bluuebunny/crossref_metadata_2025 huggingface dataset:
MiRNA Expression Patterns during Skeletal Muscle Regeneration
Authors: Chen Yongxin , Gelfond Jonathan , McManus Linda M. , Shireman Paula K.
Published: June 2021

MiRNAs regulate many biological processes. Expression of miRNAs during mouse muscle (tibialis anterior) regeneration was analyzed by qPCR‐array at baseline and days 1, 3, 4, 7, 21 post‐cardiotoxin injury. Based on histology, satellite cell proliferation occurred at days 1–4 after injury and myoblast differentiation began at day 3, with overlap of both events at days 3–4. Over 100 miRNAs changed >2‐fold (p<0.01) after injury compared to baseline.

miRNA Fold Change

Day
1
3
4
7
21

miR‐1
−104
−94
−28
−4.6
−1.6

miR‐133a
−32
−127
−34
−5.2
−1.7

miR‐682
7.4
5.2
1.5
2.8
1.8

Expression of muscle‐specific miR‐1 and −133a significantly decreased immediately after injury and returned to baseline, a pattern consistent with a role in maintaining muscle differentiation. Conversely, another subset of miRNAs, including miR‐682, significantly increased at early days after injury, suggesting regulatory roles in satellite cell proliferation. In addition, some miRNAs were up‐regulated in both early and late days after injury. Interestingly, the expression time course of the above 3 miRNAs was remarkably similar during in vivo muscle regeneration and in vitro myogenic progenitor cell proliferation and differentiation. These studies define miRNA expression patterns that may regulate muscle repair; manipulation of specific miRNAs may improve muscle regeneration. Supported by the Veterans Administration HL07446 and HL074236.
YKL‐40 glial expression may impact neuronal trophic support in neurodegeneration and neurological conditions
Authors: Bonneh‐Barkay Dafna , Figley Matthew , Zou Huichao , Starkey Adam , Wang Wanda , Bissel Stephanie , Wagner Amy K , Wiley Clayton A
Published: June 2021

YKL‐40 is up‐regulated in various inflammatory conditions and cancers. We have previously reported that YKL‐40 levels were elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis. Immunohistochemical analysis of YKL‐40 in lentiviral encephalitis showed that YKL‐40 was associated with astrocytes. In the current study YKL‐40 transcription and expression were evaluated in a variety of human neurodegenerative diseases (e.g. Alzheimer's disease (AD), Multiple sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS)) as well as other neurological conditions including brain infarction and traumatic brain injury (TBI). CSF analysis of patients with these conditions revealed mild YKL40 elevation with aging, MS, AD, ALS, stroke and brain trauma. In situ hybridization showed that YKL‐40 transcription is mostly associated with reactive astrocytes in regions of neuroinflammation. Time‐course analysis of YKL‐40 expression in an acute model of TBI in rats showed that YKL40 expression in astrocytes was induced one day after injury, remains elevated for several days and then declines by day 12. These findings demonstrate that YKL‐40 is induced in astrocytes during acute and chronic neuroinflammation. Given the potential of YKL40 to block trophic factors, the biological role of this dynamic production in early glial scar may impact regenerative response to neuroinflammation.
ACE 2: A potential therapeutic target for Angiotensin II‐mediated insulin resistance and glucose intolerance
Authors: Chhabra Kavaljit H. , Xia Huijing , Lazartigues Eric
Published: June 2021

Over activation of the renin‐angiotensin system (RAS) plays a key role in diabetes. ACE2 has been reported to counteract the pathophysiological effects of RAS over activity. To clarify the role of ACE2 in glucose homeostasis, ACE2 KO and control mice (5 months old, n=5/group) were infused with AngII (osmotic pump: 600 ng/kg/min); insulin levels (ng/ml) and glucose tolerance (AUC: mg/dl*120) were assessed after 14 days infusion. KO mice manifested significantly (P<0.05) higher hyperinsulinemia (6.2 ±0.49 vs. 4.5 ±0.23) and glucose intolerance (55300 ±1387 vs. 43550 ±1735) compared to controls, suggesting their hypersensitivity to diabetes. To assess the therapeutic potential of ACE2, βTC‐ 3 cells (mouse insulinoma) were subjected to glucose stimulated insulin secretion assay (2.8, 16, 25 mM glucose) in which AngII significantly (P<0.05) decreased insulin secretion vs. control group (1.01 ±0.01 vs. 1.84 ±0.12; 1.57 ±0.15 vs. 2.78 ±0.05; 1.7 ±0.08 vs. 2.64 ±0.05 ng/ml, respectively). In cells pretreated with an adenovirus over expressing ACE2, insulin secretion was restored (1.75 ±0.14; 2.81 ±0.22; 3.25 ±0.22 ng/ml, respectively), suggesting ACE2 can prevent the Ang‐II mediated inhibitory effect. In conclusion ACE2 is important in the regulation of glucose homeostasis and could be a potential therapeutic target to ameliorate insulin resistance and glucose intolerance resulting from RAS over activation.

LSUHSC research enhancement fund.
Endothelium‐dependent contractions induced by aging and diet‐induced obesity are attenuated in lipocalin‐2 deficient mice
Authors: Liu Jacky TC , Xu Aimin , Man Ricky YK , Mak Tak W , Law Ivy KM , Liang CF , Vanhoutte Paul M , Wang Yu
Published: June 2021

Aim

Levels of Lipocalin‐2, a pro‐inflammatory adipokine, correlates with adiposity, dyslipidemia, hyperglycemia and insulin resistance. The present study evaluates vascular function in mice with deletion of the lipocalin‐2 gene (Lcn2‐KO).

Methods

Wild type (WT) and Lcn2‐KO mice were fed with standard or high fat diet for 3–17 weeks. Intra‐arterial blood pressure was measured. Isometric tension was measured in carotid artery rings with or without endothelium. Protein expression was measured by Western blotting.

Results

The obesity‐induced increase in systolic blood pressure was attenuated in Lcn2‐KO mice. In aged or obese WT mice, acetylcholine (ACh)‐elicited endothelium‐dependent contractions were abolished by the COX‐1 selective inhibitor SC560 and the NADH oxidase inhibitor diphenylene iodonium. These contractions were attenuated in Lcn2‐KO mice. ACh‐stimulated superoxide anion production, COX‐1 mRNA and protein expression were decreased in Lcn2‐KO arteries. ACh‐induced production of prostacyclins (PGI2) was lower in Lcn2‐KO preparations. In aged or obese WT mice, PGI2 caused contractions in rings without endothelium but these contractions were reversed to relaxations in Lcn2‐KO arteries.

Conclusions

Lipocalin‐2 plays an important role in obesity‐induced hypertension and endothelial dysfunction through production of superoxide anions and COX‐1 expression.
Transgenic expression of CYP7A1 in the liver prevents high fat diet‐induced obesity and insulin resistance in mice
Authors: Li Tiangang , Owsley Erika , Matozel Michelle , Hsu Peter , Chiang John Y.L.
Published: June 2021

Bile acid‐activated FXR plays a key role in lipid, glucose, drug and energy metabolism. Cholesterol 7á‐hydroxylase (CYP7A1) catalyzes the rate‐limiting step in the conversion of cholesterol into bile acids in the liver. The mechanism of bile acid‐mediated regulation of glucose and lipid metabolism remains unclear. The objective of this study is to investigate the role of CYP7A1 in the prevention of fatty liver, obesity and diabetes. Transgenic mice carrying an ApoE3‐CYP7A1 coding sequence (Cyp7a1‐tg) were used to study high fat diet (HFD) induced obesity, fatty liver, and insulin resistance. Cyp7a1‐tg mice had a ~ 3‐fold higher bile acid pool size than wild type mice. When fed a HFD, Cyp7a1‐tg mice were resistant to weight gain, fatty liver and insulin resistance, and had decreased fat mass and hepatic triglycerides and cholesterol content than wild type mice. Cyp7a1‐tg mice showed increased hepatic VLDL secretion but maintained plasma triglyceride and cholesterol homeostasis. Indirect calorimetry revealed that HFD fed Cyp7a1‐tg mice had increased energy expenditure and higher respiratory quotient than wild type mice. This study suggests that maintaining bile acid homeostasis is important in regulating whole body lipid, glucose and energy homeostasis. Bile acids and derivative may be potential therapeutic agents for treating metabolic disorders such as fatty liver diseases, obesity and diabetes in humans.
Inhibition of NADPH oxidase restores vasoreparative function in diabetic CD34+ cells
Authors: Jarajapu Yagna PR , Caballero Sergio , Li Qiuhong , Grant Maria
Published: June 2021

We have previously showed that inhibition of NADPH oxidase corrected diabetic migratory defect in circulating putative endothelial progenitor cells (CD34+) by decreasing the production of reactive oxygen species (ROS) thereby increasing intracellular nitric oxide (NO). In this study, we evaluated the effect of NADPH oxidase blockade on the vascular engraftment potential of diabetic CD34+ cells. Peripheral blood CD34+ cells were isolated from healthy and Type 1‐diabetic individuals by immunomagnetic selection. Endothelial nitric oxide synthase (eNOS) activity, intracellular NO and cGMP production were quantified. Vascular engraftment potential of CD34+ cells was evaluated in a mouse model of retinal ischemia/reperfusion injury. Activation of eNOS in response to stromal derived factor‐1 (SDF1, 100 nM) was lower in diabetic compared to non‐diabetic cells. Treatment with apocynin or gp91ds‐tat has no effect on eNOS activity but increased intracellular NO levels and cGMP production in response to SDF1. In mouse retinal I/R injury model, vascular incorporation of diabetic CD34+ cells followed by intravitreal injection was significantly lower compared to that with non‐diabetic cells and was significantly increased after ex vivo treatment with apocynin or gp91ds‐tat. These results suggest that ex vivo NADPH oxidase inhibition increases the vascular engraftment potential of diabetic CD34+ cells.
Inhibition of Vasoconstriction by Amlodipine in Human Internal Mammary Arteries Used as Bypass Grafts
Authors: HE Guo‐Wei , BAI Xiao‐Yan , LIU Xiao‐Cheng , JING Wen‐Bin , FAN Li , YANG Qin
Published: June 2021

Graft spasm remains challenging in coronary artery bypass grafting (CABG). We studied the inhibitory effect of a dihydropyridine calcium antagonist amlodipine in 70 human internal mammary artery (IMA) segments from 34 patients undergoing CABG in organ baths in two ways: the relaxing effect of amlodipine on vasoconstrictor‐induced precontraction by potassium chloride (KCl), human urotensin II (hU‐II), and U46619 and the depressing effect of amlodipine on the contraction. Amlodipine caused full relaxation in KCl (98.0±2.1%)‐ and hU‐II (98.5±2.4%)‐ but not in U46619‐contracted IMA rings (n=8) with 15.5‐fold higher potency to KCl than to hU‐II (EC50: −8.17±0.28 vs. −6.98±0.01 logM, p<0.001). Pretreatment of IMA with plasma‐concentrations of amlodipine (−7.6 and −6.6 logM, n=6) significantly depressed subsequent contraction to KCl (from 20.8±2.5 to 16.6±2.6 mN and from 20.8±2.5 to 7.6±3.0 mN, p<0.05) and hU‐II (from 14.1±4.2 to 11.4±3.7 mN and from 14.1±4.2 to 3.8±2.0 mN, p<0.05) but not to U46619. Thus, in human IMA, amlodipine has a selective inhibitory effect on the vasoconstriction mediated by vasoconstrictors with high potency on KCl and hU‐II and amlodipine may be effectively used in CABG patients for treatment and prevention of graft spasm.

Supported by Tianjin Municipal Sci & Tech Com (09ZCZDSF04200); Ministry of Sci & Tech, China (2009DFB30560); Providence SVMF, OR & Hong Kong GRF 465107, 478909.
The anti‐atherogenic potential of C‐type natriuretic peptide: Disparate regulation of endothelial and vascular smooth muscle cell proliferation via natriuretic peptide receptor‐C
Authors: Khambata Rayomand S , Panayiotou Catherine M , Xiao Fang , Hobbs Adrian J
Published: June 2021

C‐type natriuretic peptide (CNP) is an endothelium‐derived mediator that exerts an anti‐atherosclerotic influence on the blood vessel wall. Herein, we examined whether the cytoprotective activity of CNP might be mediated, in part, via opposing effects on endothelial and vascular smooth muscle cell proliferation.

CNP increased human umbilical vein endothelial cell (HUVEC) proliferation but inhibited rat aortic smooth muscle cell (RAoSMC) growth; both effects were blocked by the selective natriuretic peptide receptor (NPR)‐C antagonist M372049 and the extracellular signal regulated kinase (ERK) 1/2 inhibitor PD98059. The effects of CNP on cell proliferation were also lost in endothelial and vascular smooth muscle cells from NPR‐C knockout mice. In both HUVEC and RAoSMC, CNP caused ERK 1/2 phosphorylation in a M372049‐inhibitable manner. In HUVEC, CNP‐dependent ERK 1/2 activation resulted in enhanced expression of the cell cycle promoter, cyclin D1, and concomitantly decreased expression of the cell cycle inhibitor, p21. In contrast, in RAoSMC ERK 1/2 activation by CNP was accompanied by increased expression of the cell cycle inhibitors p21 and p27.

These data show that CNP can up‐regulate endothelial cell proliferation yet inhibit vascular smooth muscle cell growth via the same receptor, NPR‐C. Such findings highlight further the anti‐atherogenic ability of CNP.

Funded by the Wellcome Trust
The Chemical Chaperone Tauroursodeoxycholic Acid Alleviates Cardiac Contractile Dysfunction in Obesity: Role of Endoplasmic Reticulum‐Stress in Cardiac Functionality
Authors: Isik Asli Fahriye Ceylan , Ren Jun , Sreejayan Nair
Published: June 2021

Endoplasmic reticulum (ER)‐stress has been implicated in the pathophysiology diabetes and obesity. The aim of this study was to investigate whether ER‐stress is involved in the obesity‐induced cardiac dysfunction (CD) and to assess the effect of an ER‐chaperone tauroursodeoxycholic acid (TUDCA). Seven‐week old obese (ob/ob) and their lean counterparts were randomly received TUDCA (50mg/kg, orally, 5 weeks) or vehicle. Oral glucose tolerance test (OGTT), echocardiographic analysis and cardiomyocyte function were assessed. Sarco(endo)plasmic reticulum calcium (SERCA) activity measured by 45Ca2+ uptake. Expressions of markers of ER‐stress and insulin signaling pathway were determined. TUDCA‐treated ob/ob mice displayed lower blood glucose levels following OGTT vs. vehicle treatment. Ob/ob mice exhibited increased diastolic wall thickness, diastolic and systolic diameters and decreased fractional shortening vs. lean mice, all of which were attenuated by TUDCA. TUDCA treatment alleviated the compromised cardiomyocyte mechanical functions and SERCA activity observed in ob/ob mice. Markers of ER‐stress and insulin signaling pathway were altered in the hearts of ob/ob mice vs. lean controls which were attenuated by TUDCA. Our data suggest that ER‐stress plays a pivotal role in CD and attenuating ER‐stress may represent a potential therapeutic strategy in obesity‐induced CD.
Drug Rediscovery: Glybenclamide Exhibits Thromboxane Receptor Dependent Antithrombotic Activity
Authors: Ting Harold J , Khasawneh Fadi
Published: June 2021

Occlusive disorders such as myocardial infarction and ischemic stroke require platelet activation, a central axis being the synthesis and binding of thromboxane A2 (TXA2) to its receptor (abbreviated as TPR). There are currently no TPR antagonists available for clinical use and there are profound obstacles in translating novel formulations into a therapeutic. One solution is to investigate new uses for currently prescribed drugs, an approach termed “drug rediscovery”. To that aim, it was noticed that the sulfonylurea, glybenclamide, appears to share several pharmacophores with the TPR antagonist SQ29,548. Thus, we predicted it would interact with TPRs. It was found that glybenclamide: 1) inhibited human platelet aggregation induced by the TPR agonist U46619 (1 uM) and the TXA2 precursor arachidonic acid (0.5 mM); 2) concentration‐dependently (1–10 uM) displaced SQ29,548; 3) failed to inhibit aggregation stimulated by 15 uM ADP, or the thrombin receptor activating‐peptide 4 (40 uM); 4) failed to raise cAMP levels; 5) selectively (10mg/kg) blocked mouse TPR‐mediated aggregation ex vivo; 6) prolonged the tail bleeding time in mice; and 7) prolonged the time for occlusion in a mouse carotid artery thrombosis model. These findings indicate that glybenclamide can exert an inhibitory effect on platelet function by specifically interacting with TPR and thus has potential in the management of thrombotic disorders.
Celecoxib administration interferes with the anti‐platelet effect of low dose aspirin in canines
Authors: Lauver D Adam , Frieler Ryan A , Smith William L , Lucchesi Benedict R
Published: June 2021

The purpose of this study was to determine whether simultaneous treatment with celecoxib (CEL) interferes with the anti‐platelet effects of aspirin (ASA). Patients at risk for adverse cardiovascular events often are prescribed a low dose (81 mg) of ASA to selectively inhibit the cyclooxygenase (COX)‐1 dependent synthesis of thromboxane A2 in platelets. Many of these patients are simultaneously administered the COX‐2 specific inhibitor, CEL, for pain management. Recent in vitro reports demonstrated the ability of CEL to bind to COX‐1 thereby slowing the rate of acetylation of the critical Ser530 by ASA without otherwise affecting the ability of the enzyme to metabolize arachidonic acid (AA). Therefore, it is important to understand whether CEL might adversely affect the beneficial cardiovascular effects of ASA in vivo. To test this question, dogs were administered ASA (1.16 or 4.64 mg/kg), CEL (1.43 mg/kg, bid) or the combination of both ASA and CEL for a period of at least three days. At the conclusion of each regimen, blood was collected and ex vivo platelet aggregation responses were recorded. As expected, AA‐induced platelet aggregation was inhibited by both doses of ASA. CEL administration alone had no effect on platelet aggregation. Simultaneous administration of CEL with low dose, but not high dose ASA interfered with the inhibitory effect of ASA on platelet aggregation. The results highlight the complex function of these enzymes in the hemostatic process.
12‐Lipoxygenase plays a significant role in regulation of human platelets activation
Authors: Apopa Patrick Leo , Vesci Joanne , Boutaud Olivier , Jadhav Ajit , Simeonov Anton , Rai Ganesha , Maloney David J , Holman Ted , Kenyon Victor , Holinstat Michael
Published: June 2021

Platelets play a critical role in both hemostasis and thrombosis. Arachidonic acid (AA), a precursor to eicosanoid formation is one of the polyunsaturated fatty acids found on the platelet cell membrane. Lipoxygenase enzymes metabolize AA to different active metabolites. This study was undertaken to elucidate the effects of Lipoxygenase inhibition on human platelets function. Human platelets were stimulated with different agonists in the presence and absence of a Lipoxygenase‐specific inhibitor. Following stimulation, platelets aggregation and dense granule release was monitored using a lumni‐aggregometer. We show that inhibition of 12‐LOX completely blocks granule release without affecting platelets aggregation. Further analysis showed that 12‐LOX inhibition completely blocked 12‐HETE formation while having only minor effects on TxA2 levels upon stimulation with different agonists. Flow cytometry assay indicated that the 12‐LOX inhibitor significantly inhibited both GPIIbIIIa activation and α‐granule secretion and biochemical analysis show effects of 12‐LOX inhibition on Rap1 activation. Taken together, these results suggest that 12‐LOX inhibition has the potential of selectively inhibiting 12‐HETE formation and activity without affecting TxA2 activity and TxA2 receptor and has the potential to act as an important therapeutic target in the management of thrombosis.
Thalidomide produces vascular relaxations
Authors: Docherty James R , Seto Sai Wang
Published: June 2021

We have found that thalidomide produces hypotension in conscious and anaesthetised rats, and have investigated the mode of action. In telemetry experiments, thalidomide (50 mg/kg, s.c.) produced significant falls in both systolic and diastolic pressures in conscious male Wistar rats. In anaesthetised rats, thalidomide (1 mg/kg, i.v.) produced significant falls in blood pressure. In rat tail artery, thalidomide (10–100 uM) produced significant relaxations of phenylephrine contracted tissues. In calcium re‐addition studies, in which calcium is restored in the presence of KCl or phenylephrine, thalidomide (100 uM) inhibited contractions to calcium restoration only in the presence of phenylephrine. Relaxant actions of thalidomide in vitro do not involve alpha1‐adrenoceptors, caffeine sensitive calcium stores, glibenclamide sensitive potassium channels, PKC or P38 MAP kinase. Thalidomide also inhibited nerve‐evoked contractions of epididymal, but not prostatic, portions of rat vas deferens, an action shared with the T‐type calcium channel blocker NNC 55‐0396. Like thalidomide, NNC 55‐0396 inhibited contractions to calcium restoration only in the presence of phenylephrine. It is concluded that thalidomide produces vascular relaxations by a mechanism that may be similar to that of NNC 55‐0396.
Cardiac‐specific overexpression of catalase prolongs survival and attenuates paraquat‐induced myocardial contractile dysfunction
Authors: GE WEI , MA HENG , ZHANG YINGMEI , HAN XUEFENG , REN JUN
Published: June 2021

Paraquat, a quarternary nitrogen herbicide, is highly toxic for humans and animals via generation of reactive oxygen species. This study was designed to examine the influence of cardiac‐specific overexpression of catalase (CAT), an antioxidant enzyme responsible for detoxification of H2O2 on murine myocardial contractile function following the challenge of the pro‐oxidant paraquat. FVB and CAT mice were administrated paraquat (75 mg/kg) and myocardial function was monitored with echocardiography and edge‐detection. Cardiomyocyte mechanical indices analyzed included peak shortening (PS), time‐to‐PS (TPS), time‐to‐90% relengthening (TR90), and maximal velocity of shortening/relengthening (+/‐dL/dt). Our results revealed that CAT mice displayed a significantly improved survival against paraquat‐induced death than FVB mice as evaluated by the Kaplan‐Meier curve. The CAT transgene itself did not alter cardiac function. Paraquat treatment for 48 hours enlarged end‐systolic diameter, reduced fractional shortening, PS and +/− dL/dt in FVB group. Interestingly, all these defects were nullified or significantly attenuated by CAT. Taken together, our data revealed that CAT overexpression may rescue paraquat‐induced decrease in survival, myocardial contractile dysfunction.
PSD 95 scaffolds associated with vascular KV1 channels are post‐transcriptionally down‐regulated in cerebral arteries during hypertension
Authors: Joseph Biny K , Rusch Nancy J , Rhee Sung W
Published: June 2021

Recently we reported that a transcriptional down‐regulation of pore‐forming α‐subunits of the Shaker‐type voltage‐gated K+ (KV1) channels may contribute to a vasodilator defect in cerebral arteries of hypertensive rats. The presence of a class 1 PDZ binding motif in the C‐terminus of the α‐subunits (α1.2 and α1.5) of KV1 channels raises the possibility that these subunits bind to PDZ domains of the molecular scaffold PSD 95 (Post Synaptic Density 95). The current study examined the possibility that a loss of KV1 channels may directly mediate the down‐regulation of PSD 95 scaffolds in rat cerebral arteries. First, reciprocal co‐immunoprecipitation studies revealed that α1.2 associates with PSD 95. Second, Western blotting and real‐time PCR analysis indicated a post‐transcriptional down‐regulation of PSD 95 in cerebral arteries of genetically hypertensive rats and aortic‐banded rats compared to normotensive controls. Third, α1.2 and/or α1.5 siRNAs reduced the expression levels of the pore‐forming α1.2 and α1.5 subunits and concomitantly reduced PSD 95 in rat cerebral arteries in short‐term culture. Taken together, our results suggest a previously uncharacterized function for vascular KV1 channels. We propose that they directly regulate the expression of the PSD 95 scaffolding protein in rat cerebral arteries.
Alteration in contractile mechanism of rat cavernosal smooth muscle induced by different class‐diuretics
Authors: Claudino Mário Angelo , Takeshi Fábio Ikeda , Priviero Fernanda Bruski Marinho , Antunes Edson , Lopes Anibal , De Nucci Gilberto
Published: June 2021

We assessed the involvement of different diuretics in the contractile mechanisms of rat corpus cavernosum smooth muscle (CC). In CC from male Wistar rats, concentration‐responses curves to phenylephrine (PE) and potassium chloride (KCl) and neurogenic contractile responses induced by electrical‐field stimulation (EFS) were obtained in absence (control; CTL) or in presence of furosemide (FUR; loop diuretic), hydrochlorothiazide (HTZ; thiazide) and amiloride (AMI; potassium‐sparing diuretic). PE contracted CC with a potency (pEC50) of 5.51 ± 0.02. FUR and HTZ did not change the pEC50 of PE (5.54 ± 0.06 and 5.58 ± 0.07, respectively), but significantly increased the maximal responses (EMAX; 2.97 ± 0.22 and 3.65 ± 0.39 mN, respectively; vs CTL: 2.41 ± 0.10 mN). AMI reduced the pEC50 of PE (5.37 ± 0.08) with no changes in EMAX (2.48 ± 0.29 mN). KCl contracted CC with a pEC50 of 1.35 ± 0.10. FUR and HTZ did not alter the pEC50 of KCl (1.37 ± 0.03 and 1.31 ± 0.08, respectively) but increased EMAX (1.24 ± 0.16 and 1.41 ± 0.06 mN, respectively, vs CTL: 0.65 ± 0.05 mN). AMI decreased pEC50 of KCl (1.06 ± 0.04) with no changes in EMAX (0.84 ± 0.16 mN). EFS induced frequency‐dependent contractions in CC which were enhanced by FUR and HTZ. AMI reduced EFS‐induced contractions at the highest frequencies (16–32Hz). In conclusion, different diuretics distinctively alter the CC contractions possibly by changing [Ca2+]i and [Cl−]i content. FAPESP
Hemeoxygenase‐carbon monoxide signaling in the rostral ventrolateral medulla is implicated in ethanol‐evoked sympathoexitation and pressor response in the SHR
Authors: Nassar Noha N , Li Guichu , Abdel‐Rahman Abdel A
Published: June 2021

Reported studies including ours have identified the rostral ventrolateral medulla (RVLM) as a major brainstem area for ethanol‐evoked sympathoexcitation, which elicits greater pressor response in spontaneously hypertensive rats (SHRs) via activation of NE‐containing neurons in the RVLM. Recent evidence supports a critical role for carbon monoxide (CO) in brainstem control of blood pressure, sympathetic activity RVLM neuronal activity. HO is the rate‐limiting enzyme responsible for the catabolism of heme into biliverdin, CO, and iron. Hence, we hypothesized a role for brainstem HO‐CO signaling in the central cardiovascular actions of ethanol. To this end, 30 male SHR and 30 male WKY rats were divided into three groups (n=10, each group for each strain) receiving (i) aCSF as control; (ii) ethanol (10ug) following either a) hemin (1nmol) or b) aACSF. Microinjection of ethanol elicited greater and longer lasting increase in mean arterial pressure (MAP) and NE (measured by in‐vivo electrochemistry) in SHRs compared than (WKY). Hemin microinjection elicited significantly greater and longer‐lasting reductions in BP and RVLM NE in SHRs. Moreover, HO activity was significantly increased and reduced in SHRs following hemin and ethanol treatment respectively. On the other hand animals receiving ethanol following hemin pretreatment exhibited attenuated HO activity compared to hemin treated rats but higher activity compared to ethanol treated animals. These findings implicate RVLM HO‐CO signaling in ethanol induced increases in RVLM presympathetic neuronal activity and blood pressure in the SHR.
Enhanced Hemeoxygenase‐1 Signaling in the Rostral Ventrolateral Medulla Mediates a Critical Protective Role Against Genetically Elevated Blood Pressure in SHR Rats
Authors: Nassar Noha N , Li Guichu , Abdel‐Rahman Abdel A
Published: June 2021

Heme oxygenase (HO)‐derived carbon monoxide (CO) contributes to central regulation of blood pressure (BP). However, the few reported studies demonstrating reductions in BP following microinjection into the nucleus tractus solitarius of the HO substrate hemin, were carried out in anesthetized normotensive rats. We hypothesized that HO‐CO signaling is enhanced in the RVLM of conscious SHRs to prevent exacerbation of hypertension in this genetic model of hypertension. To test this hypothesis, we investigated the effects of intra‐RVLM hemin on BP, HR and RVLM neuronal norepinephrine (index of sympathetic activity; measured in real‐time by in vivo electrochemistry) in SHRs and their normotensive control WKY rats. Furthermore, the type of the specific HO isoenzyme implicated in the hypotensive response was investigated by administering the selective HO‐1 inhibitor zinc protoporphyrin IX (ZNPPIX) 30 minutes prior to hemin. Intra‐RVLM hemin elicited greater and longer‐lasting reductions in BP and RVLM NE in SHRs. These hemodynamic and electrochemical response, which suggest enhanced HO‐CO signaling in the RVLM of SHRs, were confirmed by the greater enzymatic activity of HO in SHRs than in WKY rats, Moreover, ZNPPIX, not only abolished the hemin‐evoked responses in either strain, albeit to a greater extent in SHRs, but also caused a marked increase in baseline pressure in SHRs. The findings suggest an exaggerated HO‐CO signaling, due to higher HO‐1 activity, in the RVLM of SHR, a brainstem area that controls sympathetic outflow. Such enhancement of HO‐CO signaling seems to serve as a protective mechanism that opposes further increases in BP in the SHR.
Effect of streptozotocin on neurogenic M2 and M3 muscarinic receptor‐mediated contractions in mouse urinary bladder
Authors: Pak Kirk J , Ehlert Frederick J
Published: June 2021

We studied the effect of streptozotocin (STZ) (125 mg/kg, 20 – 24 weeks prior to experiments) on the contractile roles of M2 and M3 muscarinic receptors in the electrical‐field‐stimulated mouse urinary bladder. Electrical‐field stimulation for 5 sec (20 Hz, 0.5 msec, 100 V) elicited contractions that were inhibited by the muscarinic antagonist, N‐methyl scopolamine approximately 50% in urinary bladder from both STZ‐ and vehicle‐treated, wild type mice. In bladder from M2 KO mice, STZ‐treatment caused a large inhibition of contraction compared to that measured in vehicle‐treated mice. The relaxant effect of isoproterenol on the electrical‐field‐stimulated contraction was similar in wild type urinary bladder from vehicle‐ and STZ‐treated mice. In contrast, the relaxant effect of isoproterenol was much greater in urinary bladder from M2 KO mice treated with STZ as compared to that from vehicle‐treated mice. Our data show that the contractile role of the M2 receptor in response to acetylcholine release is increased in urinary bladder following STZ‐treatment and that this mechanism may be critical for sustaining contractile function following the substantial loss of M3 receptor function induced by STZ. Supported by the MSTP grant, the ARCS Foundation, and NIH grants GM069829, UTN‐37775 and DK081289.
Famous & Outstanding Anatomy Professors I Have Known or with Whom I Have Taught
Authors: Clemente Carmine
Published: June 2021

The first two outstanding anatomists I wish to discuss are William Frederic Windle and Horace Winchell Magoun, both students of Stephen Walter Ranson in Chicago.

I became acquainted with Bill Windle 60 years ago in 1949 while he was Chairman of Anatomy at the University of Pennsylvania. He interviewed me before my being accepted into graduate school at Penn and his principal question was, “Are you a good student?” I told him that I received A's and B's during my four years at Penn. He replied that is not good enough. He required all A's during the first two graduate school years. His next question was, “Do you have a microscope?” My brother had graduated from the Penn Medical School and I said I'm sure I could get his microscope. Then Dr. Windle said, “Come around in September. We will find a place for you.”

I will talk also about my interaction with Professor Horace Magoun who offered me my first job at UCLA. He became my close friend, often advising me through the early years of my academic life.

Giving my first paper to the AAA in Detroit, Michigan in 1951 after my second graduate school year, I was nervous and scared. When I started my talk. I noticed that in the audience was the famous Horace Magoun, the discoverer of the Reticular Activating System in the brain stem, responsible for maintaining wakefulness behavior. Later that day I was stunned when he asked me if, when I finished at Penn, I would consider joining him at Los Angeles where UCLA had just opened its medical school.

Over the years I also met or taught with such famous professors as John Boilieu Grant, Raymond Jack Last and Oscar Vivian Batson.
The central antidepressant‐like and antinociceptive effects of a delta opioid receptor agonist SNC80 in rats
Authors: Chen Chang‐Ming , Cheng Chia‐Chen , Su Pin‐Yen , Ko Mei‐Chuan
Published: June 2021

Animal studies have indicated that systemic administration of delta opioid receptor (DOR) agonists or tricyclic antidepressants (TCA) produced both antidepressant‐like and antinociceptive effects. The aim of the study was to investigate the role of DORs in the supraspinal regions by comparing behavioral effects of DOR agonist SNC80 and TCA amitriptyline in rats. The forced swim test and formalin‐induced flinches were used to quantify the antidepressant‐like and antinociceptive effects of test compounds, respectively. Following intracerebroventricular (ICV) administration, both SNC80 and amitriptyline dose‐dependently decreased immobility in the rat forced swim test, indicating of antidepressant‐like effects. ED50 values of ICV SNC80 and amitriptyline for decreasing immobility were 5.1 and 38.1 μg, respectively. In addition, ICV SNC80 dose‐dependently produced antinociception, but ICV amitriptyline tested up to 100 μg failed to attenuate formalin‐induced nociceptive responses. More importantly, pretreatment with a DOR antagonist naltrindole (1 mg/kg, SC) only blocked antidepressant‐like effects produced by ICV SNC80, but not those by ICV amitriptyline. These results suggest that central DOR is involved in the regulation of both mood and pain, and that DOR agonists may have the therapeutic potential as antidepressants or/and analgesics (Supported by NSC grant NSC‐97‐2628‐H‐004‐089‐MY2).
Effects of intradermal administration of endogenous opioid peptides, β‐endorphin and dynorphin‐A, on scratching behavior in mice
Authors: Hu Eric , Cremeans Colette M , Husbands Stephen M , Ko Mei‐Chuan
Published: June 2021

Several studies have indicated that mu (MOR) and kappa opioid receptors (KOR) have opposite actions in itch scratching responses. The aim of the study was to investigate the functions of MOR‐ and KOR‐preferring endogenous ligands (β‐endorphin vs. dynorphin‐A) in the peripheral nervous system in modulating itch sensation in mice. Two known pruritogens (histamine and serotonin), β‐endorphin, and dynorphin‐A were administered intradermally in the rostral part of the mouse back. The scratching bouts by hind paw motions directed toward the injection site were scored by experimenters who were blinded to dosing conditions. All four ligands dose‐dependently elicited scratching responses following intradermal (ID) administration within a dose range of 0.1 and 100 nmol/site. The analgesic morphine 1 mg/kg attenuated part of ID β‐endorphin‐ and dynorphin‐A‐induced scratching, but it did not affect ID histamine‐ and serotonin‐induced scratching. In contrast, the antipruritic nalfurafine 0.01 mg/kg attenuated part of ID histamine‐induced scratching, but it was not effective in attenuating other three ligand‐elicited scratching. These results suggest that ID histamine elicited itch sensation manifested as scratching responses in mice. However, ID β‐endorphin‐ and dynorphin‐A‐induced scratching may not entirely be related to itch sensation (Supported by grant DA‐013685 and grant NSC‐96‐2413‐H‐004‐019).
Pro‐depressant‐like effects of CB1 receptor inverse agonists/antagonists in male Sprague‐Dawley rats
Authors: Jutkiewicz Emily M , Makriyannis Alexandros , Vemuri Kiran , Bergman Jack
Published: June 2021

Cannabinoid CB1 antagonists such as the inverse agonist rimonabant (SR141716A) or the neutral ligand AM 4113 may have therapeutic utility in the management of obesity and other disorders. However, adverse effects including depression may hinder their application, and preclinical means for predicting such effects have not been previously developed. In the present study, the effects of SR141716A and AM4113 were examined in a modified rat forced swim test that was optimized to measure increases in immobility (pro‐depressant‐like effects) in rats. In initial tests, reserpine and amphetamine withdrawal increased immobility, confirming clinical observations of pro‐depressant‐like effects. In subsequent testing, 10 mg/kg i.p. of SR141716A but not AM 4113, significantly increased immobility and decreased swimming in the forced swim test. To our knowledge, this is the first preclinical demonstration of pro‐depressant‐like effects with CB1 antagonists. These data also provide a preliminary suggestion that differences in pro‐depressant‐like effects of CB1 antagonism may be related to efficacy. Supported by USPHS grant DA19205.
Cognitive deficits associated with chronic cocaine self‐administration in monkeys
Authors: Gould Robert W , Nader Michael A
Published: June 2021

In humans, cocaine dependence is associated with neurobiological, behavioral, and cognitive deficits when compared to non‐drug abusers. In monkeys, cocaine self‐administration (SA) induces similar neurobiological changes, yet cocaine‐induced cognitive deficits have not been extensively studied. We compared cognitive performance using CANTAB touch‐sensitive computer apparatus to assess measures of learning, memory and behavioral flexibility in age‐matched experimentally naïve rhesus macaques (n=4) and monkeys with a >5 year (>1.5 g/kg lifetime intake) cocaine SA history (n=4). Preliminary data suggests monkeys with a cocaine SA history require a greater number of trials to acquire stimulus discrimination and reversal and set shifting tasks (intradimensional/extradimensional shift). Similar response and reinforcer retrieval latencies across trials suggest differences are not a result of deficits in motor coordination or reinforcer devaluation compared to cocaine. Previous studies demonstrating specific CNS structures responsible for accurate performance on these tasks suggest cocaine SA is associated with deficits in prefrontal and orbitofrontal cortical activity, consistent with neurological assessments in human studies. Future studies will examine the ability of potential treatment medications to improve cognitive declines associated with chronic cocaine use. DA10584, DA06634.
Alterations in β‐arrestin expression in guinea‐pig ileum and colon following morphine tolerance
Authors: Kang Minho , Payne Hollis M , Dewey William L , Akbarali Hamid I
Published: June 2021

Repeated administration of morphine results in the development of tolerance to its antinociceptive effects. We have previously shown that tolerance also occurs to morphine‐induced inhibition of neurogenic contractions in the mouse ileum but not in the colon. The aim of the present study was to examine the development of morphine tolerance in the guinea‐pig ileum and colon and determine the role of G‐protein coupled receptor kinase 2 (GRK2) and β‐arrestin. Isolated guinea‐pig ileum longitudinal muscle‐myenteric plexus (LMMP) and colonic muscle strips were set‐up in the organ bath. Field stimulated contractions were dose‐dependently inhibited by morphine (10nM −10 μM) in both ileum and colon. Incubation with 10 μM morphine for 1 hr induced tolerance to subsequent administration in the ileum but not in the colon. The expression and interaction of GRK2 and β‐arrestin with the μ opioid receptor (MOR) were determined by Western blots. Immunoprecipitation of MOR with GRK2 and β‐arrestin was enhanced after morphine treatment in both tissues. The expression ratio of GRK2/GAPDH was similar, however, that of β‐arrestin/GAPDH was significantly less in the ileum (50 ± 20%) compared to the colon. Morphine treatment (10 μM for 1hr) did not alter the expression levels of GRK2 in either tissues, but markedly reduced β‐arrestin expression by approx. 30% in the ileum. In stably MOR transfected CHO cells, overexpression of GRK2 increased MOR internalization. These studies suggest that an enhanced expression of β‐arrestin in the colon may preclude development of morphine tolerance. Supported by NIH DA024009.
Effects of wheel running duration on osteoarthritis‐induced shifts in hind limb weight bearing in rats
Authors: Jezierski Justin , Dunbar Catherine , Luginbuhl Amy , Benoit Laura , Schuenke Mark , Bilsky Edward J , Stevenson Glenn W
Published: June 2021

We are developing methods to characterize the potency/efficacy of chronic pain states to suppress feeding, locomotor activity and wheel running. The present study describes a serendipitous finding that wheel running, when manipulated as an independent variable, attenuates osteoarthritis‐induced shifts in hind limb weight bearing. Rats were given intra‐articular saline or monosodium iodoacetate (MIA, 3.2mg) into the left hind knee. Rats were divided into three groups. Group 1 had 24 hr voluntary access to running wheels for 42 consecutive days (21 days acquisition followed by 21 days post‐MIA). Group 2 had 24 hr voluntary access to running wheels for 21 consecutive days (0 days acquisition followed by 21 days post‐MIA). Group 3 had no access to running wheels for 21 consecutive days (21 days post‐MIA). Weight bearing was measured on post‐MIA days 3, 7, 14 and 21. MIA produced robust and reliable shifts in weight bearing in non‐runners. In contrast, runners demonstrated a duration‐dependent attenuation of MIA‐induced shifts in weight bearing. These results suggest that voluntary wheel running may be protective against osteoarthritis pain. Parallel experiments are being conducted to determine the effects of wheel running duration on MIA‐induced damage to cartilage, subchondral bone and muscle. Funded by NIAMS grant R15 AR054975‐01 (GWS).
Constitutive PKC phosphorylation of the D1 dopamine receptor inhibits receptor signaling
Authors: Rankin Michele Lynn , Sibley David R
Published: June 2021

The D1 dopamine (DA) receptor (D1) is phosphorylated by protein kinase C (PKC), yet the role of PKC in D1 signaling is undefined. We investigated PKC effects on the D1 using transiently transfected cells. Activation of PKC by phorbol esters (PE) increased D1 phosphorylation. Co‐transfection of the D1 with PKC isoforms α, βI, γ, δ, ε, and λ resulted in greater D1 phosphorylation, whereas PKC isoforms μ, ν, η, and ζ were without effect. Activation of an M1 muscarinic receptor resulted in increased D1 phosphorylation, indicating heterologous phosphorylation by PKC. PKC effects on D1 signaling were evaluated using cAMP assays. Surprisingly, both activation and inhibition of PKC were found to potentiate DA‐stimulated cAMP accumulation. The potentiation is specific to D1 activation, since PE‐treatment had no effect on forskolin‐stimulated cAMP levels. To focus on D1‐specific PKC effects, we performed site‐directed mutagenesis of the D1 in order to identify the PKC phosphorylation sites. Five sites were identified: S259, S397, S398, S417, and S421. Simultaneous mutation of these sites eliminated PKC phosphorylation of the D1 resulting in increased G protein coupling and cAMP accumulation which was no longer potentiated by PKC inhibitors. These data suggest that constitutive PKC phosphorylation of the D1 regulates the signaling tone of the receptor.
Omega‐3 fatty acid mediated phosphorylation of the short and long human GPR120 receptor isoforms
Authors: Burns Rebecca L. , Moniri Nader H.
Published: June 2021

The orphan G protein‐coupled receptor, GPR120, has recently been thrust to the forefront of diabetes research due to its ability to stimulate release of the insulin secretagogue glucagon‐like peptide 1. While the molecular aspects involved in GPR120 signaling remain elusive, it has been shown that long chained free fatty acids, including the omega‐3 fatty acids α‐linolenic acid (ALA) and docosahexaenoic acid (DHA), are agonists of GPR120. We have recently cloned the human and rat GPR120 genes and have discovered that the human receptor can be alternatively spliced, yielding distinct short and long isoforms. Interestingly, we have identified that GPR120‐long contains an additional 16 amino acids within the third intracellular loop, and importantly, this region contains four phospho‐labile residues. Based on this distinct difference, we hypothesized that the long and short GPR120 isoforms have differing phosphorylation and desensitization profiles. Here, we present data which compares the phosphorylation of both variants upon agonism with various omega‐3 fatty acids. We also present data regarding the molecular mechanisms of GPR120 phosphorylation. Our data, taken together with the variation of the sequences encoding the third intracellular loop, suggest that the two GPR120 isoforms have distinct desensitization profiles. This project is funded by the Diabetes Action Research and Education Foundation.
A functional genomics approach identifies GPCR endocytosis‐regulating kinases
Authors: Hein Peter , Herzig Stefan , von Zastrow Mark
Published: June 2021

Signalling fidelity of G protein‐coupled receptors is controlled by the amount of receptors present at the cell membrane. Upon agonist stimulation, GPCRs traffick via clathrin‐coated pits to endosomes, from where they either recycle back to the cell membrane or are targeted to a degrading lysosome. There are a number of proteins known in this process; however, those have been identified mostly by looking for direct binding to receptors. We employed an unbiased genomics approach to identify kinases previously not inferred to be involved in receptor trafficking. For kinase knockdown, HEK293 cells stably expressing the β2‐adrenergic receptor fused n‐terminally to the SuperEcliptic GFP variant were transduced with shRNA‐containing viruses. Cells were stimulated or not with agonist for 30 minutes, and then analyzed for surface fluorescence using a flow cytometer. siRNA‐mediated knockdown of candidate genes was used to further confirm a role of those genes in receptor trafficking. Taken together, our approach identifies protein kinases that either inhibit or enhance receptor endocytosis, and that may play a crucial role in determining the output of GPCR‐linked signalling systems.
Inhibition of LIMK activity by β‐arrestins: A mechanism for cofilin activation
Authors: Lin Alice , DeFea Kathryn
Published: June 2021

β‐arrestins are mediators involved in activation and termination of GPCR signaling pathways without G‐protein coupling. Since ~40% of currently used therapeutics target GPCRs, it is important that our lab elucidate the role of β‐arrestins in protease‐activated‐receptor‐2‐induced actin reorganization. My project focuses on LIM kinase 1 (LIMK1), a key regulator of actin dynamics related to tumor cell invasion and metastasis, and dendritic spine formation through inhibiting the actin filament severing protein, cofilin. We have shown that LIMK1 directly interacts with β‐arrestin‐1(β1), and siRNA knockdown of β1 enhances both LIMK1 activity and the phosphorylation of cofilin. Here, we demonstrate the following 1) β1 directly binds LIMK and this primarily involves residues 164–418 of β1 and residues 285–638 of LIMK1, as determined by sandwich immunoassay and Bioluminescence Resonance Energy Transfer (BRET); 2) β1 directly binds and inhibits LIMK1 activity in vitro (as determined by cofilin phosphorylation), and 3) the inhibitory activity of β1 is mediated primarily through residues 1–163. These data suggest that β‐arrestins may facilitate cofilin activation by blocking the ability of LIMK to phosphorylate and inactivate cofilin. This represents a novel mechanism for the regulation of actin filament severing by β‐arrestins.