bluuebunny/crossref_metadata_2025 huggingface dataset:
Nutritional interventions protect against age‐related deficits in behavior: From animals to humans
Authors: Shukitt‐Hale Barbara , Miller Marshall G.
Published: June 2021

Aged rats show impaired performance on motor and cognitive tasks. Similar changes in behavior occur in humans with age, and the development of methods to retard or reverse these age‐related neuronal and behavioral deficits could increase healthy aging and decrease health care costs. In the present study, men and women, 21–75 years of age, were recruited to investigate declines in mobility and cognition. Balance and gait were measured using a treadmill instrumented with an array of high‐density pressure sensors. Spatial learning and memory were measured using a virtual version of the Morris water maze and executive function measured using the trail‐making test. Results show that postural sway increased while preferred walking speed, executive function, and spatial navigation decreased with increasing age. We have shown that dietary interventions with fruits and vegetables high in antioxidant and anti‐inflammatory activity are able to prevent and reverse the neurochemical and behavioral changes that occur in aging when fed to rats from 19–21 months of age. The test battery employed in the present study shows changes parallel to what we see in rodent studies; therefore, it presents a useful model for assessing the effect of dietary interventions in older adults.
Effects of supplementation of a cranberry extract enriched in A‐type proanthocyanidins on intestinal bacterial composition of pigs fed obesogenic diets
Authors: Monagas Maria , Molokin Aleksey , Lakshman Sukla , Bruna Gonzalo , Urban Joseph , Solano‐Aguilar Gloria
Published: June 2021

Proanthocyanidins, which are amongst the most bioactive polyphenols in our diet, are catabolized by intestinal microbiota. The effect of cranberry extract containing ~7% proanthocyanidins (CEPA), on weight gain and composition of intestinal microbiota was evaluated. Young pigs fed 14 mg of CEPA/kg /day added to a basal (10% kcal from fat) or high fat (HF) (35% kcal from fat) diet for 12 weeks were compared to pigs fed similar diets without CEPA. DNA from fecal samples, collected at day 0 and weeks 2, 4, 6, 10, and contents from proximal and distal colon collected at necropsy were processed. Feeding CEPA decreased body weight gain only in pigs fed a basal diet. Total bacteria measured by RT‐PCR, was not affected by the diet or CEPA. Certain Lactobacillus species (spp) were significantly increased in feces from pigs fed the HF diet (3.2–20 fold) at weeks 4 and 6, and in proximal and distal contents. CEPA supplementation significantly increased (2 fold) the abundance of certain Lactobacillus spp at week 4 in both basal and HF diets. The abundance of E. coli spp increased (3–20 fold) in pigs fed the HF diet starting at week 2, and in the proximal and distal contents. In contrast, Bifidobacterium spp were decreased (42 fold) in distal contents of pigs fed the HF diet. In summary, these results showed that CEPA consumption did not induced major changes in certain bacterial species that may explain reduction in weight gain.
ARTEVISININ INFLUENCE ON MEMBRANOUS PHOSPHOLIPIDS OF BRAIN AND LIVER TISSUES OF WHITE RATS AT INTOXICATION BY CARBON TETRACHLORIDE
Authors: Kazbekova Ainagul , Tuleshova Gulnara , Seitembetov Talgat , Dalenov Erbolat
Published: June 2021

The aim of work is to study influence peculiarities of natural sesquiterpene lactone of artevisinin from Artemisia annua L., which is spread on the territory of Kazakhstan, in comparison with hepatoprotector carsil on a dynamics of phospholipids composition of brain and liver tissues of white rats at toxic damages of liver by carbon tetrachloride in vivo.

It is known, that acute liver intoxication causes a growth of glycerophosphates, lysophosatidylcholines, phosphotidylserines, polyglycerophosphotides, phosphotide acids and decrease of sphingomyelins, phosphatidylcholines and phosphotidylethanolamines. When carbon tetrachloride affects a brain, a total quantity of acidic phospholipids increases by 5,1 times and when it affects a liver, a total quantity of acidic phospholipids increases by 1,8 times in the relation of the total sum of neutral phospholipids. The preventive introduction of artevisinin reduces these values up to 1,5 and 0,3 times accordingly.

For the first time it was calculated thermodynamic indicators ‐ entropy and redundancy for brain and liver phospholipids, which reflect a level of good organization and order of membranous phospholipids in good conditions and pathology. It is revealed, that entropy increases and redundancy decreases by influence of carbon tetrachloride. The preventive introduction of artevisinin reduces entropy by the growth of redundancy.
Comparing the antioxidant capacity of various proteins using the oxygen radical absorbance capacity (ORAC) assay
Authors: Butteiger Dustie N. , Krul Elaine
Published: June 2021

The oxygen radical absorbance capacity (ORAC) assay is a widely used method for assessing antioxidant potential of foods, food ingredients and biological samples. Various protein sources have been shown to possess differences in antioxidant capacity. Soy protein's antioxidant properties may be derived from endogenous peptides as well as its associated isoflavones. To investigate this further, we employed the ORAC assay to assess the antioxidant capacity of the aqueous soluble fraction of various proteins. Soy protein isolates (SPI), milk whey protein (WP), sodium caseinate (CAS), and soy‐dairy protein blend powders were solubilized in phosphate‐buffered saline (PBS) and the soluble fractions (250 μg/mL protein) were tested for their ability to inhibit the oxidation of fluorescein in a standard ORAC assay using fatty acid free BSA (FAF‐BSA) as a reference control. FAF‐BSA had the lowest ORAC value (~ 3000 uM Trolox equiv/mg protein). Milk whey protein (WP) had similar radical scavenging activity to that of FAF‐BSA (~ 2800 uM Trolox equiv/mg protein). Several SPI preparations had significantly higher ORAC values compared to CAS (SPI was 50–80% higher), while other SPI preparations had ORAC values similar to CAS (~ 8400 uM Trolox equivalents/mg protein) and both were significantly higher than the ORAC values for WP (p<0.05). A protein blend of SPI, WP, and CAS (25%:25%:50%, respectively) was similar to CAS and significantly lower than SPI but significantly higher the WP alone (p<0.05). In summary, SPI, on average, has a significantly higher antioxidant capacity compared to dairy CAS and WP as determined by the ORAC assay. CAS has intermediate ORAC values which are significantly higher than those of WP. The addition of SPI in protein blends increases their antioxidant properties and may enhance their potential health benefits. Solae, LLC funded research.
DNA Methylation and Histone Modifications of Wnt Genes by Genistein During Colon Cancer Development
Authors: Zhang Yukun , Li Qian , Chen Hong
Published: June 2021

The current study elucidates the epigenetic mechanisms by which genistein maintains a normal level of WNT genes during colon cancer development. We have shown that SPI and GEN diets repressed WNT signaling, which correlated with the reduction of preneoplastic lesions in rat colon. Here we hypothesized that SPI and GEN induced epigenetic modifications on WNT genes, therefore suppressed the gene expression induced by AOM. We identified that in the post‐AOM period the acetylation of histone H3 was downregulated by SPI and GEN at the promoter of selected WNT genes, which is in parallel with the reduced binding of RNA Polymerase II at the same region. Meanwhile, nuclear histone deacetylase 3 was enhanced by SPI and GEN. The diets suppressed the trimethylation of histone H3 Lys 9 and the phosphorylation of Ser 10. Methylation of the specific region of Sfrp2, Sfrp5, and Wnt5a genes was increased by SPI and GEN, which was correlated with the reduction of gene expression. Bisulfite sequencing confirmed that dietary genistein induced DNA methylation of the promoter of Sfrp5, closely correlated with the reduced histone modifications at the same region. We present an epigenetic mechanism by which dietary genistein controls the responses of WNT genes during carcinogen induction, involving DNA methylation and histone modifications at the regulatory region of gene.

Grant Funding Source: Illinois Soybean Association and NIH/NCI
Inhibition of VSMC Activation and Vascular Restenosis by Interleukin‐19
Authors: Ellison Stephen , Richards James , Miecyjak Alex , Gabunia Khatuna , Kelemen Sheri , Autieri Michael
Published: June 2021

This study tested the hypothesis that IL‐19, a member of the Th2 family of anti‐inflammatory interleukins, can attenuate vascular restenosis and modulate the vascular smooth muscle cell (VSMC) response to injury. Carotid artery ligation of hyper‐responsive wild‐type (wt) mice (FVB strain) injected with 10ng/g/day rIL‐19 had significantly lower neointima/media ratio (N/I) compared with PBS controls (P=0.006). Conversely, carotid artery of IL‐19 knock out (KO) mice demonstrated significantly higher N/I ratio compared with wt mice (both non‐responding C57B/6 strain) (P=0.04). Importantly, the increased N/I ratio in the KO could be rescued by injection of 10ng/g/day into the KO mouse (P=0.04). VSMC explanted from IL‐19 KO mice proliferated significantly more rapidly compared with wt (P=0.04). Addition of IL‐19 to cultured wt VSMC did not significantly decrease VSMC proliferation, but could rescue KO VSMC to wild‐type levels (P=0.02). KO VSMC expressed significantly greater levels of inflammatory mRNA including IL‐1β, TNFα, and MCP‐1 in response to TNFα stimulation (P<0.01 for all). No polarization of adaptive immunity was noted in these mice. Together, these data indicate that IL‐19 is a previously unrecognized anti‐inflammatory factor for VSMC, and plays an important protective role in vascular restenosis.
Myostatin inhibition improves insulin signaling in muscles of mice with high fat diet‐induced diabetes
Authors: Dong Yanjun , Han H.Q. , Dong Yanlan , Mitch William E , Zhang Liping
Published: June 2021

Myostatin inhibits skeletal muscle growth. Animals lacking myostatin develops significantly larger muscles, which are associated with reduced adiposity and lower blood glucose. We investigated whether myostatin inhibition would improve the metabolic profile of diabetic mice. C56/BL6 mice were fed a high fat diet (HFD) for 10 months. During the last 3 weeks, myostatin was inhibited by injections of a myostatin peptibody in mice on HFD vs PBS injected mice on a HFD. Body weight, lean mass and adipose tissue were quantified using an x‐ray imager; oxygen consumption and carbon dioxide production were measured using an animal monitoring system. Myostatin inhibition (MI) decreased blood glucose, serum insulin and increased glucose disposal in response to insulin. Also, there was increase in energy expenditure, associated with a decrease in the percent body fat plus a decrease in serum fatty acids. MI also increased p‐Akt in both muscle and white adipose tissue (WAT), responses linked to increased expression of fatty acid oxidation genes, plus an increase in AMPK activation plus expression of PPAR. We propose that myostatin, by suppressing both the AMPK and PPAR axes in skeletal muscle and adipose tissue interferes with insulin signaling and reduces fatty acid oxidation. In conclusion, targeting myostatin corrects these defects improving both insulin signaling and lipid metabolism in diabetes.
Nitric Oxide Inhibits IP3R‐mediated Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells
Authors: Gonzales Albert Louis , Earley Scott
Published: June 2021

The melastatin transient receptor potential channel TRPM4 is essential for pressure‐induced smooth muscle cell contraction and is activated by Ca2+ released from the SR via inositol trisphosphate receptors (IP3R). Endothelium‐derived nitric oxide (NO) activates soluble guanylyl cyclase (sGC) leading to the generation of cyclic guanosine monophosphate (cGMP), activation of protein kinase G (PKG), and smooth muscle relaxation. We hypothesized that NO blocks IP3R‐dependent activation of TRPM4 channels in cerebral artery myocytes. Using perforated whole‐cell patch clamp electrophysiology, we observe a dose‐dependent reduction in TRPM4 channel activity in response to the NO donor S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine (SNAP). TRPM4 channel activity is also diminished following exposure to the cell‐permeable cGMP analog dibutyryl‐cGMP. TRPM4 currents inhibited by NO or cGMP did not return following administration of the cell‐permeable IP3 analog, Bt‐IP3. In addition, the sGC inhibitor NS‐2028 prevented NO‐mediated inhibition of TRPM4 channel activity. These findings suggest that NO can relax cerebral artery myocytes by blocking IP3R‐mediated activation of TRPM4. RO1HL091905; F31HL094145–01 (AG)
Cinnamaldehyde and poly‐cinnamaldehyde micelles induce relaxation of isolated porcine coronary arteries
Authors: Raffai Gabor , Kim Byungkuk , Wu Dongmei , Khang Gilson , Lee Dongwon , Vanhoutte Paul M.
Published: June 2021

Cinnamaldehyde (CA), one of major components of cinnamon, induces the generation of ROS and exerts vasodilator and anticancer effects. However, its short half‐life limits its clinical use. Therefore, a polymeric pro‐drug that incorporates CA in its backbone [poly(cinnamaldehyde) (PCA)] and self assembles to form micelles was developed. Experiments were designed to determine whether or not PCA micelles possess vasodilator properties. Rings of porcine coronary arteries were contracted with U46619 and changes in isometric tension recorded. CA induced endothelium‐independent relaxations that were not affected by catalase or inhibitors of eNOS (L‐NAME), nNOS (SMLT), cyclooxygenases (indomethacin), soluble guanylyl cyclase (ODQ), TRP (ruthenium red) or TRPA1 channels (HC030031). Inhibition of either KATP channels (glibenclamide) or superoxide dismutase (DETC) caused a leftward shift of the concentration‐response curve to CA. PCA micelles induced only partial, endothelium‐dependent relaxations that were inhibited by L‐NAME, SMLT, ODQ, and catalase. The present findings demonstrate that micelles prepared from PCA possess vasodilator properties, but the mechanism underlying relaxation differs from that of CA, and thus may be used for therapeutic delivery. Research support: World Class University program (R31–20029), Ministry of Education, Science and Technology, South Korea.
Vascular relaxation induced by NO donor in mesenteric resistance artery from 2K‐1C rats involves reactive oxygen species and potassium channels activation
Authors: Andrade Fernanda Aparecida , da Silva Roberto Santana , Bendhack Lusiane Maria
Published: June 2021

This study aimed to evaluate the effect of the inhibition of soluble guanylyl‐cyclase (sGC) by ODQ, K+ channels by TEA and ROS production by apocynin on the relaxation to the NO donor RuBPY in mesenteric resistance artery from normotensive (2K) and 2K‐1C rats. Concentration‐effect curves to RuBPY were constructed in isolated denuded mesenteric arteries from 2K and 2K‐1C rats contracted with phenylephrine, in the absence or in the presence of ODQ, TEA, or Apocynin. The maximum relaxation induced by RuBPY was similar in 2K (77.6±8.9%, n=7) and 2K‐1C (76.9±4.5%,n=13), that was inhibited by ODQ in 2K (36.5±2.9%,n=5) and 2K‐1C arteries (24.6±5.5%, n=5). The relaxation induced by RuBPY was reduced by TEA (50.5±10.6%, n =8) and apocynin (45.7±3.9%,n=4) only in 2K‐1C arteries. These results indicate that although RuBPY induces relaxation with similar efficacy in mesenteric arteries from normotensive and hypertensive rats and that the NO released from RuBPY activates sGC, the NO signaling can differ. It involves ROS production and K+ channels activation only in 2K‐1C arteries. Supported by FAPESP and CNPq.
Inwardly rectifying potassium channels 2.x contribute to endothelial hyperpolarization and vasorelaxation to P2Y receptor stimulation in murine cerebral artery
Authors: Kochukov Mikhail Y , Marrelli Sean P
Published: June 2021

Inwardly rectifying potassium channels (Kir) 2.x are expressed in cerebral endothelial cells (EC) but little is known about their role in vascular tone control. We tested the hypothesis that endothelial Kir amplify membrane hyperpolarization and cerebral vasodilation to ATP and UTP.

Methods

Expression of Kir 2.1 and 2.2 was demonstrated in freshly isolated EC from rat middle cerebral artery (MCA) by PCR. Vasorelaxation (pressure myography) and endothelial hyperpolarization (di‐8‐ANEPPS) were measured in pressurized MCA following intraluminal delivery of UTP. Membrane current and voltage changes were recorded from mouse cerebral EC using perforated‐patch voltage clamp and current clamp techniques.

Results

Unstimulated EC showed Kir‐like (sensitive to Ba2+ and Kir 2.x blocker ML 133) and transient outward KCa‐like currents. Stimulation with ATP activated large outward currents that were sensitive to SKCa and IKCa blockers (UCL1684 and TRAM34). The ensuing membrane hyperpolarization was significantly attenuated in cells pretreated with Ba2+ or ML 133. In pressurized MCA, intraluminal application of Ba2+ significantly decreased hyperpolarization and relaxation responses to UTP.

Conclusion

Endothelial Kir2.x channels play a significant role in cerebral vasorelaxation by maintaining endothelial hyperpolarization in response to P2Y receptor stimulation. Supported by R01HL088435 to SPM
Role of smooth muscle cell hyperpolarization in modulation of endothelial cell spontaneous Ca2+ events
Authors: Beleznai Timea , Dora Kim
Published: June 2021

Smooth muscle cell (SMC) hyperpolarization and consequent vasodilatation has the potential to be associated with increased spontaneous Ca2+ events in endothelial cells (ECs). The underlying mechanisms remain to be determined, but possibilities could include an increased driving force for Ca2+ entry or the presence of hyperpolarization activated channels in the ECs. To hyperpolarize the SMCs, we used 3 μM levcromakalim (LVK) acting at ATP‐sensitive K+ channels (KATP) and 10 mM KCl. Rat cremaster arteries were isolated, double‐cannulated, tied onto glass pipettes (80 mmHg). With a confocal microscope and 40× objective we imaged the ECs loaded with Oregon Green BAPTA‐1 AM to study the intracellular Ca2+ events. LVK and KCl stimulated increased focal spontaneous Ca2+ events, which occasionally propagated as a Ca2+ wave across ECs. These Ca2+ events were significantly decreased following removal of extracellular Ca2+ or application of the KATP channel blocker glibenclamide. These data suggest that LVK and 10 mM KCl evoke increased ECs Ca2+ events, which could have physiological role in the regulation of arterial tone. The work was supported by British Heart Foundation and Wellcome Trust UK.
β‐arrestin2 expression is localized in cholinergic but not nitrergic motor neurons in the mouse longitudinal musclemyenteric plexus (LMMP)
Authors: Maguma Hercules Tarambirwa , Ali Iqbal J , Dewey William L , Akbarali Hamid I
Published: June 2021

Morphine inhibits intestinal peristalsis via its action on cholinergic enteric neurons. Tolerance to morphine does not develop in the colon, except upon deletion of β‐arrestin2 (β‐arr2), a key molecule involved in G protein–coupled receptor desensitization (Maguma et al., 2012). However, the specific localization of β‐arr2 in the myenteric plexus is not known. In this study we utilized dual‐labeling immunofluorescence to characterize myenteric neurons expressing β‐arr2 and investigated the impact of β‐arr2 abrogation on cholinergic function. Our results reveal robust expression of β‐arr2 in the soma of select neurons that stained positive for choline acetyltransferase and calretinin, a motor neuron marker. β‐arr2 was neither co‐localized with substance P, nitric oxide synthase nor calbindin. The density of β‐arr2 neurons was higher in the ileum (10.8%) vs. colon (4.3%). Deletion of β‐arr2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (− log EC50: wild type = 5.8 vs β‐arr2 knockout = 5.9). Our findings suggest specificity in the localization of β‐arr2 within cholinergic motor neurons of the myenteric plexus. β‐arr2 deletion does not directly alter cholinergic neuronal activation. Studying β‐arr2 signaling in the cholinergic motor neurons would be critical to assess mechanisms involved in reversal of opioid effect in the colon. Support: NIH grant DA024009
Rescue of lethal CaVβ2 knock‐out and identification of novel CaVβ2 variants
Authors: Weissgerber Petra Edeltraut , Roth Elisabeth , Buchholz Stefanie , Schmitz Frank , Flockerzi Veit , Kahl Sabine
Published: June 2021

Disruption of the CaVβ2 gene in mice resulted in diminished L‐type Ca currents in cardiomyocytes. Thereby heart function was compromised and embryonic death occurred. Partial rescue of the Ca channel currents by Bayk8644 significantly postponed embryonic death in CaVβ2‐deficient mice. The predominant ancillary β subunit of cardiac Ca channels is the β2 subunit and three splice variants are expressed in the heart, β2aN4>; β2aN1>;>;β2aN5. β2aN1 appears to be mainly expressed in embryonic heart and the β2aN4 in adult heart. We now rescued the lethal β2−/− phenotype by expression of a mouse β2 transgene under the control of the mouse a‐MHC promoter and generated two mouse lines: one rescued with the β2aN1 of embryonic heart, one with the β2aN4 of adult heart. Characterization of the founders and the β2−/−/tg+/‐ mice by Southern blots and Western blots show that the β2 transgenes are expressed in heart only and that the extracardial β2‐deficiency persists. Comparing β2 protein expression in adult heart and various tissues from wild‐type and transgenic mice reveals that additional and novel CaVβ2 variants are expressed in wild‐type tissues. We will characterize the novel CaVβ2 variants and the impact of β2‐deficiency in extracardial tissues. The availability of mice carrying either the N4 or the N1 β2 variant only in cardiac myocytes allows us to study the roles of either variant on L‐type Ca channels in cardiomyocytes.
Functional characterization of cannabinoid receptor‐interacting protein CRIP1a
Authors: Selley Dana E. , Jacob Joanna C. , Smith Tricia H. , Blume Lawrence C. , Shim Hoon , Straiker Alex , Mackie Ken , Howlett Allyn C. , Sim‐Selley Laura J. , Chen Ching‐Kang
Published: June 2021

G‐protein‐coupled CB1 cannabinoid receptors (CB1Rs) are highly expressed in the brain and together with the endocannabinoids, anandamide and 2‐arachidonoylglycerol (2‐AG), modulate appetite, reward, pain, memory and motor activity. Cannabinoid receptor‐interacting protein 1a (CRIP1a) is a novel regulatory protein that binds to the C‐terminus of CB1Rs but not CB2Rs. Studies in cell models in which CRIP1a was co‐expressed with CB1Rs at a molar ratio of 5:1, respectively, showed that CRIP1a did not alter CB1R expression levels but inhibited agonist‐stimulated and constitutive CB1R‐mediated G‐protein activation, as determined by [35S]GTPγS binding. Electrophysiological studies in cultured hippocampal neurons showed that CRIP1a over‐expression attenuated 2‐AG‐mediated inhibition of excitatory synaptic activity. These results suggest that CRIP1a negatively regulates CB1R signaling. To determine effects of loss of CRIP1a function, we generated Cnrip1 genetic null mice. Cnrip1 null mice are viable and do not express CRIP1a protein in the brain or spinal cord, thus providing a valuable tool to assess CRIP1a function in vivo. Supported by R21‐ DA025321 and R01‐DA030404 from NIDA and an A.D. Williams Multischool Award.
Interdependent expression of P2X receptors in the mouse kidney: P2X4‐P2X7 receptor “cross‐talk”
Authors: Birch Rebecca , Scott‐Ward Toby , Tam Frederick W K , Peppiatt‐Wildman Claire M , Unwin Robert J , Wildman Scott S P
Published: June 2021

In the renal collecting duct (CD), an ionotropic P2X4‐like receptor may act as a luminal Na sensor, regulating ENaC‐mediated Na reabsorption [1]. Several P2X receptor subunits are expressed in the CD and evidence is emerging that P2X4 and P2X7 subunits might interact [2]. P2X7 is also linked to many renal pathologies.

Here we determine whether P2X4 and P2X7 interact at the expression level in the CD. Using target‐specific primers, we quantified P2X4, P2X7 (and β‐actin) mRNA levels in mouse kidney and microdissected CD of wild‐type (WT), P2X4−/− and P2X7−/− mice by real‐time RT‐PCR.

Expression of P2X4 and, to a lesser extent P2X7 mRNA was readily detected in WT kidney (n=3), whereas P2X4 and P2X7 mRNA levels were <100‐fold lower in P2X4−/− and P2X7−/− mice respectively (n=3). Expression of P2X4 mRNA was reduced in P2X7−/− kidney (P=0.03; n=3), and a substantial decrease in P2X7 mRNA was observed in the CD of P2X4−/− mice (P<0.05; n=3).

Our studies indicate that expression levels of P2X4 and P2X7 in the collecting duct are interdependent and suggest a P2X4/7 heteromeric assembly as the luminal Na sensor.

Research supported by the Wellcome Trust
Pain‐related depression of the mesolimbic dopamine system in rats
Authors: Leitl Michael , Banks Matthew L , Negus S. Stevens
Published: June 2021

Some stressors produce prodepressant behavioral effects by activating endogenous kappa opioid systems that in turn depress mesolimbic dopamine release. This study evaluated the role of this mechanism in pain‐related behavioral depression in rats. Adult male Sprague‐Dawley rats were prepared either with intracranial electrodes targeting the medial forebrain bundle (for behavior studies of intracranial self‐stimulation; ICSS) or cannulae for microdialysis measures of nucleus accumbens dopamine (NAcc DA). Changes in ICSS and NAcc DA were examined after treatment with a chemical noxious stimulus (intraperitoneal injection of dilute acid) or an exogenous kappa agonist (U69593). Additional studies examined sensitivity of acid and U69593 effects to blockade by two analgesics (the nonsteroidal antiinflammatory drug ketoprofen and the mu opioid receptor agonist morphine) or by the kappa antagonist norbinaltorphimine (norBNI). Both acid and U69593 depressed ICSS and NAcc DA release. However, the pain‐related effects of acid were blocked by pretreatment with ketoprofen and morphine, but not by norBNI. Conversely, effects of U69593 were blocked by norBNI but not by ketoprofen, and were only attenuated by morphine. These results support a role for the mesolimbic dopamine system, but not endogenous kappa opioid systems, in mediating pain‐related depression of behavior in rats.
Effects of imidazoline I2 receptor agonists in rat models of chronic pain
Authors: Li Jun‐Xu , Thorn David A , Zhang Yanan
Published: June 2021

Recent studies demonstrate that a selective imidazoline I2 receptor ligand CR4056 has potent analgesic activity in rat models of chronic pain when the drug is administered acutely. However, because chronic pain treatment requires repeated dosing, the pharmacological consequence of repeated I2 receptor agonist treatment is unclear. This study examined the effect of acute and repeated I2 receptor agonist treatment in two rat models of chronic pain: complete Freund's adjuvant (CFA)‐induced inflammatory pain and chronic constriction injury (CCI) induced neuropathic pain. In CFA‐treated rats, morphine (0.32 – 10 mg/kg, i.p.) as well as two I2 agonists, 2‐BFI (3.2 – 17.8 mg/kg) and BU224 (3.2 – 17.8 mg/kg), produced dose‐dependent anti‐allodynia effects, and the effects of 2‐BFI were attenuated by an I2 receptor antagonist idazoxan (3.2 mg/kg). In addition, daily treatment with 10 mg/kg 2‐BFI or 32 mg/kg CR4056 for 7 days did not alter the anti‐allodynia effects of 2‐BFI and CR4056. In CCI‐injured rats, 2‐BFI (1 – 10 mg/kg) produced a dose‐dependent anti‐allodynia effect and the effect remained unchanged during 10 days of daily 10 mg/kg 2‐BFI treatment. Together, these data suggest that activation of I2 receptors have marked analgesic effects and repeated treatment produces little analgesic tolerance. Imidazoline I2 receptors may represent a novel drug target for pharmacotherapy of chronic pain conditions.
Examining the reinforcing properties of NOP receptor agonist SCH221510 and its role in attenuating mu‐receptor mediated reinforcement in a rat self‐administration assay
Authors: Sukhtankar Devki , Lagorio Carla H , Ko Mei‐Chuan
Published: June 2021

Nociceptin/orphanin FQ receptor (NOP) plays a critical role in regulation of reward and motivation neuropathways. In rodents, NOP agonists do not produce conditioned place preference and block rewarding effects of opiates and psychostimulants. We used a rat self‐administration assay to determine reinforcing properties of NOP agonist SCH221510 and its role in attenuating reinforcing effects of mu‐opioid receptor (MOP) agonist remifentanil. Rats with intravenous catheters were allowed to self‐administer SCH221510 (3–300 μg/kg/infusion) or remifentanil (0.3–10 μg/kg/infusion) under fixed (FR5) and progressive ratio (PR) schedules of reinforcement. All rats self‐administered remifentanil, generating a biphasic dose effect curve typical of intravenous drug self‐administration. SCH221510 did not maintain responding, resulting in a flat dose effect curve. Under PR, break points for SCH221510 were similar to those of vehicle, whereas remifentanil had significantly higher break points. Intracisternal (0.03–3 μg) but not systemic (1–10 mg/kg, s.c.) administration of SCH221510 decreased responding for remifentanil (3.2 μg/kg/infusion). This effect was comparable to that of MOP antagonist naltrexone (0.03– 0.3 mg/kg s.c.). Hence, our data show that NOP agonists do not function as reinforcers and may block reinforcing effects of MOP agonists (Supported by USPHS grant R01‐DA032568).
Effects of the cannabinoid 2 receptor‐selective agonist GW405833 in assays of acute pain‐stimulated and paindepressed behavior in rats
Authors: Kwilasz Andrew , Negus Steve
Published: June 2021

Cannabinoid 2 receptor (CB2R) agonists produce antinociception in many conventional preclinical assays of pain‐stimulated behavior, but effects of CB2R agonists on pain‐related behavioral depression are not known. GW405833 is a CB2R‐selective agonist with 1200‐fold selectivity for CB2R versus CB1R. This study compared effects of GW405833 in assays of acute pain‐stimulated and pain‐depressed in male Sprague Dawley rats. Intraperitoneal injection of dilute lactic acid (1.8% in 1 ml/kg) served as a noxious stimulus to stimulate a stretching response or depress intracranial self‐stimulation (ICSS) of the medial forebrain bundle. Effects of GW405833 on ICSS in the absence of the noxious stimulus were also examined as a measure of abuse liability. GW405833 (3.2–32 mg/kg IP) dose dependently attenuated both acid‐stimulated stretching and acid‐induced depression of ICSS without producing abuse‐related facilitation of ICSS in the absence of the noxious stimulus. However, significant antinociception was observed only at a relatively high dose (32 mg/kg), and in the assay of acid‐stimulated stretching, effects of GW4058333 were partially blocked by the CB1R antagonist rimonabant but not by the CB2R antagonist SR144528. These results suggest that antinociceptive effects of GW405833 in these assays were not mediated by CB2 receptors.
Strain Specific Persistent Suppression of Visceral Pain Perception After Recovery From a Bout of Colitis
Authors: Sessenwein Jessica L , Wang Lu , Wallace John L
Published: June 2021

Visceral pain is often experienced by patients with GI inflammation, and some studies suggest that such pain can persist after resolution of inflammation. Colitis was induced in male Wistar and Sprague‐Dawley (SD) rats via intrarectal administration of dinitrobenzene sulfonic acid (DNBS) in 50% ethanol, controls received saline only. At day 15, under general anesthesia, a balloon was inserted into the colon and distended (colo‐rectal distention; CRD), heart rate was recorded continuously. Cardioautonomic responses to CRD were analyzed and expressed as % change from the pre‐CRD heart rate. Colonic injury and inflammation were examined histologically and by measurement of tissue myeloperoxidase activity. By 15 days following DNBS administration, colitis had resolved. CRD caused a significant visceral pain response (bradycardia) in both strains of control rats. However, Wistar rats showed a significant suppression of pain response to CRD, whereas the response in SD rats remained the same after colitis. There was no difference in colonic compliance between control and DNBS groups or the two strains. Our results provide evidence of a strain‐dependent alteration in the pain response to CRD that persists after resolution of colitis, likely due to changes in autonomic sensory function induced by inflammation. Our results are consistent with the variability in pain perception seen in patients with GI inflammation.
HIGH‐THROUGHPUT SCREENING BY TRACTION MICROSCOPY
Authors: Park Chan Young , Burger Stephanie , Frykenberg Matthew , Tambe Dhananjay , Zhou Enhua , Krishnan Ramaswamy , Marinkovic Aleksander , Tschumperlin Daniel , Butler James , Lavoie Tera , Dowell Maria , Chen Bohao , Gardel Margaret , Green Geoffrey , Solway Julian , Fredberg Jeffrey
Published: June 2021

For discovery of novel drugs impacting contractile cells, such as human airway smooth muscle (HASM) cells, a high‐throughput screening (HTS) assay based upon contractility might be useful but no such HTS assay yet exists. To fill this gap, we have developed a new method called high‐throughput traction microscopy (HTTM). HTTM measures drug‐induced changes in contractile forces (tractions) exerted by adherent cells upon elastic substrates. Used together with soft gels in 96‐well plates and a motorized microscope, HTTM provides a HTS platform to screen large drug libraries and identify as hits those molecules that change cellular tractions. To demonstrate proof of concept, we used HASM cells and screened a library of 10,000 small molecular compounds. By screening mixtures of 8 compounds simultaneously, we were able to evaluate 2,000 compounds per week in quadruplicate with the possibility of attaining substantially higher throughputs. After retesting individual compounds from initially positive wells, we identified 8 individual compounds that relaxed HASM cells. For any cell type and disease process in which contractile forces are thought to play a functional role, HTTM provides a convenient, rapid, and inexpensive platform for drug discovery based upon a physiological endpoint, namely, cell contractile force. (NIH P50HL107171, Lung tissues are the generous gift from Gift of Hope.)
Enhanced oxygen transport by perfluorocarbon emulsion mixed with flowing human blood studied in vitro
Authors: Filho Ivo Torres , Pedro Jose Ricardo , Narayanan Srinivasan , Roseff Susan , Nguyen Nguyen , Berger Brian , Torres Luciana , Spiess Bruce
Published: June 2021

Perfluorocarbons (PFC) are compounds with high gas solubility that mixed with sickle cell disease (SCD) blood could potentially help deliver O2 to tissues, possibly compensating for SCD red blood cell's lower O2 affinity. PFC have also been suggested as adjunct therapy in certain ischemic conditions. Therefore, an in vitro system was developed for studying O2 transport using blood from 9 SCD patients and 5 healthy donors. The system included a pump, gas mixers, oxygenation and deoxygenation chambers that allowed the flowing blood to reach various levels of hemoglobin O2 saturation (SO2) and O2 partial pressures (PO2) that were measured using a CO‐oximeter and a blood gas analyzer. After addition of PFC emulsion (Oxycyte) (hematocrit = 31%, fluorocrit = 4%), the measurements suggested an increase in the transport of O2 and CO. Addition of PFC resulted in a larger PO2 gradient: from 15 ± 2 mmHg to 23 ± 2 mmHg. Using normal blood, the average O2 extraction ratio (O2ER) after PFC (0.13 ± 0.01) was significantly higher than baseline (0.10 ± 0.01, p<0.004). Using blood from SCD patients, the average O2ER after PFC (0.16 ± 0.01) was also significantly higher than baseline (0.12 ± 0.01, p<0.001). Addition of saline did not cause statistically significant changes. The data suggest increased O2 transport by this PFC emulsion in both normal and SCD blood.

Support: Virginia Commonwealth University and Oxygen Biotherapeutics, Inc.
Testosterone modulates sodium‐regulating hormones in women with PCOS
Authors: Stachenfeld Nina , Wenner Megan M , Taylor Hugh S
Published: June 2021

Polycystic Ovary Syndrome (PCOS) is associated hyperandrogenism and increased aldosterone (ALD). Testosterone (T) may modulate sodium regulation by the kidney, but interactions between T and ALD and the renin‐angiotensin‐aldosterone system (RAAS) have not been studied in PCOS. We hypothesized that T increases serum ALD (S[ALD]) and the RAAS in PCOS. For 11 days we suppressed sex hormones with a gonadotropin releasing hormone antagonist (GnRHant) in women with (PCOS, n=6, 23.2±0.7 y, 35.4±1.7 kg/m2) and without (C, n=7, 22.0±1.6 y, 34.1±1.1 kg/m2) PCOS; we added T (2.5 mg/day) for days 8–11. Pre‐GnRHant (BL) and on days 4 and 11, we measured plasma renin activity (PRA) and S[ALD]. Under all hormone conditions, PRA was greater in PCOS [1.4±0.1, 1.9±0.4, 1.8±0.4, BL, GnRHant, T] vs C (0.5±0.1, 0.7±0.2, 1.0±0.5, BL, GnRHant, T). S[ALD] was also greater in PCOS (130±26, 81±12, 112±28 for BL, GnRHant, T) compared to C (42±12, 37±12, 29±13 for BL, GnRHant, T, P <0.05) under all hormone conditions. GnRHant independently decreased S[ALD], which was partially restored with T (P <0.05) only in women with PCOS, suggesting T modulates renal sodium handling in PCOS. HL093450
From the classroom to communities: Connecting students in the sciences to real world situations.
Authors: Ntambi James Mukasa
Published: June 2021

Examples of real world impacts in the fields of biochemistry and molecular biology provide educational models where students are provided with a basis for understanding how biochemistry and molecular biology help us in learning about the real world environments in which people live. By taking the classroom out into communities, students see the relevance of what they learn on college campuses in their classes and labs. These “hands‐on” community based learning experiences are what brings science to life, which then sparks an interest in the students for delving further into these fields. They see the importance of studying these disciplines for helping them to have a positive impact on society. Through service learning, and community based research and teaching activities modeled upon the “hands on” experience course in international health and nutrition, the educational strategies help build a thriving democratic society and in the case of the scientific fields, more informed citizens who understand better and appreciate the positive and progressive impacts that science has on our daily lives. The strength of combining classroom experiences with community learning helps prepare students through science for lifelong civic engagement by building capacity, in partnership with the community, to help solve complex public health problems locally and globally.
Overexpression of glyoxalase 1 attenuates methylglyoxal‐induced peritoneal injury with suppression of epithelial mesenchymal transition pathway and neovascularization in Wistar rats
Authors: Oba Ikuko , Mori Takefumi , Ohsaki Yusuke , Sato Emiko , Nakamichi Yoshimi , Miyata Toshio , Ito Sadayoshi
Published: June 2021

Methylglyoxal (MG) is one of the causes of peritoneal injury in peritoneal dialysis. We have previously demonstrated that intraperitoneal administration of MG induced peritoneal fibrosis in Wistar rats, which was reduced in glyoxalase 1 transgenic (GLO1‐Tg) rats. Present study was designed to evaluate the contribution of Epithelial Mesenchymal Transition (EMT) in MG induced peritoneal injury.

Twenty ml of 1 mM MG solution was intraperitoneally administered daily to either GLO1‐Tg rats or control Wistar rats for two weeks. Saline was administered as sham group. At the end of the study, liver was removed for immunohistochemical analysis of the peritoneum.

α‐SMA, TGF‐β and MMP2 expressions in the peritoneum fibrotic region of Wistar rats compared to those of sham group, which was reduced in GLO1‐Tg rats. Neovascularization determined by Von Willebrand factor staining with increased VEGF expression was observed in the peritoneal fibrotic lesion of MG treated Wistar rats compared to sham group, which was reduced in those of GLO1‐Tg rats. Peritoneal GLO1 expression was increased in GLO1‐Tg rats compared to those of Wistar rats.

We conclude that MG induces peritoneal injury with enhanced EMT and neovascularization, which was attenuated by overexpression of GLO1.
Role of the collecting duct insulin receptor in fluid homeostasis in response to high‐ and low‐NaCl diets.
Authors: Li Lijun , Yunghans Allison , Ecelbarger Carolyn
Published: June 2021

Recently we showed reduced ENaC activity and blood pressure (BP) in mice lacking the insulin receptor (IR) in collecting duct (CD) principal cells by aquaporin‐2 promoter targeted Cre‐recombinase knockout (KO). Here we test whether KO mice have impaired ability to maintain homeostasis in response to chronic high‐ (HS, 5% NaCl) or low‐ (LS, 0.02% Na+) sodium diets. After 1‐week feeding, LS KO mice had 20% reduced 24‐hr urine volume, as compared to WT, and significantly reduced ratios of Na+ to Cl− and K+ in urine. Likewise, the Na+ to Cl− ratio was reduced in HS KO (relative to WT). BP (by telemetry) did not change in either genotype with HS diet, but decreased slightly by LS in both genotypes. Four weeks of HS or LS diet resulted in KO mice being modestly, but significantly (p = 0.046) lighter than WT littermates (~10%). HS diet increased kidney weight in both genotypes (~17%). Western blotting was used to evaluate principal cell proteins involved in electrolyte homeostasis. LS diet led to an increase in the α‐subunit of the epithelial sodium channel (ENaC) and the lower (75‐kDa) band of γ‐ENaC; however, band densities did not differ between genotypes. Serum and glucorticoid‐regulated kinase (sgk‐1) was increased by HS, and to a significantly greater extent in the WT mice (p = 0.038). Overall, these studies support a role for the renal CD‐IR in electrolyte homeostasis with chronic adjustments in dietary NaCl. NIH DK082507.
Elucidating the Role of a Renal Proximal Tubule‐Specific Olfactory Receptor
Authors: Shepard Blythe D , Cheval Lydie , Doucet Alain , Pluznick Jennifer L
Published: June 2021

Olfactory receptors (ORs) are seven transmembrane domain G protein‐coupled chemosensors that detect odorants in the nose. We previously reported that OR signaling plays a role in the kidney (PNAS 2009), and to date we have identified 9 renal ORs. One of these is Olfr1393, which is widely expressed in a number of tissues, including kidney (by RT‐PCR screen). Using reverse‐transcribed RNA from microdissected renal segments, we found that Olfr1393 is exclusively expressed in the proximal tubule (PT; S1, S2 and S3). Like the majority of ORs, Olfr1393 is an orphan receptor with no known ligand. To screen for ligands, we cloned this receptor from the kidney and expressed it in HEK293T cells after optimizing conditions to allow for surface expression. We comprehensively screened over 1400 chemicals, and found that Olfr1393 detects cyclic molecules containing either a carbonyl or alcohol group. We also found that when transiently expressed in MDCK cells, Olfr1393 localizes to the apical, not the basolateral PM. To determine the function of this receptor, we generated knockout (KO) mice; the first KO pups were recently born, and they are viable with no overt phenotype. In conclusion, we have shown that Olfr1393 an apically localized, PT‐specific OR which responds to cyclic molecules. Future studies will examine PT function in wild‐type and KO mice to better understand the role that Olfr1393 plays in the kidney.

Funding: NIDDK
Cl− and K+ channels in human pancreatic ductal adenocarcinoma (PDAC) cells
Authors: Sauter Daniel Rafael Peter , Pedersen Stine Falsig , Novak Ivana , Larsen Erik Hviid , Hoffmann Else Kay
Published: June 2021

The Cl‐ channel anoctamin‐1 (ANO1 or TMEM16A) is overexpressed in oral, head, prostate and colon cancer and predicted as a pharmaceutical target or biomarker for distant metastasis. The K2p K+ channel family (e.g. TREKs, TWIKs, and TASKs) is also associated with the pathogenesis of cancer. ANO1 and the K2p channels are involved in regulation of cell migration, cell volume, and apoptosis and might contribute to cancer development through modulating these processes. Here, we aim at determining the roles of ANO1 and K2p channels in pancreatic ductal adenocarcinoma (PDAC). Channel expression levels in 4 human PDAC lines (AsPC‐1, BxPC‐3, PANC‐1, MIAPaCa‐2) were compared to the normal pancreatic ductal epithelial cell line (HPDE*) by qPCR analysis. We found ANO1, TWIK‐1, TREK‐1 and TASK‐3 to be over‐expressed in PDAC cells. Whole‐cell patch‐clamp measurements of ANO1 in AsPC‐1 cells revealed a time‐, voltage‐and temperature dependent Cl‐ current with the instantaneous ICl/V relationship exhibiting GHK‐rectification. ANO1‐like currents were in our experiments not recorded in HPDE cells. We conclude that ANO1 upregulation may be a marker in pancreatic cancer cell lines, however, its function is yet unknown and ongoing studies evaluate the potential roles of ANO1 in PDAC growth and chemotherapy resistance. Financed by FP7 Marie Curie Initial Training Network “IonTraC”.
Sex(y) hormone a‐go‐go: Estrogen–dependent stimulation of hERG potassium channel via G Protein Estrogen Receptor (GPER)
Authors: Gentile Saverio , Lansu Katherine
Published: June 2021

Female gender is an independent risk factor for several channelopathies. Malfunction of the potassium channel encoded by the human ether a‐go‐go related gene 1 (hERG1) which associates with Long QT type 2 (LQT2) syndromes is more prominent in women. In addition, drug‐induced LQT2 appears to fluctuate during different phases of the menstrual cycle. This raised the question that the steroid sex hormone estrogen (E2) could play a major role in regulating the hERG channel. E2 effects on cell targets have been explained by activation of genomic mechanism; however, a non transcriptional regulation of ion channels might be responsible for sex‐dependent susceptibility to LQT. Interestingly, recent studies have demonstrated that some of the E2's effects on breast cancer cells can be mediated by the newly characterized G protein‐coupled E2 receptor, GPER.

In our present study we have characterized a non‐genomic biochemical cascade through which E2 stimulates hERG channel activity. This effect was mediated by GPER and the ser/thr protein kinase PKCalpha via direct phosphorylation of the channel. Ultimately, channel phosphorylation increased hERG membrane stability which determined an augmentation of its current activity. Understanding E2‐dependent regulation of hERG potassium channel can lead to a better understanding of complex physiological mechanisms including heart function and will help to develop better therapeutical strategies for channelopathies.