABAergic neurons were eliminated. A reduced NK1R immunoreactivity (NK1R‐ir) in the LC (72%) showed the effectiveness of the lesion. SP‐SAP lesion also caused a reduction of TH‐ir neurons (66%) and GABAergic neurons (70%). Hypercapnia caused a decreased ventilatory response in SP‐SAP group (30%), due to a reduction in tidal volume. SP‐SAP lesion in the LC did not affect MAP but caused an increase in HR during hypercapnia. The results suggest that NK1R‐ir neurons in the LC modulate hypercapnic ventilatory response but play no role in breathing control under resting conditions. Additionally, NK1R‐ir neurons in the LC seem to play no role in MAP regulation in resting conditions and during hypercapnia, but modulate HR during CO2 exposure. Part of these modulations might be due to noradrenergic and GABAergic effects. Supported by: FAPESP, CNPq.
Effects of HIF‐1α gene deletion in respiratory centers on ventilatory acclimatization to hypoxia (VAH)
Authors: Go Ariel , Fu Zhenxing , Johnson Randall S. , Powell Frank L.
Published: June 2021

HIF‐1α controls the expression of several genes that increase O2 supply. VAH is the time‐dependant increase in ventilation and O2‐ sensitivity with chronic hypoxia (CH). Previously, we found that normal VAH in mice depends on HIF‐1α expression in the CNS. To better localize and define the timing of this effect, we used the Cre‐LoxP strategy to delete HIF‐1α, in the nucleus tractus solitarius (NTS), which receives input from carotid body chemoreceptors and exhibits intrinsic CO2‐senstivity. We made stereotaxic microinjections of AAV‐Cre into the NTS of HIF‐1α floxed mice and exposed them to CH (10% O2, 2 wks). AAV‐LacZ microinjections were used for controls and localization. We measured ventilation with barometric pressure plethysmography:

Ventilation increased in normoxia but not hypoxia or hypercapnia with HIF‐1α deletion in the NTS during CH, primarily through increased fR. This suggests HIF‐1α in the NTS suppresses normoxic ventilatory drive during CH but does not affect the ventilatory chemoreflexes. Further studies are needed to quantify HIF‐1α deletion and determine its effect in unacclimatized mice, at other sites in the brain and at different times relative to CH. Supported by RO1 NIH‐HL‐081823.
Lipoxygenases mediate endothelium‐dependent vasodilation in the human coronary microcirculation
Authors: Larsen Brandon T , Mendoza Suelhem A , Campbell William B , Gutterman David D
Published: June 2021

12‐ and 15‐lipoxygenases (LOs) metabolize arachidonic acid (AA) to 12‐ and 15‐hydroxyeicosatetraenoic acids (HETEs) that function as endothelium‐derived hyperpolarizing factors (EDHFs) in some animals. EDHFs may compensate for loss of nitric oxide in cardiovascular disease. In human coronary arterioles (HCAs), we previously observed endothelial 12‐ and 15‐LO expression and vasodilation to exogenous 12/15‐HETEs; however, a physiologic role of LOs in HCAs has not been explored. We hypothesized that endothelium‐dependent dilation requires LOs and that HCAs produce HETEs. HCAs were isolated from right atrial appendages (n=13), vasomotor tone was measured by wire myography, and AA metabolism was assayed by mass spectrometry. Bradykinin (an endothelium‐dependent vasodilator) induced a concentration‐dependent relaxation of HCAs (22±6% and 82±3% at 1 and 10 μM, respectively, n=7) that was attenuated by the LO inhibitors CDC (5±5% and 75±9%, respectively, n=3) and NDGA (8±9% and 52±13%, respectively, n=4). In the presence of bradykinin, HCAs metabolized AA to 12‐ and 15‐HETE (1.26±0.24 and 0.80±0.18 pg/min, respectively, n=6), as measured in the superfusate by mass spectrometry. We conclude that HETEs are produced by HCAs and are required for endothelium‐dependent dilation. These findings support a physiologic role for HETEs as EDHFs and suggest that LOs modulate vasomotor tone in the diseased human heart.