bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanistic Role of Cytochrome P450 (CYP)1A2 in the Regulation of Hepatic and Pulmonary CYP1A1 Expression in Mice Treated with 3‐Methycholanthrene
Authors: Moorthy Bhagavatula , Maturu Paramahamsa , Wang Lihua , Chu Chun , Jiang Weiwu
Published: November 2023

3‐Methylcholanthrene (MC) is one of most potent polycyclic aromatic hydrocarbons (PAHs) present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP) 1A enzymes play key roles in the activation of PAHs to carcinogenic metabolites. In this study, we tested the hypothesis that CYP1A2 plays a mechanistic role in the regulation of hepatic and pulmonary CYP1A1 by MC. Wild type (WT) (C57B6) and Cyp1a2_null mice were divided into two groups. Group I was treated with vehicle corn oil (CO) (8 ml/kg) and group II was treated with four doses of MC (100 μmol/kg), i.p. once daily for 4 days. Four animals from each group were sacrificed at 6, 12, 24, and 48 h, 8 and 15 days after MC withdrawal. The mRNA levels, protein content and enzyme activities of CYP1A1 and 1A2 were determined at different time points. In addition, the binding of MC‐AHR‐AHR nuclear translocator (ARNT) to the AHREs on the CYP1A1 promoter region was determined by chromatin immunoprecipitation (ChIP) assay in liver and lung tissues. In WT mice, ChIP experiments indicated that transcriptional activation of CYP1A1 was most pronounced at 6 h, followed by 12 h, but declined at later time points. In Cyp1a2_null mice, on the other hand, transcriptional activation of CYP1A1 persisted till 48 h. In lungs, AHR complex binding peak was observed at 12 h and declined later, but Cyp1a2 null mice showed more persistent binding till 48 h with a peak at 24 h. On the other hand, the expression of CYP1A1/1A2 at the mRNA, protein, and enzyme levels persisted for up to 48 h both in lung and liver tissues. These results suggest a mechanistic role for CYP1A2 in the molecular regulation of CYP1A1, and that transcriptional activation of CYP1A1 at 6–12 h is sufficient to result in sustained induction of CYP1A mRNA and protein expression for up to 48 h. These studies have important implications for carcinogenesis mediated by PAHs.

Support or Funding Information

NIH Grant R01 ES009132
Effect of CYP17A1 17,20 Lyase Inhibitors on Regulation of Adrenal Androgen Biosynthesis
Authors: Pandey Amit V , Udhane Sameer S
Published: November 2023

Background

The P450c17 (CYP17A1) plays a vital role in regulating adrenal androgen production. CYP17A1 localized in the endoplasmic reticulum can catalyze both 17α‐hydroxylase and 17,20 lyase reactions. Understanding the mechanisms regulating 17,20 lyase activity is important for the understanding of hyperandrogenic disorders such as premature, exaggerated adrenarche and the polycystic ovary syndrome, and also for the design of selective 17,20 lyase inhibitors for use in hyperandrogenic states and in sex‐steroid dependent cancers. The orteronel and galeterone are known to inhibit 17,20 lyase activity; however, the precise mechanism of the CYP17A1 inhibition remains unknown. These inhibitors have been developed to treat the castration resistant prostate cancer (CRPC) but little is known about their effect on adrenal androgen biosynthesis. We have studied the specific 17,20 layse inhibitors to understand their effects on CYP17A1 and adrenal androgen biosynthesis.

Methods

We used NCI‐H295R adenocarcinoma cells to study the effect of orteronel and galeterone. We treated the H295R cells with 0–2μM orteronel and galeterone for 24 hours. Steroid production was labeled with [3H] pregnenolone for 90 min. Steroids were extracted and resolved by thin layer chromatography. For specific analysis of the CYP17A1, CYP19A1 and CYP21A2 activities, cells were treated with 1μM trilostane (a specific blocker of HSD3B) for 90 min before adding radiolabelled steroids. To study the effect of these inhibitors on steroidogenic gene expression we performed the relative quantification PCR (qRT‐PCR). Steroid metabolome of cells was determined in parallel experiments using GC‐MC/LC‐MS.

Results

The drugs orteronel and galeterone were able to inhibit CYP17A1 activity in H295R cells. Both drugs have more potency towards the 17,20 lyase activity but we also observed that they partially affected 17 alpha‐hydroxylase activity. From our results, we observed the orteronel seems to be more potent and selective towards 17,20 lyase activity than galeterone. However, we also found dihydroepiandrosterone (DHEA), cortisol and androstenedione were drastically decreased by both compounds at 1 and 2μm concentration. Further, we also studied effect of these inhibitors on genes which are involved in androgen biosynthesis. We found slight increase in the HSD3B2 gene expression under galeterone treatment but no change was observed in CYP17A1 and AKR1C3 expression.

Conclusions

Based on our results we conclude that orteronel is a more potent inhibitor of 17,20 lyase activity and also has partial effect on 17α‐hydroxylase activities.

Support or Funding Information

This work was supported by grants to Amit V Pandey from the Swiss National Science Foundation and Bern University Research Foundation.
Inhibition of Human CYP3A‐Catalyzed Lithocholic Acid 3‐Oxidation by Specific Vitamin E Isomers
Authors: Lau Aik Jiang , Chai Swee Fen , Teo Josephine Si Min
Published: November 2023

Lithocholic acid is a hepatotoxic and carcinogenic bile acid. Previous studies suggested that CYP3A4 but not CYP3A5 catalyzed the oxidation of lithocholic acid to 3‐ketocholanoic acid and that CYP3A metabolized vitamin E isomers (tocotrienols and tocopherols). Given that a substrate of an enzyme may also be an inhibitor of that enzyme, we determined whether α‐tocotrienol, γ‐tocotrienol, δ‐tocotrienol, a tocotrienol‐rich mixture (a mixture consisting of 25.7% α‐tocotrienol, 2.6% β‐tocotrienol, 28.6% γ‐tocotrienol, 8.4% δ‐tocotrienol, 25.6% α‐tocopherol, and 4.3% α‐tocomonoenol), and α‐tocopherol inhibit the oxidation of lithocholic acid to 3‐ketocholanoic acid. Enzymatic formation of 3‐ketocholanoic acid via lithocholic acid 3‐oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5 enzymes. Enzyme inhibition assay was conducted in an incubation mixture containing potassium phosphate buffer, lithocholic acid, NADPH, enzyme (human liver microsomes, recombinant CYP3A4, or recombinant CYP3A5), and a putative inhibitor (a tocotrienol, a tocotrienol‐rich mixture, or α‐tocopherol). The amount of 3‐ketocholanoic acid was quantified by ultra‐high performance liquid chromatography‐tandem mass spectrometry. Lithocholic acid was metabolized to 3‐ketocholanoic acid by human recombinant CYP3A4 and CYP3A5 enzymes and human liver microsomes. Human liver microsomal‐catalyzed lithocholic acid 3‐oxidation exhibited Michaelis‐Menten kinetics at substrate concentrations less than 100 μM. Substrate inhibition kinetics was evident at lithocholic acid concentrations greater than 100 μM. The Km, Vmax, and Vmax/Km values for human liver microsomal LCA 3‐oxidation were 26 μM, 304 pmol/min/mg, and 12 μl/min/mg, respectively. Enzyme kinetics experiments indicated that human recombinant CYP3A4 and CYP3A5 catalyzed lithocholic acid 3‐oxidation to a similar extent. α‐Tocotrienol, γ‐tocotrienol, δ‐tocotrienol, and a tocotrienol‐rich mixture, but not α‐tocopherol, inhibited lithocholic acid 3‐oxidation by human liver microsomes. Concentration‐response experiments indicated that a tocotrienol‐rich mixture and δ‐tocotrienol inhibited human liver microsomal lithocholic acid 3‐oxidation with IC50 of 6.6 ± 2.1 μg/ml and 19.0 ± 1.0 μM, respectively. Overall, lithocholic acid 3‐oxidation is catalyzed by both CYP3A4 and CYP3A5 enzymes. It is differentially inhibited by tocotrienols, but not α‐tocopherol, suggesting vitamin E isomer‐specific inhibition of human liver microsomal lithocholic acid 3‐oxidation.

Support or Funding Information

Supported by National University of Singapore Research Fund.
The N‐terminal Region of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) Plays an Essential Role in Regulating its Plasma Membrane Trafficking
Authors: Chun Se‐Eun , Thakkar Nilay , Park Ji Eun , Han Songhee , Maeng Sang Hyun , Lim Young‐Ran , Machado Daniel , Lee Wooin
Published: November 2023

Organic anion transporting polypeptide 1B3 (OATP1B3) mediates the hepatic uptake of various endogenous and xenobiotic compounds and it is shown to influence the disposition of clinically important drugs including statins and paclitaxel. Recently, we and others reported the expression of cancer‐type OATP1B3 which arises from the use of an alternative transcription initiation site and lacks the N‐terminal 28 amino acids compared to liver‐type OATP1B3. We also noted that cancer‐type OATP1B3 displays mainly a cytoplasmic expression pattern clearly different from a membranous expression pattern of liver‐type OATP1B3, in both clinical cancer tissue specimens and cancer cell lines. In this study, we set out to verify the importance of the N‐terminal region of OATP1B3 in its membrane trafficking and to identify responsible sequence motif(s) in that region. We first confirmed our previous findings by transfecting the expression plasmid for cancer‐type or liver‐type OATP1B3 fused with the C‐terminal myc tag using HCT8 and HEK293T cells. Subsequently, we prepared a number of expression plasmids harboring point mutations at various amino acid positions (K or R with a positive charge, S with potential for phosphorylation, ASSE tetrapeptide with potential for forming a beta‐turn) within the N‐terminal 28 amino acids of OATP1B3 via site‐directed mutagenesis. These constructs were transiently transfected in HEK293T cells and compared for their cellular localization by immunoblotting analyses of fractionated samples. Our results showed that various point mutations within the N‐terminal 28 amino acids do not alter the extent of membrane trafficking of OATP1B3. As an alternative approach, we prepared a construct for the N‐terminal 50 amino acid sequence of OATP1B3 fused with the C‐terminal myc tag and we found that the resulting fusion protein is effectively localized to the plasma membrane. Similar experiments were carried out for OATP1B1, a closely related member to OATP1B3 and the results showed that the importance of the N‐terminal region in regulating membrane trafficking is also applicable to OATP1B1. Further investigations are ongoing to identify essential amino acid residue(s)/motif(s) for membrane trafficking within the N‐terminal 50 amino acids of OATP1B3 and OATP1B1. Our current findings may provide important mechanistic insights into the regulation of OATP1B3 and OATP1B1 expression and membrane localization in physiological and pathological conditions.
Potent in vitro and in vivo effects of EB‐3D, a novel choline‐kinase inhibitor in breast cancer
Authors: Viola Giampietro , Mariotto Elena , Bortolozzi Roberta , Ronca Roberto , Cara Luisa Carlota Lopez , Entrena Antonio , Basso Giuseppe
Published: November 2023

Choline kinase (ChoK) is one of the most important enzymes for the generation of two major membrane phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), and subsequently for the cell division. Choline kinase is an homodimeric enzyme that catalyses the Mg. ATP‐dependent phosphorylation of choline to generate phosphocholine (PCho) as the first step in the Kennedy pathway. In mammalian cells, only the ChoKα isoform (synthesized by Chkα gene) has a central role in sustaining PCho biosynthesis, indeed ChoKβ alone cannot compensate this activity.

Furthermore, a large number of studies in cancer cells suggests that ChoKα expression and activity are directly associated not only with increased cancer cell proliferation but also with malignancy, making it a potential prognostic marker for some cancers, such as non‐small‐cell lung cancer and breast cancer as well as a potential novel target for image‐guided cancer therapy. Thus, the cholinic phenotype, characterized by increased ChoKα activity and expression, renders this enzyme an attractive new therapeutic target. The feasibility of ChoKα inhibition as antitumoral therapy is being pursued through the development of chemical inhibitors of its enzymatic activity and through ChoKα silencing by RNA interference.

In this context we have evaluated both in vitro and in vivo the activity of EB‐3D (1,1′‐(((ethane‐1,2‐diylbis(oxy))bis(4,1‐phenylene))bis(methylene))bis(4‐(dimethylamino)pyridinium) bromide) a new choline kinase inhibitor endowed with high antiproliferative activity in different tumor mammary cell lines such as the human MCF‐7, MDA‐MB231 and the murine EO771.

Interestingly we observed that the inhibition of cell proliferation is not dependent on the presence of the molecule in the incubation medium. In fact, drug wash‐out experiments highlighted that proliferation continues to be inhibited even after EB‐3D removal. These data suggests that the catalytic activity of the enzyme may be irreversibly inhibited. In addition the compound induces a strong G1 arrest of the cell cycle that occurs in a concentration dependent manner in all three cell lines tested. Along with the G1 increase, we observed a concomitant reduction in the S phase. We are actually investigating the mode of cell death induced by this compounds and the result will be presented.

To further examine the antitumorigenic potential of EB‐3D in vivo, we utilized a syngeneic orthotopical EO771‐C57BL/6 mouse model of breast cancer. Preliminary results indicated that the compound significantly reduce the tumor mass at the dose of 1 mg/Kg, with no apparent sign of toxicity. Taken together our results suggest that EB‐3D is a promising new anticancer agent for cancer treatment and worthy of further development.
Preclinical Research Strategies for Newly Approved Veterinary Drugs as Reflected in Early Publication Patterns
Authors: Köster Ursula , Nolte Ingo , Michel Martin C.
Published: November 2023

The present study has explored early publication patterns (i.e. publication up to one year after obtaining regulatory approval) for newly registered veterinary drugs, assuming that these reflect preclinical research strategies. Newly approved drugs for veterinary use were identified from the US Food and Drug Administration website for six approval years between 1991 and 2011; drugs from additional approval years are currently being retrieved. Vaccines, drugs previously registered for human use and those representing new salts or new formulations of previously approved compounds were excluded. Pre‐clinical original publications for each compound were retrieved from PubMed and their abstracts analyzed for type of study and reported data. A total of 28 veterinary drugs were identified that had a total of 284 original non‐clinical publications (median/mean of 3/10.1 publications per compound); however, number of publications per compound varied widely (0–52). The cardiovascular drug pimobendan (52), the anesthetics atipamezole (35), medetomidine (28) and xylazine (47), and the anti‐infective ivermectin (37) had the greatest number of publications and markedly skewed the overall distribution. However, this does not necessarily reflect a trend for more publications in some indications; as the anesthetics carprofen (10) and flunixin (5) or the anti‐infectives ceftiofur, dorameticin, florfenical, sarafloxacin and tilmicosin (0–4 each) had much fewer publications. The earliest non‐clinical publications were published more than 10 years before regulatory approval in 10 cases, including 3 with 20 years prior to approval. Publications most often reported on efficacy related to the target indication and on pharmacokinetics and drug metabolism. Papers using isolated tissues or cells were found only rarely, and the vast majority of publications reported in vivo data. Those in vivo studies used a wide range of species other than the intended target species, including mice, rats, rabbits, pigs, dogs and others. We conclude that number, timing and scope of early non‐clinical publications on newly approved veterinary drugs exhibit major variance, which is not easily explained by indication. While median and mean number of early preclinical publications of veterinary drug did not differ substantially from that of 142 compounds approved for human use in the same period (Köster et al., Naunyn‐Schmiedebergs Arch Pharmacol, in press), the early preclinical publications of veterinary drugs appear to use in vivo models more frequently and often have a different focus; moreover, publications often start earlier relative to approval date.
Kolaviron attenuated arsenic acid‐induced cardiovascular dysfunction by enhancing antioxidant defense system and inhibiting inflammatory and apoptotic signaling pathways
Authors: Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Olowu Ebunoluwa Racheal , Adedapo Adeolu Alex , Yakubu Momoh A
Published: November 2023

Arsenic acid is one of the abundant environmental pollutants present in soil, water and the air. Undoubtedly, it has found its way to the food chain in which humans and animals are the final targets causing arrays of disease conditions including cardiovascular and renal dysfunction. Hence, the use of phytochemicals present in medicinal plants has gained global acceptance as chemotherapeutic agents that can prevent, ameliorate, reverse or treat diseases. From our study, Arsenic acid intoxication led to significant increase in heart rate (HR), QRS, together with prolonged QT and QTc. However, Kolaviron (KV) at the dosage of 100 and 200mg/kg body weight reversed the aforementioned electrocardiographic (ECG) changes. KV pre‐treatment also ameliorated cardiorenal dysfunction via significant reduction in cardiac and renal markers of oxidative stress such as malondialdehyde (MDA), hydrogen peroxide generation (H2O2), myeloperoxidase (MPO) activity and nitric oxide (NO) contents. Similarly, antioxidant deference system was significant improved in rats pre‐treated with KV. Immunohistochemistry revealed reduction in the expression of renal C‐reactive proteins (CRP) and increase in anti‐apoptotic protein BCL2 expression in KV treated rats. Furthermore, cardiac troponin I (CTnI) expression was lower in KV treated rats. Taken together, KV mitigated arsenic‐acid induced cardiovascular dysfunction via up‐regulation of antioxidant defense system and down‐regulation of inflammatory and apoptotic signaling pathways.
Interaction of Gq‐ and Gs‐coupled Pathways in the Regulation of Ca2+ Spiking and Activity of Kv7.5 Potassium Channels in A7r5 Vascular Smooth Muscle Cells
Authors: Himes Christopher R , Condon Bridget , Rak Mariola , Brueggemann Lyubov I , Byron Kenneth L
Published: November 2023

The vasoconstrictor hormone arginine vasopressin (AVP), at picomolar concentrations, activates Gq‐coupled Va1 receptors and suppresses the activity of vascular Kv7.5 potassium (K+) channels in a protein kinase C (PKC) ‐dependent manner. The PKC‐dependent inhibition of Kv7.5 K+ current in vascular smooth muscle cells causes membrane depolarization and activation of L‐type voltage dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ concentration, contraction of the vascular smooth muscle cells, and vasoconstriction. Conversely, we found that activity of vascular Kv7.5 channels is enhanced upon activation of β‐adrenergic Gs‐ coupled receptors, in a protein kinase A (PKA)‐ dependent manner. In the present study we investigated physiological consequences of the sequential activation of the PKC‐ and PKA‐dependent pathways. The amplitude of endogenous Kv7.5 currents in A7r5 vascular smooth muscle cells was measured using whole‐cell perforated‐patch voltage clamp, and the frequency of Ca2+ spiking was measured using the Ca2+ indicator fura‐2. We found that the Kv7.5 current amplitude was reduced by 100 pM AVP by 79 ± 5%, but addition of the β‐adrenergic agonist isoproterenol (1 μM) restored the amplitude to the original level. Applying the treatments in the opposite order, isoproterenol (1 μM) enhanced the Kv7.5 current by 6.1 ± 1.5 ‐fold and this much larger current was suppressed by 40% by addition of 100 pM AVP. In contrast, Ca2+ spiking, induced by 50 pM AVP and dependent on activation of L‐type voltage dependent Ca2+ channels, was not effected by treatment with 1 μM isoproterenol, added either before or after application of AVP. The discrepancy between the effectiveness of the isoproterenol to restore Kv7.5 current amplitude and its inability to reduce the frequency of AVP‐induced Ca2+ spiking in A7r5 cells might be partially explained by an isoproterenol‐induced increase (54 ± 8%) in the amplitude of L‐type Ca2+ currents.

Support or Funding Information

Loyola University Chicago
Decreased Role for BKCa Channels in Endothelium‐Dependent Relaxation of Nitrate Tolerant Coronary Arteries
Authors: Mughal Amreen , Sane Mukta , Sun Chengwen , O'Rourke Stephen
Published: November 2023

Prolonged exposure to nitroglycerin (NTG) leads to tolerance and impaired endothelium‐dependent vasodilation. Causes of endothelial dysfunction in nitrate tolerant arteries are well studied, but little is known about mechanisms that underlie the responses to endothelium‐derived mediators in nitrate tolerant blood vessels. Here we evaluated the role of large conductance, calcium‐activated K channels (BKCa) in acetylcholine (ACh)‐induced relaxation of isolated coronary arteries. Rats (Sprague‐Dawley, 250–300g) were treated with or without transdermal NTG patches (0.6 mg/hr) for 3 days. Isolated coronary arterial rings (diameter: 150–300 μm) were mounted in a multi‐wire myograph for isometric tension recording. Relaxation responses were determined in rings contracted with 5‐HT (0.1–1μM). In some experiments, arterial segments were incubated with the selective BKCa channel inhibitor, iberiotoxin (IBTx, 100 nM), for 20 min prior to contraction. Expression of BKCa channel proteins was determined by Western blot. Nitrate tolerance was confirmed by the impaired relaxation response to NTG (1 nM – 100 μM) observed in rings from rats treated with NTG patches (pD2= 7.28 ± 0.1 vs 6.45 ± 0.2, control vs NTG patch‐treated, respectively; p<0.05). Relaxations to the endothelium‐dependent vasodilator, ACh (1 nM – 10 μM) were impaired in nitrate tolerant arteries (pD2 = 7.01 ± 0.1 vs 6.52 ± 0.1; control vs tolerant, respectively; n=8; p<0.05). Incubation with the eNOS inhibitor, nitro‐l‐arginine (30 μM), or the guanylyl cyclase inhibitor, ODQ (10 μM), markedly inhibited the response to ACh in control and tolerant arteries, consistent with the release of endothelium‐derived NO in ACh‐induced relaxation of both control and tolerant arteries. Similarly, IBTx markedly reduced the response to ACh in control and tolerant arteries; however, IBTx was a more effective inhibitor of ACh‐induced relaxation in control arteries. Relaxations to 8‐Br‐cGMP (100 nM – 300 μM), a stable, cell‐permeable cGMP analog, were inhibited by IBTx in control arteries (pD2 = 5.37 ± 0.2 vs 4.21 ± 0.2; in the absence and presence of IBTx, respectively; n=4; p<0.05), but not in tolerant arteries (pD2 = 4.45 ± 0.1 vs 4.03 ± 0.1; in the absence and presence of IBTx; n=3; p>0.05). Protein expression of BKCa α‐subunits and β‐subunits was decreased by more than 40% in nitrate tolerant arteries (n=4–5; p<0.05). Taken together, the data indicate that, under control conditions, BKCa channels play a pivotal role in endothelium‐dependent relaxation to ACh in rat coronary arteries. In nitrate tolerant coronary arteries, the response to ACh is impaired and the contribution of BKCa channels to ACh‐induced relaxation is diminished.

Support or Funding Information

Supported in part by grant HL098896 from the National Institutes of Health
Activation of CLC‐3 Chloride Channels During Myocardial Ischemia Limits Prolongation of Q‐T and Effective Refractory Period
Authors: Yu Ying , Ye Lingyu Linda , Li Yi‐Gang , Duan Dayue Darrel
Published: November 2023

Introduction

Ischemia causes myocardial infarction and arrhythmias, such as Q‐T prolongation which leads to fatal ventricular arrhythmia. Up‐regulation of cardiac CLC‐3 chloride channels is important for ischemic preconditioning‐induced second‐window protection against myocardial infarction. But its consequences in ischemia‐induced electrical remodeling are still unknown.

Hypothesis

CLC‐3 up‐regulation may shorten action potential during, Q‐T interval, and effective refractory period (ERP)under ischemic conditions.

Methods

In vivo surface electrocardiography (ECG) and intracardiac electrophysiology (ICEP) were used to compare the electrophysiological properties of age‐matched wild‐type (Clcn3+/+) and heart‐specific overexpression of human short CLC‐3 isoform (hsCLC‐3OE) mice under control and myocardial ischemia‐reperfusion conditions.

Results

Q‐T and Q‐Tc intervals of hsCLC‐3OE mice were significant shorter than those of Clcn3+/+ mice under control, ischemia and reperfusion conditions. In the ICEP, ventricular effective refractory period (VERP) of hsCLC‐3OE mice (26.7±1.7 ms, n=6) was significantly shorter than that of Clcn3+/+ mice (36.9±2.8 ms, n=8, P<0.05). Under ischemia condition, both VERP (19.8±1.3 ms) and atrial effective refractory period (AERP, 34.8±2.5 ms) of hsCLC‐3OE mice were significantly shorter than those of Clcn3+/+ mice (35.2±3.0 ms and 45.8±1.6 ms, P<0.01, respectively). Wenckebach atrioventricular block point (AVBP, 91.13±4.08 ms) and 2:1 AVBP (71.3±3.8 ms) of hsCLC‐3OE mice were significantly shorter than those of Clcn3+/+ mice (102.0±2.0 ms and 84.1±2.8 ms, P<0.05, respectively). However, no differences of ICEP parameters between hsCLC‐3OE and Clcn3+/+ mice were observed under reperfusion conditions.

Conclusions

Up‐regulation of CLC‐3 may limit the ischemia‐induced Q‐T and ERP prolongation and postpone the advancements of Wenckebach and 2:1 AVBP. CLC‐3 up‐regulation may serve as an important adaptive mechanism against myocardial ischemia.

Support or Funding Information

This study was supported by National Heart, Lung, and Blood Institute Grant R01 #HL63914, #HL113598, R21 HL106252, AHA Western State Affiliate Grant‐in‐Aid #11GRNT7610161 (to D.D.D.), and National Natural Science Foundation of China (#81070154 and #81270258 to Y.G.L.).
Carvacrol inhibits TRPM7‐like current in native myocytes from mesenteric artery of Hypertensive Rats
Authors: Silva Darizy Flavia , Reis Milena Ramos , Vasconcelos Alyne Pereira , Cruz Jader Santos , Araújo Demetrius Antonio Machado
Published: November 2023

Carvacrol, a phenolic monoterpene, is found in several plants of Origanum genus and has been described to cause vasodilatation in mesenteric artery from normotensive rats by reducing Ca2+ influx in vascular smooth muscle cells (VSMC). Then, the aim of this study was to investigate the effect of carvacrol on TRPM6/TRPM7 channels in VSMC from normotensive and hypertensive rats. Under whole‐cell voltage‐clamp, with voltage‐dependent Ca2+ and K+ channels blocked, currents generated by TRPM6/TRPM7 channels were measured by symmetrical voltage ramp commands to potentials between −100 to +100 mV, from a holding potential of 0 mV, displaying outward rectification in physiological extracellular medium. In VSMC isolated from hypertensive rats, carvacrol (100 and 300 μM) inhibit significantly both inward (−80 mV) and outward (+80 mV) currents [(current values at − 80 mV: (−3.42 ± 0.47 pA/pF; N = 5, control); (−1.07 ± 0.07 pA/pF, N = 5, p<0.05, carvacrol 100 μM); (−1.86 ± 0.34 pA/pF, N = 5, p<0.05, carvacrol 300 μM); (current values at + 80 mV: −6.21 ± 0.57 pA/pF, N = 5, control); (−3.53 ± 0.28 pA/pF, N = 5, p<0.01, carvacrol 100 μM); (−2.29 ± 0.27 pA/pF, N = 5, p<0.001, carvacrol 300 μM)]. This effect was similar to that induced by elevating extracellular Mg2+ (2.5 mM, N=5). In the presence of 2‐APB (100 μM, N=5), a TRPM7 blocker, no additional effect in TRPM7/TRPM6 whole‐cell current was observed after perfusion with carvacrol (300 μM; N=5), suggesting that probably, the effect of carvacrol is through inhibition of TRPM7‐like currents (ITRPM7) in VSMC. Additionally, since the sensitivity of TRPM7 channels to Mg2+ is an essential condition, we examined whether the above described current is inhibited by high concentrations of intracellular Mg2+. In this condition, the magnitude of TRPM7‐like current was significantly decreased inward and outward currents, in both myocytes, from hypertensive and normotensive rats, confirming that ITRPM were ITRPM6/TRPM7. In conclusion, these data demonstrate that carvacrol inhibited ITRPM6/TRPM7, in myocytes isolated from mesenteric artery of hypertensive and normotensive rats. This study provides the first electrophysiological evidence of TRPM7‐like current in native VSMC from mesenteric artery.

Support or Funding Information

Fundação de Amparo à pesquisa do estado da Bahia (FAPESB) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Effects of Nerve Stimulation in Mouse Lungs
Authors: Kieffer Cameron , Tu Yaping , Abel Peter
Published: November 2023

Mouse pulmonary airways and blood vessels are innervated by autonomic and sensory nerves. Stimulation of mouse pulmonary nerves can activate a variety of neurotransmitter systems to control airway and blood vessel diameter. The objective of this study was to identify the neurotransmitter systems causing constriction and/or dilatation of mouse airways and pulmonary arteries. Precision cut lung slices from CF‐1 mice were exposed to electric field stimulation (EFS) using parameters known to activate neurotransmitter release (1.0 ms, 50 V, 1–30 Hz). Changes in airway or pulmonary artery luminal area were captured as video images and quantified using ImageJ. EFS caused constriction in both airways and pulmonary arteries in a frequency‐dependent manner. Maximum constriction to 30 Hz EFS was 52.8 ± 3.5% in airways and 42.4 ± 3.7% in pulmonary arteries. Interestingly, the ability of the muscarinic receptor antagonist atropine to block EFS induced constriction in airways was frequency dependent. In airways, 1 μM atropine blocked 70.6 ± 19.2% of EFS induced constriction at 3 Hz, but only blocked 22.1 ± 12.6% of EFS induced constriction at 30 Hz. Treatment of pulmonary arteries with 1 μM prazosin, an α1‐adrenoceptor antagonist, reduced constriction by 62.6 ± 12.3% at 30 Hz EFS. These data are consistent with parasympathetic constriction of airways by acetylcholine and sympathetic constriction of pulmonary arteries by norepinephrine. In addition, an atropine‐resistant EFS constriction in airways and a prazosin‐resistant EFS constriction in pulmonary arteries were identified. Understanding the neurotransmitter systems causing nerve mediated constriction and/or dilatation of airways and pulmonary arteries may identify novel therapeutic targets for treatment of asthma, pulmonary hypertension, and other lung diseases.

Support or Funding Information

Supported by NIH R01HL116849 and Nebraska State LB595 to Y. Tu and P.W. Abel
Hypertension Mechanisms: Elastin Haploinsufficiency Alters Renal Structure and Function
Authors: Osei‐Owusu Patrick , Owens Elizabeth A , Jie Li , Reyes Beverly A , Van Bockstaele Elisabeth J
Published: November 2023

Hypertension is a hallmark of Williams syndrome, an autosomal‐dominant disease caused partly by loss‐of‐function mutations or gene deletions resulting in elastin (Eln) haploinsufficiency. Hypertension is a leading risk for several cardiovascular diseases including heart failure, stroke, chronic kidney disease, and renal failure. The mechanisms underlying hypertension due to elastin insufficiency are poorly understood. Because the kidney plays a critical role in long‐term blood pressure (BP) control, we determined whether Eln haploinsufficiency affects renal structure and function to contribute to hypertension development. We determined the effects of hypertension on wild type (WT) and Eln+/− mouse kidney structure using histology and electron microscopy. Kidney function was assessed by measuring renal blood flow, glomerular filtration rate (GFR), urine output, sodium and potassium excretion, and proteinuria. We found that Eln+/− mice had glomerular podocyte effacement accompanied by increased BP, renal vascular resistance (RVR), urine output and sodium excretion rate. However, GFR was only slightly elevated in Eln+/− mice. Systemic angiotensin type 1 receptor blockade with candesartan decreased RVR and BP in female Eln+/− mice to levels in WT controls. Ex vivo pressurized vessel studies showed increased myogenic tone accompanied by increased wall tension and wall strain in interlobar arteries from Eln+/− mice. We conclude that Eln haploinsufficiency causes renal vascular dysfunction, which contributes to the development of hypertension. Such a prolonged systemic BP elevation leads to glomerular structural damage due to high renal perfusion pressure. Therefore, therapies that control BP in patients with elastin deficiency may be beneficial in preventing hypertension‐evoked kidney damage.

Support or Funding Information

Margaret Q. Landenberger Research Foundation Grant
Loss of serotonin transporter function alters ADP‐mediated αIIbβ3 activation through disregulation of 5HT2A receptor
Authors: Oliver Kendra H , Hamm Heidi , Carneiro Ana M. D.
Published: November 2023

Clinical observations indicate that patients taking selective serotonin reuptake inhibitors (SSRIs) have an increased risk of bleeding. However it remains unclear how SSRIs, which inhibit the serotonin transporter (SERT), modulate hemostasis, particularly platelet activation. 5HT (serotonin, 5‐hydroxytryptamine) is taken up by platelets via SERT and activates the 5HT2A receptor, the only serotonergic receptor found on platelets. αIIbβ3 is an integrin that binds fibrinogen and plays a critical role in platelet aggregation, which is central to hemostasis and thrombosis. Futhermore, it is known that αIIbβ3 and SERT interact genetically, physically, and functionally. To gain a clearer understanding of how SERT function regulates platelet activity we examined two independent models of SERT inhibition – constitutive genetic deletion (SERT−/−) and 6‐day water citalopram administration. As observed in clinical populations, whole blood 5HT was drastically reduced in SERT−/− and 6‐day water citalopram‐treated mice. This suggests that, through SERT, platelets are the major storage site for 5HT in whole blood. In this model of altered serotonergic peripheral tone, SERT−/− platelets respond normally to PAR4‐AP activation, but ADP‐mediated αIIbβ3 activation is decreased as assessed by JON/a binding. Additionally while 5HT alone does not cause any platelet activation in SERT+/+, 5HT synergizes with submaximal ADP to potentiate αIIbβ3 activation. We next tested if the 5HT‐ADP synergy remained intact in SERT−/− platelets by adding exogenous 5HT. Strikingly, we found that 5HT was unable to potentiate ADP‐mediated αIIbβ3 activation in SERT−/− platelets. Blocking 5HT2A receptor with an antagonist, ketanserin (50nM), inhibited ADP‐mediated αIIbβ3 activation, suggesting that 5HT‐ADP synergy could be mediated through activation of the 5HT2A receptor. We hypothesized that SERT−/− platelets may not respond to 5HT due to an alteration in 5HT2A receptor and found that surface expression of 5HT2A receptor was reduced in SERT−/− platelets. SERT−/− platelet membranes incubated with a 5HT2A agonist, DOI, also exhibited reduced 35SGTPys incorporation as compared to SERT+/+. These findings suggest that long‐term inhibition of SERT leads to reduced 5HT2A receptor‐dependent potentiation of αIIbβ3 activation. Additional experiments are required to investigate the mechanism governing altered surface expression of 5HT2A receptor and ADP‐receptor status in SERT−/− platelets. However, these findings suggest a novel mechanism central to the increased bleeding observed in patients taking SSRIs and support the continued investigation of 5HT2A receptor inhibitor as anti‐platelet drugs for treatment of cardiovascular diseases.

Support or Funding Information

14PRE19640007 AHA Pre‐doctoral Fellowship
3‐O Sulfation of Heparin is Required for Hepatic Accumulation
Authors: Harris Edward N , Liu Jian , Miller Colton , Xu Yongmei , Blank Emma
Published: November 2023

Introduction

Heparin is the most common and widely used anti‐coagulant in use for surgery, dialysis, and treatment of thrombosis. Heparin, which is a polysaccharide composed of glucuronic/iduronic acid and N‐acetylglucosamine sugars that are highly sulfated, comes in three forms: unfractionated heparin (UFH) having a polydispersity of 3000–30,000 Da, low‐molecular weight heparin (2000–8000 Da), and the synthetic pentasaccharide, Fondaparinux. Due to size, UFH accumulates in liver, is degraded and then filtered out by the kidneys. In contrast, LMWHs do not accumulate in liver and are eliminated by the kidneys, thus, LMWHs may not be suitable for renal impaired patients. LMWHs are the most commonly prescribed form of the drug since it may be self‐injected by the patient at home for post‐surgical treatment. Since heparin comes from a porcine source, its availability is dependent on a healthy swine herd. The side‐effects of heparin include bleeding and heparin‐induced thrombocytopenia (HIT) which affects 2–4% of patients. The development of synthetic heparins will potentially eliminate dependence on the swine source and the monodisperse preparations will give the physician more control over the side‐effects. In this study, we used a monodisperse dodecamer (12mer) LMWH with and without specific 3‐O sulfation.

Methods

4–6 week old CD‐1 mice were subcutaneously injected with 1 mg/kg of either 35S‐12mer (containing one 3‐O sulfation) or 35S‐n12mer (not containing 3‐O sulfation) and placed in metabolic cages for a duration of up to 26 hrs. Urine was collected at various times during the incubation phase. At each time point, mice were anesthetized and blood, liver, and kidney tissue was collected and evaluated for content of the radiolabeled material.

Results

The n12mer had a shorter half‐life in blood and was excreted faster than the 12mer. The hepatic accumulation of the n12mer was low and brief, suggesting that the polymer briefly bound to the sinusoidal endothelial cells due to static charge rather than being internalized by receptors. In contrast, the 12mer had a much higher and longer duration in the liver and the amount in urine was lower and more prolonged. Degradation of the 12mer was also higher in the urine in the 3–7 hr range post‐injection, whereas, most of the n12mer was still intact.

Conclusion

Synthetic 12mer containing 3‐O sulfation, which represents only a 6% increase in overall charge of the polymer, binds to specific receptors (Stabilin‐2/HARE) in the liver sinusoidal endothelium and is degraded before release in the blood and filtered out by the kidneys.

Support or Funding Information

NIH/NHLBI 5R01HL094463‐07
Reducing Thrombosis Risk With JNK‐2 siRNA Therapeutic Nanoparticles In ApoE−/− Mice
Authors: Pan Hua , Palekar Rohun U , Bacon John , Allen Stacy , Schlesinger Paul H , Wickline Samuel A
Published: November 2023

Introduction

Macrophage foam cell formation is regulated in part by the MAPK family member, c‐Jun N‐terminal kinase, JNK2. Genetically modified ApoE−/− JNK2−/− mice are known to develop less plaque, suggesting that therapeutic strategies that inhibit JNK2 focally at the site of developing plaques could check the progression of atherosclerosis. However, whether JNK2 inhibition could modulate the risk of thrombosis in plaques is unknown. To this end, we report the utility of a new peptide‐based siRNA delivery nanoplatform that rapidly conveys serum‐stable anti‐JNK2 oligonucleotides into growing plaques (but not to or pass through normal vasculature) to inhibit plaque progression while simultaneously reducing plaque thrombotic risk.

Methods and Results

The nanoconstruct designed to condense any siRNA into a 55nm particle comprised a nontoxic amphipathic cationic 21 a.a. peptide that facilitates both endosomal escape and release of the siRNA upon endosomal acidification. ApoE−/− mice at 6 weeks old were fed a Western diet for 14 weeks prior to treatment with either anti‐JNK2 siRNA NP (0.5 mg siRNA/kg; n=16) or HBSS (control; n=8) administered by intravenous injection twice weekly for 7 total doses under continued fat diet. Focal delivery of Cy5.5‐labeled siRNA was confirmed in excised aortas by fluorescence imaging 24 hours after nanoparticles injection. Real Time RT‐PCR and Western blots confirmed JNK2 knockdown in the total aorta in treated ApoE−/− at both mRNA (treated vs untreated aorta: 20% reduction p=0.034) and protein (treated vs untreated aorta: 50% reduction p=0.041) levels. Sudan IV staining of the arch revealed a 38% reduction in plaque surface area for control (47%) vs treatment (29%) groups (p=0.002). Thrombotic risk with or without JNK2 siRNA NP treatment was quantified by carotid artery dye‐laser injury according to occlusion times, indicating that JNK2 siRNA NP treatment increased occlusion time in treated mice by ~2× over that of control treatments, which confirms reduced procoagulant risk. Thrombin staining of the aorta sections demonstrated reduced thrombin deposition in plaque from the treated mice. Safety evaluations confirmed normal routine blood tests, and innate and adaptive immune responses.

Conclusion

Anti‐JNK2 siRNA nanoparticles specifically accumulate and are retained selectively in atherosclerotic plaque, inhibit plaque growth, and remarkably reduce thrombosis risk after only 3.5 weeks of biweekly therapy. In view of the exceptional safety profile, this therapeutic siRNA approach holds promise for rapidly stabilizing vulnerable atherosclerotic plaque.
Implementing Modified Active Learning Strategies in Graduate Pharmacology Education
Authors: Ponnoth Dovenia S.
Published: November 2023

Active learning techniques and strategies are used to improve student learning while in class and can complement lecturing. Modifying select strategies and tailoring them to graduate education could be useful and creative pedagogical tools to encourage student learning and involvement in class. The aim of this study was to incorporate modified active learning approaches in several graduate pharmacology courses over a period of one year and evaluate in‐class student learning, participation and involvement. The different techniques employed included pre‐ and post‐lecture tests to solidify key concepts, team based learning to encourage discussion, increased use of the blackboard and modified concept maps to serve as learning tools. Each class (average class size was 15 graduate students) met once a week for 3 hours of contact time and along with the interactive exercises, traditional didactic lecturing was always part of the class. Other activities included journal clubs and discussion of peer‐reviewed articles. At the end of the year, an anonymous student survey was conducted not only to evaluate student learning and preferences but also use these responses to design future classes. The pre‐ and post‐tests were evaluated separately to assess learning. Each category on the survey could be scored a maximum of 4 points (strong agreement) and a minimum of 0 points (strong disagreement). The survey results showed students were not only enthusiastic about incorporating newer techniques into their learning but also had distinct preferences for particular exercises. Each category on the survey scored an average of 3.5 points, with the lowest score being 3.1 (use of the blackboard). The solving of modified concept maps was by far the most preferred activity (100% positive response, maximum average score 4) to aid learning. Based on the findings of this study, it was concluded that active learning can successfully be incorporated into graduate pharmacology education and using suitable exercises increases both student involvement and learning.

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NA
Integrating Pharmacokinetics, Pharmacodynamics, and Pharmacogenetics into a First‐year Medical School Foundational Module Using Active Learning Methodologies
Authors: Peffley Dennis Michael , Reuben Jayne , Wolff Dennis , Farrell Chris
Published: November 2023

The University Of South Carolina School Of Medicine Greenville has adopted curricular strategies that emphasize vertical and horizontal integration of basic biomedical and clinical sciences with an emphasis on active learning approaches. This poster illustrates our methodologies for integrating pharmacokinetics, pharmacodynamics, and pharmacogenetics into foundational material presented to first‐year medical students. Foundational material is presented in the Molecular Foundations of Medicine Module, the first module in the first year of medical school. Module content includes cell biology, biochemistry, genetics and principles of pharmacology. The majority of content within this module is delivered through various types of active learning methodologies. Pharmacokinetics exercises utilized dynamic web pages incorporating random number generators with automated correction such that students were able to complete problems on drug dosing, absorption and elimination as often as necessary for mastery through self‐directed learning. An Excel spreadsheet graphing a single compartment model in which students could visually assess the consequences of altering drug dose, bioavailability, absorption and/or elimination relative to the basal tablet properties or intravenous administration of the same drug dose was used to complement the web page exercises. A second Excel spreadsheet showed plots of 5 dose‐response curves that students can alter/reposition by changing potency and efficacy parameters. Participation in these self‐directed exercises was tracked on‐line. Pharmacogenetics was introduced immediately after pharmacokinetics and pharmacodynamics using a self‐directed study guide where students completed problems and other questions related to content objectives listed for this topic. The integration of pharmacogenetics with pharmacokinetics and pharmacodynamics provided an effective venue for introducing first year medical students to the concepts of drug absorption, distribution and excretion as well as phenotypic variations in drug response associated with genotypic changes in phase I and phase II enzymes. Reading assignments as well as PowerPoint presentations were posted on the Canvas Web site to assist students in the completion of self‐directed activities. Assessment of this material included an additional graded in class problem assignment as well as relevant questions on the week's formative and final summative exams. As shown through the degree of student participation in classroom exercises, the use of self‐directed learning modalities appears to be a far more effective approach compared to didactic lectures to enhance student engagement when introducing this complex foundational material to M1 students.