bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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 InformationLoyola 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 InformationSupported 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

IntroductionIschemia 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.HypothesisCLC‐3 up‐regulation may shorten action potential during, Q‐T interval, and effective refractory period (ERP)under ischemic conditions.MethodsIn 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.ResultsQ‐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.ConclusionsUp‐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 InformationThis 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 InformationFundação 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 InformationSupported 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 InformationMargaret 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 Information14PRE19640007 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

IntroductionHeparin 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.Methods4–6 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.ResultsThe 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.ConclusionSynthetic 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 InformationNIH/NHLBI 5R01HL094463‐07