bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased renal protein expression of adrenomedullin and RAMP2 is associated with impaired pressure‐natriuresis in human GRK4γ 142V transgenic mice (1136.4)
Authors: Zhou Donghai , Jones John E. , Asico Laureano , Jose Pedro A. , Wang Xiaoyan
Published: June 2021

We have reported that the expression of the human GPCR kinase 4γ142V variant (hGRK4γ142V)in mice causes hypertension, related in part to the dysfunction of renal GPCRs, such as impaired D1 and D3 dopamine receptor function and increased angiotensin type 1 receptor (AT1R)function. The natriuretic and diuretic effects of the D1‐like dopamine receptor agonist fenoldopam are impaired in hGRK4γ142V, relative to hGRK4γ wild‐type (hGRK4γWT) transgenic mice. GRK4 has also been reported to negatively regulate adrenomedullin (ADM), a natriuretic hormone like dopamine. Therefore, we evaluated the expression of sodium transporters, ADM and components of its receptor, RAMP2 (receptor activity modified protein), CLCR (calcitonin‐like calcitonin receptor) and ADMRL2 (ADM receptor L2) in the kidneys of hGRK4□γ142V mice on normal NaCl (0.8%)diet.Blood pressure was elevated in hGRK4γ142V transgenic (129.3±10.4, mm Hg) but not in GRK4γWT (101.7±6.3), relative to non‐transgenic (NT) (99.8±1.8) mice(n=4/group).Theplot of urine sodium excretion and blood pressure in anesthetized GRK4γ142V transgenic mice was shifted to the right of GRK4γWT□transgenic mice.Protein expressions of ADM (‐37±4%) and RAMP2 (‐36±16%) were decreased in hGRK4γ142V transgenic mice, although these remained unchanged in GRK4γWTtransgenic mice, relative to NT mice. CLCR and ADMRL2 werenot altered. Of the 8 sodium transporters, only NKCC2 was increased in GRK4γ142V transgenic mice (+297±72%). ADM immunofluorescence, which was mainly observed in the renal cortex, was decreased in GRK4γ142V transgenic relative to NT mice. Our data suggest that GRK4γ142V and/or the dysfunction of the dopamine and AT1 receptors caused by this variant decreases ADM/RAMP2 expression and increases NKCC2 expression in the kidney.These changes may contribute to the impaired sodium excretion and increased blood pressure in GRK4γ142V transgenic mice.

Grant Funding Source: Supported by the National Institutes of Health (HL068686,HL023081,HL074940,HL092916 and DK090918)
A novel mouse model for determining the role of FKBP12 and calcineurin inhibition in tacrolimus‐mediated hypertension and hyperkalemia (1136.6)
Authors: Lazelle Rebecca , Meermeier Nick , Yang Chao‐Ling , Ellison David
Published: June 2021

The calcineurin inhibitor tacrolimus is widely used to prevent transplant organ rejection. Its use, however, is complicated by hypertension, hyperkalemia, and kidney failure. Tacrolimus suppresses the immune system by binding to an endogenous protein, FKBP12, inhibiting the phosphatase calcineurin. We reported previously that tacrolimus treatment activates the renal sodium chloride cotransporter (NCC), an event necessary to cause hypertension. It is unclear whether tacrolimus activates NCC in the kidney by 1) blocking calcineurin‐independent effects of FKBP12 or 2) inhibiting calcineurin.

To address this, we used the Pax8 CRE/LOX system to delete FKBP12 from the renal tubules of mice, upon treatment with doxycycline. Kidney‐specific FKBP12 knockout alone did not increase the abundance of phosphorylated NCC (active), or cause hypertension or hyperkalemia. In contrast, phosphorylated NCC was significantly lower in FKBP12 knockout mice than in wildtype mice (un‐induced), after tacrolimus treatment. These results suggest that tacrolimus inhibition of FKBP12 does not activate NCC directly; instead the drug must bind to FKBP12 and inhibit calcineurin along the nephron to activate NCC. This implies that calcineurin inhibitors that do not bind FKBP12 retain the potential to cause hypertension and hyperkalemia. Understanding how calcineurin regulates NCC, however, may lead to safer immunosuppressive drugs.

Grant Funding Source: Supported by RO1 DK095841 02
Purinergic signaling is enhanced in the absence of UT‐A1 and UT‐A3 (1137.10)
Authors: Rogers Richard , Himmel Nathaniel , Redd Sara , Blount Mitsi
Published: June 2021

The extracellular nucleotide ATP is an important paracrine regulator of renal tubular transport of water and urea. P2Y2, the predominant P2Y receptor expressed in the medullary collecting duct, mediates the action of ATP and thus, is important for urinary concentration. To investigate the role of purinergic signaling in the absence of urea transport in the collecting duct, we housed wild type (WT) and UT‐A1/A3 null (KO) mice in metabolic cages to monitor urine output. Not only did we confirm that KO mice are polyuric, we also observed that urinary cAMP was lower (WT: 3100 ± 467 vs KO: 699.3 ± 95 pmol/ml/creatinine) despite elevated circulating AVP (WT: 8.3 ± 1 vs KO: 28.4 ± 5 pg/ml) in KO mice. Because P2Y2 regulates AVP‐stimulated transport by damping cAMP synthesis, we suspected that, similar to other animal models of AVP‐resistant polyuria, purinergic signaling is increased in the KO mice. In fact, urinary levels of ATP are higher in KO (21.34 ± 5.3 nM/creatinine) compared to WT (5.6 ± 3.5 nM/creatinine) mice and purinergic‐mediated prostanoid (PGE2) production was elevated (WT: 12.4 ± 2.5 vs KO: 32.7 ± 7.1 pg/ml/creatinine). The mRNA expression of P2Y2 was not significantly different in the inner medullas of WT and KO mice which, given the resistance of the KO mouse collecting duct to AVP, was to be expected. Collectively, our data suggest that the reduction in medullary osmolality due to the lack of UT‐A1 and UT‐A3 induces an AVP‐resistant polyuria that is exacerbated by elevated purinergic signaling.

Grant Funding Source: Supported by NIH‐NIDDK grants K01‐DK082733 and R03‐DK091501
Increase in collecting duct basal:apical AQP2 protein expression ratio with increasing depth along the corticopapillary axis positively correlates with maximum urine concentrating capacity in the kangaroo rat and Sprague‐Dawley rat (1137.2)
Authors: Espineira Madeline , Pannabecker Thomas
Published: June 2021

Our goal is to investigate the role of aquaporin 2 (AQP2) membrane expression in inner medullary (IM) collecting ducts (CD) relative to the urine concentrating mechanism by comparing basal‐to‐apical (B:A) AQP2 ratios of kangaroo rats (KR) with control and water‐restricted Sprague‐Dawley (SD). Previous studies have shown that vasopressin increases IMCD transepithelial water permeability, with apical AQP2 considered rate‐limiting, while in MCDK cells AQP2 is translocated to the basal membrane with hypertonicity and forskolin. The latter is seemingly inconsistent with the known role of AQP2. IM transverse sections at 1000‐µm intervals throughout the corticopapillary axis were labeled by immunohistochemistry for AQP2. The B:A AQP2 ratio at increasing 1000‐µm intervals from the IM border to papilla tip is ~2‐fold greater at each level in KR CD (.71, 1.23, 1.46, 1.18, 1.40, 1.51) relative to SD control (.46, .62, .65, .80, .88, 1.08) and SD water‐restricted (SDWR)(.41, .54, .54, .64, .54, .77). These results correlate with prior studies by others indicating peritubular hypertonicity elevates CD water permeability and B:A AQP2 ratios. Immunohistochemistry suggests a steady presence of basal AQP2 in KR and SDWR. Our data suggest a more significant role for basal AQP2 than presently realized. NIDDK DK083338, NSF 1OS095285, APS/NIDDK STEP‐UP, WAESO
Estrogen increases expression of aquaporin‐2 protein in the inner medullary collecting duct (1137.3)
Authors: Irsik Debra , Hyndman Kelly , Marks Eileen , Boesen Erika
Published: June 2021

Previous studies of sex differences in renal water handling have focused on vasopressin and the V2 receptor rather than possible differences in their downstream target, aquaporin‐2 (AQP2). We report that inner medullary glycosylated and non‐glycosylated AQP2 protein levels are approximately 30‐40% lower in male (0.69±0.04 and 0.62±0.07 arbitrary units, a.u., n=5) compared to female C57BL/6 mice (1.00±0.08 and 1.00±0.16 a.u., n=4; P<0.05). Immunofluorescent staining and Western blot analysis revealed that freshly isolated collecting duct cells and mpk‐CCD‐clone II cells express estrogen receptor (ER) β, the ER splice variants ERα36 and ERα46, but little if any full‐length ERα66. Exposure of mpk‐CCD‐clone II cells to 10 nM estrogen for 24 h significantly increased AQP2 protein expression (2.6±0.4 vs 0.9±0.2 a.u. for vehicle, n=3; P<0.05), whereas exposure to the selective ERβ agonist DPN at 10 nM had no significant effect. Inner medullary AQP2 mRNA levels were not significantly different between male and female C57BL/6 mice (2‐ΔΔCT = 1.03±0.12 and 1.09±0.08, n=6 per group; P>0.05). Thus our data show that estrogen increases collecting duct AQP2 protein levels, potentially via a mechanism independent of gene transcription.
Identification of vasopressin‐responsive miRNAs and AQP2‐targeting miRNAs in kidney collecting duct cells (1137.6)
Authors: Kim Jae‐Eun , Jung Hyun Jun , Choi Hyo‐Jung , Lim Jung‐Suk , Kwon Tae‐Hwan
Published: June 2021

MicroRNA (miRNA), a small RNA synthesized from non‐coding RNA, acts as an important post‐transcriptional regulator. We aimed to identify the vasopressin‐responsive miRNAs from kidney inner medullary collecting duct (IMCD) cells, and particularly to demonstrate aquaporin‐2 (AQP2)‐targeting miRNAs. Microarray chip assay (Affymetrix GeneChip miRNA 3.0 Array) was carried out in IMCD tubule suspension of rat kidney in the absence or the presence of dDAVP stimulation (10‐8 M, 2 h). The results demonstrated 19 miRNAs, including both precursors and mature miRNAs, which showed 1.3‐fold changes in the expression in response to dDAVP stimulation (P<0.05). Additionally, vasopressin‐responsive miRNA networks were predicted by in silico analysis, which exhibited potential target genes. To identify AQP2‐targeting miRNAs, in silico analysis using TargetScan 6.2 was performed. Four miRNAs (miR‐32, miR‐137, miR‐216a, and miR‐216b) targeting 3’UTR of rat AQP2 mRNA were predicted. Target seed regions of miR‐32 and miR‐137 were conserved at the 3’UTR (476 ‐ 483) of rat AQP2 mRNA. RT‐qPCR and immunoblot analysis demonstrated that dDAVP‐induced AQP2 up‐regulation was significantly attenuated in mpkCCDc14 cells, when the cells were transfected by miRNA‐mimic of miR‐32 or miR‐137. This study provides a novel insight on the regulation of AQP2 protein expression via RNA interference, i.e., AQP2‐targeting miRNAs.
A mathematical model of rat proximal tubule and loop of Henle (1137.9)
Authors: Weinstein Alan
Published: June 2021

PT and LOH function are coupled, with proximal transport determining loop fluid composition, and loop transport modulating glomerular filtration via tubuloglomerular feedback (TGF). To examine this interaction, we began with published models of superficial rat proximal convoluted tubule (PCT) (including flow‐dependent transport in a compliant tubule), and rat thick ascending Henle limb (AHL). This PCT was scaled down to represent outer medullary proximal straight tubule (PST), which was connected to thick AHL via a short descending limb. Superficial PT was scaled up to a juxtamedullary nephron, and connected to thick AHL via outer and inner medullary descending limbs, and inner medullary thin AHL. Medullary interstitial solute concentrations were specified. End‐tubule hydrostatic pressure was required to conform to a distal nephron flow resistance. The TGF signal was represented as a linear function of end‐AHL cytosolic Cl concentration. These two distal conditions required iterative solution of the model. Calculations revealed Na, K, and HCO3 reabsorption in all segments, with thin limb transport driven by the increases in lumen concentrations produced by osmotic water reabsorption. PST was a critical locus for NH4 secretion, due to high interstitial NH4, and cellular uptake via the Na,K‐ATPase. Late AHL cytosolic Cl was a sigmoidal function of flow, and appears suitable to represent the TGF signal.

Grant Funding Source: Supported by NIH RO1‐DK29857
Prophylactic effects of melatonin with treadmill exercise on cartilage damage of rats with collagenase‐induced knee (1139.15)
Authors: Hong Yunkyung , Jin Yunho , Choi Jeonghyn , Hong Yonggeun
Published: June 2021

The purpose of this study is to evaluate effect of melatonin and exercise on extracellular matrix synthesis against cartilage damage in rats with collagenase‐induced knee laxity. Melatonin was injected twice/day subcutaneously, and treadmill exercise was conducted for 1‐hr/day. Intervention did not alter joint parameters, of which were significantly augmented in vehicle. Serum IL‐6 and TNF‐α was upregulatedin vehicle, which was attenuated by intervention. Melatonin combined with exercise decreased catabolic mediators but also cellular death with lower mineralization. TGF‐β1, which was reduced in vehicle, was augmented by intervention at early phase. At advanced phase, melatonin alone promoted mineralization in the cartilage as well as apoptosis, and melatonin blocked bone erosion and osteophyte formation. Combined intervention increased IL‐6, HIF‐2α, and MMP‐13 with COL2A1. Histological images exhibited rounded chondrocyte shape, smooth cartilage surface, and clear tidemark in combined intervention. Inhibition of subchondral bone erosion was detected in micro‐CT 3D image. Co‐treatment did exert non‐significance of trabecular parameters between both limbs. We suggest that melatonin with treadmill exercise may havepreventive and synergistic effect on cartilage degeneration, of which was more effective at initial phase.
Lack of reversal from lead acetate‐induced hepatotoxicity, free radical generation and oxidative stress in Wistar rats (1139.17)
Authors: Oyagbemi Ademola , Saba Adebowale , Omobowale Temidayo , Akinrinde Akinleye , Ogunpolu Blessing , Daramola Oluwabusola
Published: June 2021

Lead is a ubiquitous metal and also one of the most abundant elements present on earth. Both the developing and the developed countries have been contaminated by this element. This study was designed to evaluate possible toxic effects of lead acetate (PbA) exposure and subsequence withdrawal in male Wistar rats. Control group (group I) received normal saline while groups II, III and IV received oral administration of 0.25, 0.5 and 1.0mg/ml PbA respectively for the period of 6 weeks. One half of the population of the rats was sacrificed at 6 weeks of PbA exposure. PbA was similarly withdrawn from the remaining rats for another 6 weeks. Exposure of rats to PbA led to significant decline (p<0.05) in Glutathione peroxidase (GPx), Glutathione S‐transferase (GST), Catalase (CAT), Superoxide dismutase (SOD), and Reduced glutathione (GSH) content. Similarly, Malondialdehyde (MDA) and H2O2 concentrations were significantly (p<0.05) elevated. In the same vein, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP activities also increased significantly in exposed rats. Animals from the withdrawal period exhibited similar pattern of alterations, with significant (p<0.05) reduction in GSH, GST, GPx and SOD and significant elevation in MDA. Serum ALT, AST and ALP however remained persistently high. In conclusion, experimental animals exposed to PbA could not recover from hepatotoxicity and oxidative stress. This might be due to significant disruption of antioxidant defence system and free radical generation by PbA.
Clinical and epidemiological characteristics and condition of the hormonal status in women with androgenetic alopecia (1139.3)
Authors: Batpenova Gulnar , Dzhetpisbaeva Zulfiya , Tarkina Tatiyana , Kazieva Aliya , Abduldayeva Aigul
Published: June 2021

The aim is to study the clinical and medical history, and hormonal characteristics in women with androgenetic alopecia.Since 2007, we observed 12 patients with androgenetic alopecia in age from 20 to 45 years, which the diagnosis was based on medical history and trihoscopy data. The average age of women was 25,3±3,27. On examination, women complained of increased hair loss, changes in their structure, which is more pronounced thinning. Analysis of anamnesis data revealed that most part of women‐66.7% of the 12 linked the onset of the disease with the onset of puberty, 16.7% of pregnancy and childbirth, 2 with nothing connected. As a result of dermatological examination of the predominant number of patients‐57.3% only androgenic alopecia was diagnosed, and in 41.7%, combined with other skin symptoms of androgens. So, for a combined androgenic alopecia seborrhea was observed in 60% patients, and the combination of alopecia with acne‐in 40%. In the study of the menstrual cycle character it was revealed disruption of the menstrual cycle in 33.3% patients, of whom 3‐proceeds as oligomenorrhea, and at 1‐was found infertility.It is established that the finding in this category of the patients other symptoms of hyperandrogenism of skin and gynecological system causes the need for further investigation of of steroid hormones concentration in the blood serum.
Effect of purified poloxamer 188 and various dextrans on erythrocyte sedimentation rate in healthy subjects and patients with sickle cell disease (1139.6)
Authors: Hoppensteadt Debra , Emanuele Marty , Molnar Joann , Iqbal Omer , Iqbal Omer , Fareed Jawed
Published: June 2021

Introduction: Purified poloxamer188 (P188) (Mast Therapeutics) is a non‐ionic, linear block copolymer composed of a central chain of hydrophobic polyoxypropylene and two flanking chains of hydrophylic polyoxyethylene (MW 8.5 kDa). Dextrans represent branched polysaccharides of 10‐70 kDa that have been used as antithrombotic agents and plasma expanders. This study was designed to compare the effect of P188 and dextrans on ESR’s in blood obtained from healthy subjects and patients with sickle cell disease who were seen at Loyola University Medical Center clinics.

Material and Methods: Whole EDTA blood collected from normal individuals (n=8) and sickle cell patients confirmed by electrophoresis (n=11) were supplemented with P188 or dextran 10K, 18K , 40K and 70K at various concentrations (or saline control). ESR was measured using standard laboratory technique.

Results: The ESR’s for sickle cell patients (26.4 ± 7.1 mm/hr) were significantly higher in comparison to the ESR’s for healthy subjects (14.6 ± 2.1 mm/hr). Supplementation of P188 decreased ESR’s in both populations. Normal blood ESR’s decreased to 9.1 ± 1.3 mm/hr (38%), whereas the sickle cell patient values decreased to 14.1 ± 4.6 mm/hr (47%). At comparable concentrations, none of the dextrans changed ESR’s in healthy subjects or patients with sickle cell disease.

Discussion: These results demonstrate that ESR in SCD patients are elevated compared to healthy subjects. P188 supplementation decreased (up to 50%) ESR’s in both the healthy subjects and sickle cell patients.
Molecular effects of Ang II‐mediated differential ACE2 expression in cultured rat astrocytes (1140.2)
Authors: Vudhya Gowrisankar Yugandhar , Clark Michelle
Published: June 2021

Objective: Angiotensin (Ang) II effects on Angiotensin Converting Enzyme 2 (ACE2) in spontaneous hypertensive rats (SHR) was determined. Background: ACE2 is a negative regulator of the renin angiotensin system catalyzing the conversion of Ang II to Ang‐(1‐7), a peptide that counteracts Ang II effects. Methods: Brainstem (BS) and cerebellum (CB) astrocytes isolated from SHR and Wistar rats were used in this study. Cells were treated with 100nM Ang II at various times and ACE2 protein, and mRNA levels measured using Western blot and qPCR techniques, respectively. Results: Ang II showed a differential pattern to affect ACE2 protein levels in Wistar and SHR astrocytes. Most treatment groups showed ACE2 protein levels significantly reduced compared to basal ACE2 protein. There was a significant difference in Ang II‐mediated ACE2 protein levels in Wistar versus SHR astrocytes. Similarly, Ang II treatment had a differential pattern to modulate mRNA levels of ACE2 in these cells. In SHR CB astrocytes, the pattern of Ang II effects on ACE2 mRNA was significantly different compared to Wistar astrocytes. However, the effects of Ang II were mostly similar in BS Wistar and SHR cells. Conclusion: These findings suggest that the hypertensive state in the SHR may significantly affect Ang II’s ability to influence ACE2 mRNA expression but this effect did not translate into differences in ACE2 protein expression.

Grant Funding Source: Supported by Nova Southeastern University Chancellor's Faculty Research & Development Grant # 335872
Long‐lasting inhibitory effect of electroacupuncture in hypertensive patients ő role of catecholamine, renin and angiotension (1140.3)
Authors: Li Peng , Cheng Ling , Liu Dongmei , Painovich Jeanette , Vinjanury Sivarama , Tjen‐A‐Looi Stephanie , Longhurst John
Published: June 2021

Acupuncture and electroacupuncture (EA) are being used to treat hypertension (HTN). However, its mechanisms are unknown. The present study apply EA in patients with mild to moderate HTN (BP 145‐180/90舉110 mmHg). A group of 42 hypertensive patients without medication were assessed with 24 hr BP monitoring, EA at P 5‐6 and S 36‐37 once weekly for 30 min for eight weeks. After eight weeks treatment, 31 (71%) patients demonstrated significant (P<0.05) reduction at peak (12 mmHg) and average (8 mmHg) SBP/24hrs. The peak and average DBP/24hrs decreased 6 and 5 mmHg,respectively (P<0.05). Heart rate was unchanged. After termination of EA treatment, SBP remained reduced for four weeks but after eight weeks returned to higher levels. Twelve patients (29%) did not respond to the EA treatment. Both SBP and DBP/24hrs were not reduced in 33 other patients treated with control acupoints (LI 6‐7+G 37‐39). This beneficial effect of EA was slow in onset but long‐lasting. After eight weeks of EA treatment, the responders (n=10) plasma concentrations of nor‐sepinephrine decreased from 378±20pg to 278±30pg/ml; renin decreased from 0.568±0.098 to 0.292±0.071 ng/ml.hr, and angiotensin decreased from 0.568±0.098 to 0.262±0.075 ng/ml.hr (All P<0.05). The non‐responders have no significant decrease. Thus, the sympathetic function and renin‐angiotensin system were related to the long‐lasting effect of EA.

Grant Funding Source: DANA Foundation, Adolph Coors Foundation, Susan‐Samueli Center for Integrative Medicine and National
Effect of inhibiting the synthesis of Ang II in a model of preeclampsia (1140.5)
Authors: Rosas Paula , Tufiño Cecilia , Bobadilla Rosa
Published: June 2021

The renin angiotensin system (RAS) plays an important role in the physiological adaptations of pregnancy, so that alterations in the functioning of this system may contribute to the development of hypertensive disease of pregnancy or preeclampsia. The aim of this study was to determine the contribution of the RAS in a model of preeclampsia. To meet the objective was reduced uterine circulation during pregnancy with coarctation of the aorta at the level subrenal in Wistar rats aged 2 months. Captopril was administered (5mg/kg vo) for 1, 2 or 3 weeks. Joined weight, glucose and PA. There were concentration‐response curves to angiotensin II and phenylephrine (Fen) in isolated aortic rings using a conventional preparation of isolated. The results show that the model alters glucose concentrations and that the tx with captopril (2 to 3 weeks) and reduces the increase in PA, while a significant increase in the vascular response to tx Fen with captopril (3 weeks). These results allow us to conclude that the RAS is involved in both vascular response as metabolic alterations related to the pathophysiology of preeclampsia pregnancy from the start.
Evidence for an angiotensin‐(1‐7) neuropeptidase in the brain medulla of sheep (1140.6)
Authors: Marshall Allyson , Pirro Nancy , Rose James , Diz Debra , Chappell Mark
Published: June 2021

Angiotensin‐(1‐7) [Ang 7] is an alternative product of the renin‐angiotensin system (RAS). In contrast to Ang II, Ang 7 exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We identified a thiol‐sensitive peptidase that metabolized Ang 7 to Ang‐(1‐4) (Ang 4) in CSF of adult sheep. This activity was 2.5‐fold higher in sheep with antenatal glucocorticoid exposure while CSF Ang 7 levels were 50% lower compared to controls. To characterize this peptidase, we purified the protein 1400‐fold from the brain medulla by multiple chromatography steps monitored by 125I‐Ang 7 conversion to Ang 4. Chelating agents o‐phenanthroline and EDTA, as well as the mercury compounds APMA and PCMB, but not the cysteine protease inhibitor E64 blocked peptidase activity. Specific inhibitors to neprilysin, neurolysin, or thimet oligopeptidase were ineffective while the activity was abolished by the metallopeptidase inhibitor JMV 390 (Ki = 0.7 nM). HPLC analysis confirmed the processing of unlabeled Ang 7 to Ang 4 by the peptidase, but revealed <5% hydrolysis of Ang II or Ang I and no hydrolysis of neurotensin, bradykinin or apelin. Kinetic studies using 125I‐labeled Ang 7 and Ang I show similar Kms (2.6 and 4.3 μM), but a higher Vmax for Ang 7 (72 vs. 6 nmol/min/mg for Ang I, p<0.01, n=3). We conclude that evidence for an Ang 7 neuropeptidase may portend a novel pathway to influence actions of the peptide within the brain.

Grant Funding Source: Supported by HD47584
CYP3A5 genotype impacts maraviroc pharmacokinetics in healthy volunteers (1141.15)
Authors: Lu Yanhui , Hendrix Craig , Bumpus Namandjé
Published: June 2021

We have demonstrated in vitro that the polymorphic enzyme cytochrome P450 3A5 (CYP3A5) plays a major role in the oxidative metabolism of the anti‐HIV drug maraviroc. The present study evaluated the impact of CYP3A5 genotype on the pharmacokinetics of maraviroc. Of the sixteen subjects enrolled, 8 subjects carried the CYP3A5*1 allele (3 CYP3A5*1 homozygous and 5 heterozygous) and 8 subjects did not carry the CYP3A5*1 allele. Subjects received an oral dose of 300 mg maraviroc and blood was sampled over a period of 32 hr. Plasma maraviroc concentrations were determined using liquid chromatography‐mass spectrometry and pharmacokinetic parameters including area under the plasma concentration‐time curve (AUCinf), peak concentration (Cmax), and clearance were calculated. The pharmacokinetic parameters did not differ between the group carrying the CYP3A5*1 allele versus those not carrying a CYP3A5*1 allele. However, the CYP3A5*1 homozygous group exhibited 55% and 52% lower AUCinf, 97% and 84% higher apparent clearance, and 65% and 59% lower Cmax, respectively, as compared to the subjects that were heterozygous for CYP3A5*1 and those not carrying the CYP3A5*1 allele. In conclusion, Cmax and AUCinf were decreased while clearance was increased in subjects carrying two CYP3A5*1 alleles. The results indicate maraviroc has the potential to be used as a CYP3A5 clinical phenotyping probe.

Grant Funding Source: Supported by The Johns Hopkins ICTR ATIP Grant
Altered expression of small heterodimer partner governs CYP2D6 induction during pregnancy in CYP2D6‐humanized mice (1141.2)
Authors: Jeong Hyunyoung , Pan Xian , Koh Kwihye , Yu Ai‐ming , Isoherranen Nina
Published: June 2021

Hepatic drug metabolism plays a key role in determining drug response and safety. Over the decades, substantial progresses have been made in our understanding of the regulation of genes encoding drug‐metabolizing enzymes, providing critical information in developing dosing guidelines. However, current knowledge is insufficient to support dosing guidelines for pregnant women. Specifically, substrates of a major drug‐metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6‐humanized mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, small heterodimer partner (SHP) was downregulated during pregnancy. SHP repressed transactivation of the CYP2D6 promoter by hepatocyte nuclear factor (HNF) 4α. Finally, retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy. This study provides key insights into mechanisms underlying altered CYP2D6‐mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.

Grant Funding Source: Supported by Grant HD065532 and Fellowship K12HK055892
Biological relevance of PPM1B phosphatase regulation of PXR‐mediated CYP3A expression in hepatocytes (1141.9)
Authors: Pondugula Satyanarayana , Apte Udayan , Flannery Patrick , Abbott Kodye
Published: June 2021

Variations in pregnane xenobiotic receptor (PXR)‐mediated cytochrome P450 3A (CYP3A) expression can alter therapeutic response to a variety of drugs. It was previously shown that Mg2+/Mn2+‐dependent phosphatase 1B (PPM1B) levels positively regulate human PXR (hPXR) transactivating function and negatively regulate proliferation of HepG2 cells. In the current study, we sought to determine the biological relevance of this regulation. In COS‐7 cells, PPM1B increased hPXR transactivation of CYP3A4 promoter activity and coimmunoprecipitated with hPXR. Elevated PPM1B levels in HepG2 cells led to a significant attenuation of hPXR inhibition by TNFα. This finding, together with the previous finding of PPM1B attenuation of hPXR inhibition by CDK2, suggests that PPM1B counteracts PXR inhibition by proliferation‐dependent signaling pathways in hepatocytes. In mice regenerating livers, gene expression of PPM1B and Cyp3a11 (ortholog of human CYP3A4) was found to be downregulated, although PXR levels were marginally upregulated. Similarly, PPM1B and CYP3A4 gene levels were downregulated in human hepatocellular carcinoma livers with no significant change in hPXR gene levels. Together, these results show that PPM1B interacts with PXR, and increases PXR function by counteracting proliferation‐dependent PXR inhibitory signaling pathways in hepatocytes. Furthermore, downregulation of PPM1B levels in regenerating and hepatocellular carcinoma livers may in part explain a mechanism for repression of PXR‐mediated CYP3A levels in these liver pathophysiologies.

Grant Funding Source: Supported by Auburn University Animal Health and Disease Research grant and Start Up funds
Rolipram rescues oxidative stress‐induced premature phenotypes by SIRT6‐dependent NF‐kappaB inhibition (1142.11)
Authors: Wang Zhao , Liang Yaru
Published: June 2021

Cyclic adenosine monophosphate (cyclic AMP) is a key mediator of metabolic regulation and its signalling pathway is highly complex. Recent studies showed that resveratrol activates SIRT1 by increasing cyclic AMP signalling pathway to ameliorate aging‐related metabolic phenotypes. However, whether cyclic AMP itself could also rescue aging‐related functional declines remains unknown. We suppose that cyclic AMP may also contribute to other Sirtuin family members. We investigated the physiological and metabolic changes in oxidative stress‐induced premature mice, which were treated with experimental galactosemia and developed glycometabolism dysfunction. Here, we demonstrated that Rolipram, the specific PDE4 inhibitor, could increase cyclic AMP level to rescue the pathophysiology in premature mice. The mice treated with Rolipram showed increased SIRT6 and reduced acetylated NF‐kappaB protein level. However, cyclic AMP lost its inhibition effect on NF‐kappaB when SIRT6 was knocked down. Therefore, administration of Rolipram may protect against premature phenotypes in galactosemia or other glycometabolism dysfunction.
Association between decreased expression of GLT‐1 derived spinal glutamate accumulation and oxaliplatin‐induced mechanical allodynia (1143.1)
Authors: Ushio Soichiro , Mitsuyasu Shohei , Masuguchi Ken , Ozawa Nana , Ono Yuko , Kawashiri Takehiro , Egashira Nobuaki , Masuda Satohiro
Published: June 2021

Oxaliplatin (L‐OHP) is a key drug in the treatment of colorectal cancer, but it causes acute and chronic neuropathies in patients. We reported that NMDA receptors in spinal cord is involved in mechanical allodynia induced by L‐OHP (4 mg/kg, i.p., twice a week). In this study, we investigated the involvement of spinal glutamate in L‐OHP‐induced mechanical allodynia more closely. The L‐OHP‐induced mechanical allodynia was reversed not only by a NMDA receptor antagonist MK‐801 but also by an AMPA receptor antagonist NBQX. In vivo microdialysis, formalin‐induced nociception (5% formalin, 50µL, s.c.) sustainably increased extracellular glutamate concentration in the spinal cord in L‐OHP‐treated rats compared to vehicle‐treated rats, whereas tactile stimuli (brushing stimulation) increased extracellular glutamate concentration in the spinal cord in L‐OHP‐treated rats alone. Moreover, GLT‐1 (Glutamate transporter 1) but not GLAST (Glial glutamate transporter) and EAAC1 (Excitatory amino‐acid carrier 1), was decreased in the spinal cord dorsal horn in L‐OHP‐treated rats. These results suggest that L‐OHP inhibits uptake of extracellular glutamate by down‐regulation of GLT‐1 expression and increased accumulation of glutamate in the spinal cord.
The effect of pentadecapeptide BPC 157 in metamphetamine‐induced dopaminergic neurotoxicity (1143.11)
Authors: Suran Jelena , Kolenc Danijela , Prevendar Crnic Andreja , Radic Bozo , Drmic Ivan , Seiwerth Sven , Sikiric Predrag
Published: June 2021

We investigated the effect of stabile gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419), an original antiulcer peptide studied in inflammatory bowel disease and now multiple sclerosis clinical trials (LD‐1 not achieved, no side effects reported), in methamphetamine (METH)‐induced neurotoxicity, representing an animal model of monoamine disruption.

METH dose of 40 mg/kg b.w. (s.c.) was administered to Wistar rats 24 h prior to random assignment to control (saline, 1 ml/200 g b.w., i.p.) or BPC 157 (10 µg/kg, i.p.) group. Rats were monitored (phenotype, locomotor activity) for 7 days and histopathological analysis of striata was performed.

All animals had neurodegenerative phenotype, but these traits became more pronounced in controls compared to BPC 157 rats. Gradual decline in locomotor activity in BPC 157 rats was similar to behavior of healthy rats, while there was a marked hyperactivity in controls 7 days after METH administration. A higher number of red neurons counted in control rat striata and significantly higher number of normal‐appearing neurons in BPC 157 rats imply its beneficial effect on striatum. Therefore, we propose BPC 157as a potential therapeutic agent in monoamine disorders.

Grant Funding Source: Supported by Grant 108‐ 1083570‐3635, Croatia
Intracellular chloride concentration and its impact on dichloroacetate metabolism (1143.2)
Authors: Jahn Stephan , Zhong Guo , Smeltz Marci , Rowland‐Faux Laura , Stacpoole Peter , James Margaret
Published: June 2021

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase and an investigational drug used to treat mitochondrial disorders, pulmonary arterial hypertension, and cancer. The enzyme that metabolizes it, glutathione transferase Z1‐1 (GSTZ1‐1), is irreversibly inactivated upon metabolism of DCA. This leads to decreased clearance of subsequent doses and a buildup of the toxic physiological substrates of GSTZ1‐1, maleylacetone and maleylacetoacetate, possibly causing the observed side effect of peripheral neuropathy upon chronic administration. We have observed that the extent of this inactivation is dependent on chloride ion concentration, with high concentrations providing a protective effect. Here, we present data measuring intracellular [Cl‐] in human tissues and the effects that it has on the mechanism of inactivation, adduct formation. These results have important implications in the use of DCA, including predicting patient response, and may provide information to advance the use of DCA in patients in which GSTZ1‐1 inactivation leads to adverse effects.

Grant Funding Source: This work was supported by the US Public Health Service RO1 GM099871.
Comparison of the norepinephrine transporter blockers cocaine and desipramine in α1‐adrenoceptor studies of rat vas deferens (1145.5)
Authors: Docherty James
Published: June 2021

A wide range of prazosin pA2/pKB values have been reported in rat vas deferens: varying between 8.32 and 9.35, and the receptors present have been identified as α1A‐, α1D‐ in addition to α1A‐, or α1L‐adrenoceptors (see 1). Studies differ in choice of NET blocker: cocaine or desipramine (DES). We have now investigated the effects of NET blockers on the potency of prazosin at antagonizing contractions to norepinephrine (NE) in epididymal portions of rat vas deferens.

Cocaine significantly increased NE potency by revealing the α1D‐adrenoceptor‐mediated component. However, in the presence of cocaine (10 µM), DES (0.1‐10 µM) produced concentration‐dependent inhibition of the response to NE. Prazosin pKB values against NE varied from 8.32 (control), and 8.50 (DES 0.1µM), to 9.05 (cocaine 3 µM), with the lower values indicative of α1A‐adrenoceptor potency, and the higher value indicative of α1D‐adrenoceptor potency. Cocaine and DES (both 0.3 μM) potentiated the contractile response to nerve stimulation, but higher concentrations of DES inhibited the response.

Prazosin potency against NE was increased in the presence of NET blockade by cocaine. However, DES does not increase the potency of prazosin as it acts as an α1‐adrenoceptor antagonist over the concentration range that it blocks NET. DES may not be a suitable NET blocker for use in α1‐adrenoceptor studies.

(1) Docherty JR (2013). J Auton Autacoid Pharmacol 33, 49‐57.
The endothelium‐dependent, soluble guanylyl cyclase‐dependent augmentation caused by thymoquinone in isolated porcine coronary arteries is mediated by cIMP (1146.6)
Authors: Detremmerie Charlotte , Leung Susan , Xu Aimin , Alkharfy Khalid , Gao Yuangsheng , Vanhoutte Paul
Published: June 2021

Aim: Experiments were designed to determine the mechanism underlying the endothelium‐dependent, soluble guanylyl cyclase‐dependent augmentation caused by thymoquinone in isolated arteries.

Methods: Rings, with or without endothelium, of porcine coronary arteries were suspended in conventional organ chambers for isometric tension recording. Certain rings were incubated with inhibitors of nitric oxide (NO) synthase inhibitor (L‐NG‐nitroarginine methyl ester, L‐NAME) or soluble guanylyl cyclase (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, ODQ). They were contracted with phenylephrine (rat arteries) or prostaglandin F2α (porcine coronaries) and exposed to increasing concentrations of thymoquinone. Cyclic nucleotides were measured by HPLC‐MS/MS.

Results: Thymoquinone caused a sustained further increase of tension in rings with endothelium. This augmentation was prevented by endothelium‐removal, L‐NAME and ODQ. Incubation with the NO‐donor detaNONOate in L‐NAME‐treated rings restored and even increased the contractile response to thymoquinone. Administration of cIMP restored contractions to thymoquinone in rings without endothelium. By contrast; treatment with pyrophospate did not restore the augmentation by thymoquinone. HPLC‐MS/MS measurements revealed that the latter compound increased the production of cIMP while reducing that of cGMP.

Conclusion: The endothelium‐dependent augmentation caused by thymoquinone requires endothelium‐derived NO, activation of soluble guanylyl cyclase and production of cIMP but not the presence of either pyrophosphate or cyclic GMP.
PMA suppresses breast cancer cell proliferation via PKCε‐mediated P‐Rex1 downregulation (1148.5)
Authors: Wong Chuu‐Yun , Xie Yan , Abel Peter , Scofield Margaret , Tu Yaping
Published: June 2021

Upregulated phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) promotes tumorigenesis and metastasis of various cancers, and thus could be a potential therapeutic target. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. Western blot analysis showed that PMA treatment caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6 %, respectively, at a concentration of 10 ng/ml. Interestingly, either silence of P‐Rex1 by siRNA or PMA treatment inhibits breast cancer cell proliferation by over 70%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 cells express PMA‐sensitive PKCα, δ, ε, and η. The PKC general inhibitor Gö6983, but not the PKCα‐selective inhibitor Gö6976, blocked the PMA down‐regulation of P‐Rex1 expression and breast cancer cell proliferation. Overexpression of a constitutively active PKCε mutant suppressed P‐Rex1 expression and breast cancer cell proliferation, which was also blocked by Gö6983 treatment. In contrast, overexpression of wild‐type PKCε, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell proliferation via PKCε‐mediated P‐Rex1 down‐regulation. Since P‐Rex1 is highly overexpressed in human breast cancers with estrogen receptor (ER) expression, our study may provide a novel strategy for development of chemotherapeutic agents for patients with ER positive tumors that develop resistance to anti‐estrogen therapies.

Grant Funding Source: Supported by the National Institutes of Health (CA125661 and 5P20GM103489) and Nebraska State LB595
Role of different cathepsins in the invasion of MDA‐MB‐231 human breast cancer cells (1148.6)
Authors: Gautam Jaya , Kim Jung‐Ae
Published: June 2021

For the progression of tumor towards the malignancy such as invasion and metastasis, proteolytic activity is required for the degradation of the basement membrane and extracellular matrix. Matrix metalloproteinases (MMPs) were reported to play a major role in the proteolytic action. In addition to MMPs, cathepsins have emerged as an important molecular target for the management of cancer. They play a vital role in extracellular matrix degradation which accelerates the process of cancer cell invasion. Although cathepsin D (catD), a aspartyl protease, is overexpressed and secreted by cells of various tumor types including breast carcinomas, we found that cysteine proteases (catB, catK, catL and catS) rather than the aspartyl proteases participates in the invasion when they were blocked by siRNA transfection and specific inhibitors. In addition, combination of catB and catS inhibitor showed an additive effect on the inhibition of in vitro matrigel invasion. The result was correlated when both catB and catS were silenced by siRNA. Our data suggest that among the cysteine proteases, catB and catS are the key target enzymes for the invasion of highly metastatic breast cancer cells (MDA‐MB‐231). CatS has been suggested as a target for management of autoimmunity‐related disease such as arthritis without adverse effect (Nakagawa et al., 1999). Taken together, our results implicate that catS may be a good candidate target for new drug development for the management of human breast cancer metastasis.
Expression of hyperoxic bradycardia during hyperbaric exposure in rats prenatally exposed to hypoxia (1150.3)
Authors: Rozloznik Miroslav , Molcan Lubos , Svitok Pavel , Balestra Costantino , Zeman Michal
Published: June 2021

Intermittent prenatal exposure to hypoxia is associated with postnatal alteration in cardiac and endothelial functions. Hyperoxic exposure has significant effects on the cardiovascular system including bradycardia, vasoconstriction and endothelial dysfunction. The impact of intermittent prenatal hypoxia on cardiac rhythm control during mild hyperoxic conditions is unclear. Thus, expression of hyperoxic bradycardia during normobaric and hyperbaric hyperoxia was investigated telemetrically (PA‐C40, Data Sciences Int) in rats prenatally exposed to hypoxia.

Six male Wistar rats prenatally exposed to 10% hypoxia between gestation day 19‐21 (H) and 5 control (C) Wistar rats were exposed for 30 minutes to mild hyperbaric hyperoxia (ppO2 63 kPa, pabs 300 kPa and ppO2 108kPa, pabs 300 kPa) and normobaric hyperoxia (ppO2 67 kPA, pabs 100 kPa).

Hyperoxic exposure immediately triggered a bradycardic response in all animals. The bradycardic response was significantly more pronounced (p<0.05) during the hyperbaric exposure to ppO2 108kPa in H rats compared to C rats. Ambient pressure change significantly alternated baroreflex sensitivity (p<0.01) during ppO2 63 kPa exposure in H rats.

We conclude that hyperbaric exposure to mild hyperoxia indicates a developmental alteration in cardiac rhythm control in rats prenatally exposed to hypoxia and represents a perspective model to study cardiac rhythm alterations.

Grant Funding Source: Supported by grants VEGA 1/0686/12, 2/0107/12 and NOREG and FP7‐PEOPLE‐2010‐ITN
Glucagon like peptide‐1 augments cardiac output during regional myocardial ischemia via increases in ventricular preload, independent of changes in cardiac inotropy (1150.6)
Authors: Goodwill Adam , Casalini Eli , Conteh Abass , Noblet Jill , Sassoon Daniel , Tune Johnathan , Mather Kieren
Published: June 2021

This study examined the cardiovascular effects of GLP‐1 (7‐36) vs. (9‐36) in normal and ischemic hearts. Swine received systemic infusions of GLP‐1 (7‐36 or 9‐36) at 1.5, 3.0, and 10.0 pmol/kg/min in sequence for 30 min at each dose. Subsequent to this dose response, vehicle or GLP‐1 infusions were maintained at 10.0 pmol/kg/min and the left circumflex artery was ligated to induce regional ischemia. GLP‐1 (9‐36) had no effect on coronary flow, blood pressure, heart rate or indices of cardiac function before or during regional myocardial ischemia. GLP‐1 (7‐36) dose‐dependently increased myocardial glucose uptake (1.3 + 0.8 to 5.5 + 1.9 μmol/min/g; p=0.05), but exerted no other cardiovascular effects prior to ischemia. During ischemia, GLP‐1 (7‐36) increased cardiac output by 2.4 L/min relative to vehicle‐controls (p=0.003). Left ventricular pressure‐volume loops measured during graded IVC occlusion revealed marked increases in end diastolic volume (74 + 1 to 92 + 5 mL;p=0.03) and calculated ventricular volume at zero pressure (8 + 2 to 26 + 8; p=0.05) with GLP‐1(7‐36), while the slope of the end systolic pressure volume relationship was unchanged compared to vehicle and GLP‐1 (9‐36) treatments (p=0.98). These findings indicate that GLP‐1 (7‐36) but not (9‐36) significantly augments cardiac output during regional myocardial ischemia via increases in ventricular preload, independent of changes in cardiac inotropy.

Grant Funding Source: Supported by NIH grants HL117620 & T32HL079995. Additional support provided by AHA 13POST1681001813
Effect of berberine on the atrial natriuretic peptide secretion in cardiac atria (1150.9)
Authors: Yoo Hye Rim , Kim Hye Yoom , Kwon Oh Jeong , Cho Kyung Woo , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Effect of Berberine on the Atrial Natriuretic Peptide Secretion in Cardiac Atria

Hye Rim Yoo 1,2, Hye Yoom Kim 1,2, Oh Jeong Kwon 1,2, Kyung Woo Cho 2, Dae Gill Kang 1,2, and Ho Sub Lee 1,2*

1College of Oriental Medicine and Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570‐749, Republic of Korea; 2Hanbang Body‐fluid Research Center, Wonkwang University, Shinyong‐dong, Iksan, Jeonbuk, 570‐749, Republic of Korea

The aim of the present study is to identify the effects of berberine on the secretion of atrial natriuretic peptide (ANP) from the atrium. Experiments were performed in perfused beating rat left atria paced electrically. Atrial dynamics, atrial pulse pressure, was measured with physiograph. ANP was measured using radioimmunoassay. As a result, berberine increased ANP secretion and atrial dynamics in a concentration‐ dependent manner. Atropine‐non‐selective muscarinic receptor antagonist, methoctramine‐selective M2 muscarinic receptor antagonist, and hemicholinium 3‐inhibitor of choline transporter which inhibits acetylcholine synthesis had no effects on the berberine‐induced changes in ANP secretion and pulse pressure. Berberine is known to activate AMP‐activated protein kinase (AMPK), experiments were performed to identify involvements of this possibility on the berberine‐induced changes in ANP secretion and pulse pressure. Compound C, an inhibitor of AMPK activation, slightly inhibited the berberine‐induced increase in ANP secretion and pulse pressure. These findings suggest that berberine increases ANP secretion and atrial pulse pressure via, at least in part, activation of AMPK signaling.