bluuebunny/crossref_metadata_2025 huggingface dataset:
Methamphetamine‐Induced Changes to Serotonergic Markers in the Frontal Cortex
Authors: Cordie Rebecca , Livermont Tamee , Johansen Andrew , McFadden Lisa M
Published: November 2023

Methamphetamine use is associated with a variety of negative health outcomes including psychosis. Alternations in the frontal cortex including changes in serotonin (5HT) 2A receptors are thought to contribute to psychosis‐like behaviors. Previously we have reported that methamphetamine self‐administration changes serotonin in the frontal cortex of rats. The purpose of this study was to investigate changes in serotonergic markers in the frontal cortex following methamphetamine self‐administration and how these changes relate to hallucination‐like behavior. Male and female rats were allowed to self‐administer either methamphetamine or saline and were sacrificed either one hour after the last self‐administration session or following extinction training. Serotonin transporters (SERT) and 5HT2A receptors were assessed in the frontal cortex via autoradiography. No changes in SERT autoradiography was observed in the orbitofrontal, prelimbic, or infralimbic cortices one hour after self‐administration or after extinction training. However, preliminary findings suggest time dependent changes in 5HT2A receptors in the frontal cortex. One hour after the last self‐administration session 5HT2A receptors were increased in the orbitofrontal cortex. Further, there was a trend towards an increase in prelimbic 5HT2A receptors in rats that self‐administered methamphetamine. However, no changes were found in male rats following extinction training. To assess the functioning of the 5HT2A receptors, the 5HT2A/2C agonist, DOI, was administered in a separate group of animals following extinction training. Preliminary results suggest that rats with a history of METH self‐administration have reduced hyperthermia following the administration of DOI. Efforts are underway to quantify DOI‐induced head twitches which are thought to reflect 5HT2A receptor function in the brain and hallucination‐like behavior. Overall, results of the current study suggest that methamphetamine self‐administration causes time dependent changes in 5HT2A receptors but not SERT in the frontal cortex. Ongoing studies will examine how methamphetamine‐induced changes in the frontal cortex may result in changes in behaviors associated with psychosis such as hallucinations.

Support or Funding Information

Funding for this study was provided the NIH grant DA036012.
Mass Spectrometric Imaging of Brain Lipid Alterations Following Exposure to Cocaine
Authors: Pati Sumitra , Sun Min , Angel Peggi , Drake Richard , Wagner John , Cummings Brian
Published: November 2023

Although much research has focused on characterizing the various mechanisms involved in cocaine‐induced alterations of brain function, few studies have assessed the effect of cocaine exposure on brain lipid metabolism. Interestingly, recent studies have demonstrated relationships between brain lipid alterations and neurological dysfunction, and here we have investigated the long‐term effects of cocaine on lipidomic endpoints in the brain. Mass spectrometric imaging (MSI) of tissues permits structure‐specific lipid analysis, which can provide essential information regarding the localization of lipid remodeling. We utilized MSI to investigate brain lipid alterations in Sprague‐Dawley rats following non‐contingent cocaine administration. Two‐dimensional matrix‐assisted laser desorption/ionization (MALDI) Fourier‐transform ion cyclotron resonance (FT‐ICR) imaging mass spectrometry analysis was performed on sagittal brain sections from saline‐ and cocaine‐treated adult rats. 2‐D MSI indicated specific lipid alterations in neuroanatomical substructures including the hippocampus, striatum, cerebellum, thalamus, and corpus callosum. In particular, cocaine exposure decreased the relative abundance of various phospholipid species such as phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, and sphingolipids. We attempted to identify a potential mechanism mediating these changes by examining the expression of the lipid synthesis protein phosphocholine cytidylyltransferase (CCT), including both isoforms alpha (CCTa) and beta (CCTb), using immunohistochemistry. Little, if any staining of CCTα was detected. In contrast, CCTβ expression was detected in the Purkinje cells of the cerebellum and its expression appeared to be altered following cocaine exposure. These data demonstrate cocaine‐induced brain lipid remodeling in a region‐specific manner through heterogeneous alterations in phospholipid species, and further indicate a region‐specific expression of CCTβ in the rat brain.
Dose Effects of Synthetic Cathinone Mephedrone on Cocaine Reinstatement in Rats
Authors: Saber Iman A , Ward Sara J , Rawls Scott , Walker Ellen A
Published: November 2023

Mephedrone is a recreational synthetic cathinone and a major component of psychoactive ‘bath salts.’ In previous in vitro studies, mephedrone increases dopamine (DA) and serotonin (5‐HT) release and inhibits DA and 5‐HT synaptosomal uptake similar to other abused stimulants.

The purpose of this study was to determine the capacity of mephedrone to reestablish on cocaine self‐administration when a history of cocaine is already established in rats. We predict that rats will readily engage in cocaine‐seeking behavior after the extinction of cocaine self‐administration, when presented with a mephedrone‐induced prime injection. If mephedrone activates the mesolimbic DA system and produces a rewarding effect through DA mechanisms, then mephedrone will reinstate the extinguished cocaine‐seeking behavior in a pattern comparable to cocaine. Male Sprague‐Dawley rats were allowed to self‐administer 0.375 mg/kg/inf of cocaine for two hours for a total of 10–14 days. Following 8–20 days of extinction, rats were reinstated with either i.p. injection of 10 mg/kg cocaine, 1, 5, or 10 mg/kg mephedrone.

After acquisition and extinction, a high dose of 10.0 mg/kg cocaine produced reinstatement of cocaine‐responding. A moderate dose of 5.0 mg/kg mephedrone decreased amount of infusions administered during reinstatement. However, a low and high dose of 1.0 and 10.0 mg/kg mephedrone produced an increased amount of infusions, but not higher than cocaine. In addition, only 4 rats failed to reinstate cocaine responding after cocaine‐prime but 10 rats failed to reinstate after any dose of mephedrone‐prime. These preliminary results suggest that after a history of cocaine self‐administration, cocaine reinstates cocaine responding more fully than mephedrone. This may be due to mephedrone's dual dopaminergic and serotonergic mechanism.
A role for GPR83, a newly deorphanized G protein‐coupled receptor, in stress‐induced neuroimmune modulation
Authors: Lueptow Lindsay Mariah , Gomes Ivone , Fakira Amanda , Devi Lakshmi A
Published: November 2023

GPR83 is a recently deorphanized G‐protein coupled receptor (GPCR) found to be widely expressed throughout the brain and immune system of mice. Analysis of expression reveals high levels in discrete brain regions, such as nucleus accumbens, hypothalamus, amygdala and hippocampus, regions known to be involved in stress modulation, anxiety, depression, and memory. Previous studies have reported that GPR83 is regulated by the stress hormone corticosterone and that GPR83 knockout mice are resistant to stress‐induced anxiety, despite having similar increases in plasma corticosterone as wild type mice. Because stress is known to have a profound effect on immune function, we have recently begun to explore a role for GPR83 in neuroimmune interactions during stress. Using flow cytometry, we have identified the immune cell types that express GPR83. We find GPR83 expression on CD4+ and CD8+ T‐cell populations, as well as a small portion of B cells and natural killer cells. To explore the neuronal pathway that activates GPR83 in these cells, we sought to map the projections from the brain to the spleen and thymus. For this, we injected attenuated pseudorabies virus tagged with GFP into the spleen and thymus and examined viral expression following 72 hrs of retrograde transport. Expression was observed in portions of the reticular formation (notably gigantocellular reticular nucleus), nucleus raphe magnus, dorsal raphe nucleus, and periaqueductal grey. There was strong expression in the paraventricular hypothalamus and in the gray matter cells of the subthalamus, known as the zona incerta. Most interestingly, projections to the central amygdala and anterior insular cortex were also observed. Since many of these brain regions are involved in somatic regulation, fear learning and response to stress, as well as pain modulation, these results are consistent with a role for the GPR83‐PEN receptor system as a mediator of neuroimmune interactions.

Support or Funding Information

Funding: NS026880 and DA019521 to L.A.D and T32 grant DA007135 to L.M.L.
AMPA receptor RNA editing may alleviate the long‐term effects of prenatal stress.
Authors: Bristow Greg , Sodhi Monsheel
Published: November 2023

Background

A post‐transcriptional process known as RNA editing regulates the trafficking and function of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid glutamate receptors (AMPARs), which are critical for synaptic plasticity, learning and memory. These processes are impaired in psychiatric disorders that are exacerbated by stress, such as schizophrenia. RNA editing of AMPARs has been associated with several neurological disorders, including epilepsy and schizophrenia.

Methods

We exposed mice to environmental stress during gestation i.e. prenatal stress (PRS). In adulthood, mice were treated with haloperidol (1mg/kg), clozapine (5mg/kg) or saline twice daily for 5 days. 16hrs after the final injection, mice were assessed for social interaction behavior and locomotor activity. The hippocampus of each mouse was analyzed for the expression of the RNA editing enzymes (ADARs 1–3) in addition to tagged next‐generation sequencing analysis of the RNA editing levels of 24 genes, including the AMPA glutamate receptor subunits.

Results

PRS mice exhibited reduced social interaction behavior. This abnormal behavior was associated with reduced hippocampal RNA editing of several ADAR targets, including the AMPAR subunits GluA2‐4, the potassium channel Kv1.1, and the serotonin2C receptor (5‐HT2CR). Treatment of the PRS mice with clozapine, but not haloperidol, eliminated abnormal social interaction, along with elimination of abnormal GluA2 RNA editing. Measurement of the RNA editing enzymes revealed increased ADAR3 expression in the clozapine‐treated PRS mice, indicating that PRS and clozapine both alter transcriptional pathways associated with RNA editing in the hippocampus.

Conclusions

Our data show that PRS produces long‐term changes in behavior that may be due to abnormal development of the hippocampus. Social interaction behavior was correlated with the hippocampal RNA editing of several ADAR targets. Only GluA2 RNA editing appeared to be ‘normalized’ by clozapine treatment, along with the elimination of abnormal social interaction in PRS mice. Therefore the long‐term behavioral effects of prenatal stress may be alleviated by clozapine through a molecular pathway involving GluA2 RNA editing.

Support or Funding Information

Funded by a Hans W. Vahlteich Scholar Award (MS), the Chicago Biomedical Consortium and a University of Illinois Centre for Drug Discovery seed grant (MS). Work completed in collaboration with Dr. Erbo Dong, Dr. Alessandro Guidotti (UIC), Dr. Evelyn Nwabuisi‐Heath and Michelle Vierra.
GENDER DIFFERENCES IN THE INTERACTION OF CATHINONE WITH CAFFEINE ON LOCOMOTOR ACTIVITY IN THE RAT
Authors: Docherty Jim , Alsufyani Hadeel A.
Published: November 2023

Leaves of Khat (Catha edulis Forsk) contain the β‐keto‐amphetamine cathinone that has MDMA‐like actions (Kalix, 1990). The actions of cathinone are largely indirect by release of monoamines to act both centrally and peripherally (Alsufyani & Docherty, 2015). Gender differences occur in the use and effects of stimulants, including Khat, in humans, but in basic research, female animal models have been rarely studied. We have investigated gender differences in the effects of cathinone (the major stimulant from Khat) and the interaction with caffeine on locomotor activity in Wistar rats. Indeed, Khat users often combine coffee with the stimulant. Animals were implanted with telemetry probes under isoflurane anaesthesia, and 7 days later, locomotor activity was recorded in conscious unrestrained animals. Following an equilibration period, sampling was commenced and at 5 min, caffeine (10 mg/kg) or vehicle, and 30 min later, cathinone (5 mg/kg) or vehicle were injected subcutaneously in male and female rats. Recording continued for a further 240 min. Time course of effects was investigated at 5 min intervals. Caffeine alone produced only small increases in total locomotor activity, but even these small effects were significantly greater in female rats (one way Anova of total activity over the whole sampling period, P<0.05). Cathinone produced significant and marked increases in locomotor activity, beginning soon after injection, and the response to cathinone was significantly greater in female animals (two way Anova of time course of effect, P<0.05). The combination of caffeine and cathinone causes a transient potentiation, followed by a prolonged inhibition, of the locomotor response to cathinone. Hence, the peak response to injection of cathinone was significantly increased, but for much of the next 120 min, the response to cathinone was significantly reduced by the presence of caffeine, especially in female rats. In conclusion, cathinone causes greater increases in locomotor activity in female than in male rats, but although the acute effects of cathinone are potentiated by caffeine, the maintained effects of cathinone on locomotor activity are, perhaps surprisingly, reduced by caffeine in both male and female rats. These differences highlight the need to carry out gender studies of the actions and interactions of stimulants in basic studies.

Alsufyani HA, Docherty JR (2015). Eur J Pharmacol 758: 142–146 Kalix P (1990) Pharmacol Ther, 48:397–416.

Support or Funding Information

HAA is funded by a scholarship from the Saudi Government Ministry of Higher Education, KAU.
The role of estradiol on social stress induced depressive‐like behavior and neuroinflammation in females
Authors: Finnell Julie E , Lombard Calliandra M , Moffitt Casey M , Padi Akhila R , Wood Christopher S , Wilson Marlene A , Wood Susan K
Published: November 2023

It has long been established that repeated exposure to social stress can lead to the development of psychosocial disorders, such as depression. Strikingly, women are more sensitive to stress‐related disorders, such as depression compared to men. This enhanced stress sensitivity in females begins at the onset of puberty and ends during menopause, suggesting that ovarian hormones could be a large contributing factor to this phenomenon. However, the mechanism by which ovarian hormones could be driving this enhanced stress sensitivity is unknown. Using a modified social defeat paradigm which consists of a rat bearing witness to an aggressive social defeat encounter, we have previously shown that witness stress produces depressive‐like anhedonia and enhanced pressor and tachycardic responses selectively in intact cycling females while females that are ovariectomized (OVX) are resistant, making this an ideal model to study mechanisms of stress susceptibility in females. We recently demonstrated that neuroinflammation is crucial in the development of depressive‐like anhedonia in socially stressed males. The purpose of this study was to determine the effects of estradiol on social stress‐induced neuroinflammation within the central amygdala (CeA) and locus coeruleus (LC) of witness stressed females. Study 1 determined the differential neuroinflammatory consequences of witness stress in intact cycling vs. OVX females. Much like our behavioral data suggests, intact cycling females show enhanced neuroinflammation in the CeA compared to OVX females. Interestingly, neuroinflammation was not elevated in the LC in response to witness stress, but rather OVX females demonstrated stress‐induced reductions of IL‐1β within the LC vs. controls. In order to determine the discrete role of estradiol in these neuroinflammatory responses to stress, study 2 determined cytokine levels in OVX females that received estradiol or placebo replacement. While estradiol replacement did not result in the emergence of stress‐induced depressive‐like anhedonia, it did promote anxiety‐like behavior as evidenced by increased burying during witness stress exposure. Based on this enhanced anxiety‐like response, we predict concomitant increases in neuroinflammation within the CeA, a brain region known to contribute to behavioral stress responses. Therefore, the current study suggests that ovarian hormones, potentially estrogen, may mediate enhanced stress sensitivity in females by promoting stress‐induced inflammation in critical brain regions involved in the behavioral stress response, providing novel therapeutic targets to study female stress resiliency and susceptibility.

Support or Funding Information

ASPIRE18080‐16‐41861, 15SDG22430017, and P20GM103641
The Role of Estradiol in the Behavioral and Cardiovascular Consequences of Social Stress in Females
Authors: Padi Akhila R , Finnell Julie E , Lombard Calliandra M , Wilson Marlene A , Wilson L Britt , Wood Susan K
Published: November 2023

Repeated exposure to social stress results in the emergence of psychosocial disorders, which significantly increases the risk of cardiac morbidity. Depressed women are at a greater risk for the development of comorbid cardiovascular disease in comparison to depressed men. Furthermore, increased risk in females begins during puberty and ends following menopause, suggesting that ovarian hormones play a role. In this study, female rats were subjected to witnessing a social defeat encounter between an aggressive male resident and a male intruder. Female rats were implanted with a cardiovascular telemetry device and either ovariectomized (OVX) or sham OVX (intact). In study 1, stress‐induced cardiovascular consequences were measured in female intact vs OVX witness rats compared with controls. In study 2, all females were OVX and replaced with estradiol (E) or vehicle (V). Each witness was paired with an intruder, and placed behind a plexi‐glass partition in the resident cage for the 5 daily 15 min intruder defeat exposures. Cardiovascular telemetry from Study 1 indicated higher heart rate, blood pressure, and the occurrence of premature ventricular contractions (PVCs) and behavioral anhedonia during stress in intact female witnesses compared to OVX witness and controls. This suggests that ovarian hormones increase sensitivity to stress‐induced cardiac dysfunction and stress‐induced depressive‐like behavior. Study 2 identified a higher presence of the same cardiac dysfunction factors during stress in OVX+E witnesses versus OVX+V witnesses. This demonstrates that E plays a role in the increased vulnerability to stress‐induced cardiac and behavioral dysfunction. Ongoing studies are assessing the mechanism whereby, when combined with stress, E increases susceptibility to cardiac dysfunction through analyses of heart rate variability. These findings recapitulate the increased vulnerability to cardiovascular dysfunction in an intact, cycling female population that suffers from comorbid depressive disorders. These studies seek to identify why females who undergo stress are more vulnerable to the development of co‐morbid depression and cardiovascular disease, thereby allowing for the development of novel targeted therapeutics.

Support or Funding Information

ASPIRE18080‐16‐41861, 15SDG22430017, and P20GM103641
The Timing of Weaning in Rats Has a Long‐Lasting Impact on the Vulnerability to Stress‐Induced Gastric Erosion in Both Sexes
Authors: Filaretova Ludmila , Vataeva Ludmila , Zelena Dora
Published: November 2023

Weaning is actually a maternal separation, characterized by maternal reduction of resources, therefore is both psychologically and energetically stressful to the offspring. Weaning is an important period of life and its timing may influence the resilence for later stress. One of the most important stress‐related disorder is gastric ulceration. Weaning is actually a maternal separation, characterized by maternal reduction of resources, therefore is both psychologically and energetically stressful to the offspring. Therefore we aimed to investigate the sensitivity of gastric mucosa to cold (at 14°C) water immersion stress (WIS for 3h) in adult (75‐day‐old) female and male rats after weaning them at different timepoints (at 17, 21, 30, 36 or 42 postnatal days). The connection with stress was studied by comparing control groups to those underwent WIS at the time of weaning and measuring corticosterone levels at the time of collecting the stomach samples. The timing of weaning has strong impact on all studied parameters. Stress‐induced erosion development was the smallest in rats weaned at 36‐day independently from preconditioning with WIS at weaning, or sex, despite a clear sex‐effect on blood corticosterone levels and body weight. WIS at weaning influenced only the body weight in adult rats weaned at 30‐day, being higher in stressed than in control groups. Erosion area significantly correlated with blood corticosterone level at weaning non‐stressed females. Taken together our results confirm that the timing of weaning has long‐lasting impact on the resiliance of gastric mucosa to ulcerogenic stressful events. The postnatal day 30–36 seems to be the optimal for weaning as both earlier and later weaning increased vulnerability. In conclusion, in contrast to the general practice, weaning at the postnatal day 30–36 would be optimal for rats leading to maximal mucosal resistance. Additional stress at weaning does not make too much difference.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790. (Filaretova L.: who was responsible for general experimental design, performing experiments in adult rats and analysis of the results).
Altered Kisspeptin Responsiveness in Bmal1 Knockout Females
Authors: Tonsfeldt Karen J , Schoeller Erica , Brusman Liza , Mellon Pamela
Published: November 2023

The use of the hypothalamic peptide kisspeptin is a novel treatment for hypothalamic infertility. The preovulatory luteinizing hormone (LH) surge occurs in the hypothalamic‐pituitary‐gonadal axis to initiate ovulation. Estrogen primes kisspeptin (Kiss1) neurons in the anteroventral periventricular nucleus (AVPV) to secrete kisspeptin, which potently activates gonadotropin‐releasing hormone (GnRH) neurons to release a pulse of GnRH, eliciting a surge of LH to prompt ovulation. A critical component of this preovulatory surge is temporal gating. Knockout of Bmal1 ‐ a critical component of the molecular circadian clock ‐ produces arrhythmic and infertile animals. Female Bmal1 KOs show no detectable LH surge but normal pituitary responsiveness, suggesting hypothalamic dysfunction. Our study sought to characterize neuroendocrine function in these mice, particularly responsiveness to kisspeptin. Corroborating previous studies, we found no difference in the LH response to 1 μg/kg GnRH 10 minutes after administration between wildtype (WT) and Bmal1 KOs (LH increase of 2.61 ± 0.25 ng/μl vs 2.51 ± 0.50 ng/μl, n=6). Interestingly, Bmal1 KO female mice had a significantly increased response to 30 nmole kisspeptin after 10 minutes (LH increase of 1.19 ± 0.34 ng/μl vs. 3.08 ± 0.56 ng/μl, n = 6, p<0.05). In addition to having an increased LH response to a kisspeptin challenge, the knockout mice showed greater area under the curve in response to kisspeptin during a 45‐minute time course (38.99 ± 4.69 vs 163.30 ± 16.88 ng/min/μl, n=3–4, p<0.005). A dose response curve indicated no difference in ED50 (0.144 ± 0.048 vs. 0.085 ± 0.048 mg/kg, n=4) despite the increased maximal effect, suggesting kisspeptin is more efficacious in eliciting LH in Bmal1 knockout females. Since the sex‐specific expression of Kiss1 in the AVPV is critical for regulating ovulation in females, we hypothesized that this population may be absent in Bmal1 KO females. However, we found no differences in Kiss1 mRNA expression between WT and Bmal1 KO females (1.08 ± 0.25 vs 1.03±0.29‐fold change, n=4). Overall, our results suggest disruption of the hypothalamic and pituitary regulation of fertility in the Bmal1 KO females, specifically in responsiveness to kisspeptin. Our findings suggest that circadian cues are important for regulating the temporal release of hypothalamic peptides that regulate fertility, and these findings may help direct kisspeptin treatment in women with circadian‐derived hypothalamic amenorrhea.

Support or Funding Information

NIH R01 HD072754, R01 HD082567, P50 HD012303, T32 HD007203
Increased α‐tubulin Acetylation Translocates Gαs out of Lipid‐rafts: a Novel Mechanism for Antidepressant Effects of HDAC‐6 Inhibitors
Authors: Singh Harinder M , Schappi Jeffrey , Rasenick Mark M
Published: November 2023

Histone deacetylase‐6 (HDAC‐6) enzymes deacetylate α‐tubulin and have been shown to be upregulated during neuropsychiatric disorders. HDAC6 knockout or HDAC6 inhibitors displayed an antidepressant profile in animal models. While, a possible role for HDAC6 inhibitors in treatment of depression exists, the potential mechanism for this remains elusive. Previously, our group has shown that treatment of rats or C6 glioma cells with several classes of antidepressants result in facilitation of the G‐protein, Gαs, activity by translocating it from lipid‐rafts inducing a sustained elevation in cAMP production. Studies have also demonstrated interplay between tubulin and Gαs in lipid‐rafts. Once freed from lipid‐raft domains, Gαs, couples with adenylyl cyclase (AC). Although Gαs interacts directly with tubulin to modify microtubule dynamics, tubulin also acts as an anchor for Gαs in lipid rafts. Based on HDAC‐6 roles in tubulin deacetylation and our data showing Gαs complexes with tubulin in lipid‐rafts, we hypothesized that acetylation of α‐tubulin disrupts tubulin‐Gαs anchoring, rendering Gαs free to activate AC. To test this, C6 glial cells were treated with the HDAC‐6 inhibitor, tubastatin‐A. Three‐day treatment with tubastatin‐A not only increased acetylation of α‐tubulin but also caused translocation of Gαs out of lipid‐rafts. Fluorescence Recovery After Photobleaching (FRAP) on C6 cells stably expressing GFP‐Gαs, was conducted and cells treated for three days with tubastatin‐A showed an “antidepressant signature”. Finally, sustained elevation of cAMP was revealed by increased cAMP response element binding protein (CREB) phosphorylation and increased expression of brain derived neurotrophic factor (BDNF). An increase in acetylated α‐tubulin prevented the formation of tubulin‐Gαs‐complexes in lipid rafts. This did not appear to be a mechanism shared with other classes of antidepressants. These findings suggest HDAC6 inhibition shows incomplete commonality with traditional antidepressants. Nonetheless, it is possible that compounds that decrease tubulin‐Gαs interactions by increasing acetylation of a α‐tubulin may show promise for antidepressant therapy.

Support or Funding Information

NIH R01 AT 009169 (MMR) VA BX 001149 (MMR) AHA Postdoctoral Fellowship (HS)

NIH T32 MH0667631 (JS)
High Concentration of Rosiglitazone Reduces Low‐density Lipoprotein Receptor‐Related Protein‐1 (LRP1) Levels Via Lysosomal Degradation in HepG2 Cells
Authors: Rondón‐Ortiz Alejandro Nico , Gugnani Kuljeet , Maher Timothy J. , Pino‐Figueroa Alejandro
Published: November 2023

LRP1 is a transmembrane receptor involved in the internalization of molecules by either transcellular transport or lysosomal degradation via clathrin. PPARγ is a nuclear receptor that transcribes several target genes involved in cell differentiation, lipid metabolism and glucose homeostasis. PPARγ activation has been shown to upregulate LRP1 mRNA and protein levels in HepG2 cells. However, overactivation of this nuclear receptor has exhibited a reduction in LRP1 protein levels. The aim of this research was to evaluate the effect of rosiglitazone (RGZ), a known PPARγ agonist, on LRP1 protein levels in HepG2 cells. Additionally, the LRP1 protein reduction mechanism in RGZ treated‐cells studying PPARγ activation, ubiquitin proteasome system and lysosomal degradation were evaluated.

HepG2 cells were treated with RGZ (0.003 – 30 μM) to show no increase on LRP1 protein levels as reported in the literature. However, cells incubated for 24 and 48 hours with 30 μM of RGZ reduced LRP1 protein levels by 40 and 90%, respectively. PPARγ antagonism and ubiquitin proteasome system inhibition were studied in RGZ treated‐cells demonstrating no effect on LRP1 reduction. Bafilomycin A1, a lysosome degradation inhibitor, increased LRP1 protein levels up to 4‐fold in RGZ treated‐cells.

These results suggest that overactivation of PPARγ reduces LRP1 protein levels via lysosome degradation. Further experiments are required to evaluate the mechanism involved in the effect of RGZ on LRP1 protein expression.
Involvement of KCa2.3 Channels in Relaxation of Erectile Tissue Is Altered in Type 2 Diabetic Mice
Authors: Steffensen Simon Gabriel Comerma , Mogensen Susie , Beck Lilliana , Simonsen Ulf
Published: November 2023

Introduction

Activation of endothelial small conductance calcium‐activated K+ channels (KCa2.3 or SK3) and intermediate conductance calcium‐activated K+ channels (IK or KCa3.1) leads to vascular relaxation. Our previous studies have shown that KCa2.3 down‐regulation diminishes the normal erectile function; this channel it is the most expressed KCa channels in the endothelium of corpus cavernosum. In the present study we hypothesized that KCa2.3 channel function is altered in erectile tissue of type 2 diabetic animals.

Materials and Methods

Erectile function was measured in diabetic db/db mice was compared to heterozygous control animals. Corpus cavernosum strips were mounted for isometric tension recording and processed for qPCR or Western Blot.

Results

In anesthetized diabetic db/db mice erectile function was diminished compared to control animals. In isolated corpus cavernosum strips from db/db mice, concentration‐dependent contractions to noradrenaline were incremented compared to strips from control mice. Apamin, a blocker of KCa2 channels blocked acetylcholine relaxation in corpus cavernosum from db/db and control animals, being less effective in db/db compared to control animals. NS309 (0.5 μM), an activator of KCa2 and KCa3.1 channels, potentiated acetylcholine concentration‐dependent relaxations in corpus cavernosum from control, but this was not the case in corpus cavernosum from db/db mice. SNP relaxation was similar in corpus cavernosum from db/db and control animals. KCa2.3 gene and protein expression was incremented in corpus cavernosum from db/db mice, nevertheless protein expressed in the aorta was diminished.

Conclusions

Our results suggest that in type 2 diabetes KCa2.3 channel expression is down‐regulated in the systemic circulation, and upregulated in erectile tissue. In erectile tissue noradrenaline contraction is increased and the effect of NS309 reduced which may be related to impaired KCa2.3 channel function. These changes may contribute to impairment of erectile function in diabetes.

Support or Funding Information

We acknowledge Aarhus University, Novo Nordisk and Consejo de Desarrollo Cientifico y Humanistico de la Universidad Central de Venezuela (CDCH‐UCV) for their support.
Thiol‐Oxidation Impairs Alpha2‐Adrenergic Receptor Mediated Inhibition of N‐type Ca2+ channels and Results in Receptor Mislocalization
Authors: Mui Ryan K , Fernandes Roxanne N , Feng Huijie , Galligan James J
Published: November 2023

Introduction

Oxidative stress and sympathetic hyperactivity contribute to hypertension. In sympathetic neurons, activation of alpha2‐adrenergic receptors (α2AR) decreases nerve terminal release of norepinephrine (NE) by activating Gαi/o proteins to inhibit N‐type voltage‐gated Ca2+channels. We have shown in DOCA‐salt hypertensive rats that α2AR‐mediated inhibition of Ca2+channels is impaired in sympathetic neurons, resulting in increased NE release. Furthermore, blood pressure and α2AR function are preserved following treatment with clodronate (macrophage depletion agent) and apocynin (NADPH oxidase inhibitor). Oxidative stress is associated with adrenergic dysfunction and Gαi/o are targets of oxidative stress. Inhibition of α2AR‐Gαi/o coupling following pertussis toxin (PTX) treatment occurs due to ADP‐ribosylation on the C‐terminal cysteine (position 351) of Gαi/o. It is not known if thiol‐oxidation of this residue results in similar uncoupling. Therefore, we tested the hypothesis that oxidizing agents impair α2‐AR function by thiol‐oxidation of Cys351 on Gαi/o.

Methods

These studies used cultured sympathetic neurons, G1A1 cells (HEK cells stably expressing N‐type Ca2+ channels), and HEK 293T cells. Cell lines were co‐transfected with α2AR and either WT‐Gαo or PTX‐insensitive Gαo (PTXi‐Gαo, Cys351Gly). Oxidative stress was induced with H2O2, t‐BOOH, or diamide (thiol‐specific oxidant). Whole cell patch clamp was used to detect inhibition of Ca2+ current using NE to activate α2AR and GppNHp to inhibit Ca2+ current independent of α2AR activation. Immunohistochemistry was used to localize α2AR and Gαo following oxidant treatment before and after activation of α2AR with NE.

Results

In sympathetic neurons, α2AR‐mediated inhibition of Ca2+ current was reduced following H2O2, t‐BOOH or diamide treatment vs. untreated neurons. GppNHp inhibited Ca2+ current equally in all neurons independent of oxidative stress. Oxidative stress impaired α2AR‐mediated inhibition of Ca2+ current similarly in G1A1 cells co‐transfected with WT‐Gαo or PTXi‐Gαo. Oxidative stress had no effect on cell capacitance or Ca2+ channel activity in neurons and G1A1 cells. In HEK 293T cells, oxidative stress did not affect co‐localization of α2AR and Gαo, however, oxidative stress resulted in greater cytoplasmic localization of α2AR.

Conclusions

Thiol‐oxidation results in impaired α2AR‐mediated inhibition of Ca2+ current. Thiol‐oxidation does not affect Gαo coupling to α2AR. Dysfunction may be attributed to thiol‐oxidation of cysteine residues on α2AR, which do not affect Gαo coupling, but may alter structural motifs that could affect receptor localization to the cell membrane.

Support or Funding Information

Supported by NIH 2P01HL070687 and American Heart Association (15PRE25090218)
Ellagic Acid Mediated Attenuation of Store Operated Calcium Entry Alters Cytokine Expression in Jurkat T Cells
Authors: Murphy Matthew , Quin Xia , Kaul Shashank , Mathias Clinton , Thomas David , Bose Diptiman
Published: November 2023

Calcium (Ca2+) is a ubiquitous second messenger regulating a myriad of cellular processes including proliferation, gene expression, and apoptosis. Agents that can modulate Ca2+ homeostasis are being extensively explored as potential anti‐inflammatory agents. Ellagic acid (EA), a polyphenolic compound found in many fruits and plant extracts, has been known to possess anti‐inflammatory properties. EA is thought to modulate Ca2+ homeostasis by enhancing sarcoplasmic endoplasmic reticulum ATPase pump (SERCA) activity, however effects of EA on Ca2+ signaling is not clearly understood. In order to study this relationship, we measured changes in Ca2+ transients in Jurkat T‐cells pretreated with EA (0–60 μM, 12 h). EA‐treated Jurkat T‐cells were challenged with SERCA inhibitor, thasipargin (TG) (2 μM). The amplitude of TG‐induced Ca2+ transient in the EA‐treated cells was 30% lower than vehicle control cells. The depletion of ER‐stores signals activation of store‐operated Ca2+ entry (SOCE) channels, resulting in Ca2+ influx. EA‐pretreatment induced a concentration‐dependent attenuation in SOCE‐mediated Ca2+ influx in Jurkat T‐cells. EA pretreatment also resulted in disruption of puncta formation in HEK293 cells overexpressing ORAI1 channels. The expression of certain cytokines is directly dependent on SOCE mediated Ca2+ influx. We demonstrated that EA‐treatment resulted in a significant decrease in IL‐2 and INFγ expression. Taken together, these results indicate that the anti‐inflammatory effects of EA could be attributed to its ability to attenuate store operated Ca2+ influx.
ADP Ribosylation Factor 6 (Arf6) Modulates the Cell Surface Expression of the Cardiac Acetylcholine‐Activated Inward Rectifier Potassium Current (IKACh) Proteins
Authors: Alhadidy Mohammed M , Reiser Michelle , Szekeres Charles , Cha Byeong , Noujaim Sami F.
Published: November 2023

Introduction

It has been suggested that the ADP ribosylation factor 6 (Arf6) plays a role in the trafficking of Kir3.1 and Kir3.4 proteins ‐ the molecular correlates of the cardiac acetylcholine‐activated inward rectifier potassium current (IKACh). However, further details need to be elucidated. Therefore, we investigated the effects of Arf6 activity on the localization of Kir3.1 and Kir3.4 proteins using flow cytometry, Arf6 activation pull‐down assay, and live confocal microscopy. We tested the hypothesis that Arf6 cycling between the GDP‐ and GTP‐bound forms modulates the cell surface expression of IKACh proteins.

Methods

Studies were performed in a human embryonic kidney (HEK293) cell line stably expressing Kir3.1 and Kir3.4 (GIRK cells). The IKACh proteins at the cell surface were labelled with TertiapinQ‐ATTO‐488 and quantified using flow cytometry in GIRK cells transiently transfected with Arf6WT (wild type), Arf6T27N (dominant negative, GDP‐bound form), or Arf6Q67L (constitutively active, GTP‐bound form). Additionally, GIRK cells were treated for 30 minutes with 10 μM Chloroquine or Primaquine, then cell surface IKACh proteins were quantified using flow cytometry, and their internalization was studied using live confocal microscopy. Arf6 activation by Chloroquine was assayed using purified Arf6 protein and human atrial tissue samples in pull‐down experiments. Results are reported as average ± standard error.

Results

In flow cytometry experiments, both Arf6T27N (dominant negative) and Arf6Q67L (constitutively active) showed significant increase in IKACh proteins expression at the cell surface compared to Arf6WT (Arf6T27N, 14% ± 3, p<0.01; Arf6Q67L, 28.5% ± 4, p<0.01). This suggested that interference with Arf6 cycling between the GDP‐ and GTP‐bound forms leads to cell surface accumulation of the IKACh proteins. Moreover, treatment of GIRK cells with 10 μM Chloroquine for 30 minutes significantly decreased cell surface IKACh proteins (8% ± 1.7, p<0.01). Conversely, treatment with Primaquine, a structurally similar 4‐aminoquinoline, did not show the same effect. In live confocal microscopy studies, Chloroquine promoted the internalization of IKACh proteins compared to control. Arf6 activation pull‐down experiments using purified Arf6 protein and human atrial tissue samples suggested that Chloroquine, but not Primaquine, increased Arf6 activity.

Conclusion

Expression of the IKACh proteins at the cell surface is, in part, modulated by an Arf6‐dependent pathway. Constitutive interference with Arf6 cycling between GDP‐ and GTP‐bound forms increases the cell surface density of IKACh proteins, while transient pharmacological activation of Arf6 could lead to their internalization.

Support or Funding Information

Funded in part by NIH Grant R01HL129136 to SFN
Pannexin‐1 Channel Activity and Associated ATP Release in Podocytes in Response to Adipokines
Authors: Li Guangbi , Zhang Qinghua , Li Ningjun , Ritter Joseph K. , Li Pin‐Lan
Published: November 2023

Pannexin‐1 (PANX1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether PANX1 channel is present in podocytes and thereby mediates ATP release leading to glomerular inflammation or fibrosis. In the present study, we characterized the PANX1 channel in podocytes and examined the possibility of this channel in mediating ATP release induced by adipokines. By real time RT‐PCR, Western blot analysis and immunohistochemistry, we demonstrated that among 3 different pannexins, PANX1 is mostly enriched in podocytes either cultured or in situ in mouse glomeruli. Using patch clamp technique, we recorded a large voltage‐gated outward current under the Cs+in/Na+out gradient, which was 290.2±8.7 pA at +80 mV. Na‐gluconate or Na‐asparate substitution for NaCl in the bath solution blocked voltage‐gated outward currents and shifted the reversal potential of PANX1 currents to right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage‐gated outward currents were substantially attenuate by either carbenoxolone (CBX) or probenecid (PBNC), the specific PANX1 channel inhibitors (to 23.8±2.5% and 21.4±1.7% of control, respectively). Given the stimulatory role of extracellular ATP in inflammation and chronic low‐grade inflammation as a hallmark of obesity, we tested whether adipokines from fat cells activate PANX1 channel activity to release ATP. Adipokine, visfatin was shown to remarkably enhance pannexin‐1 channel activity in podocytes (to 252.5±7.7% of control), which was completely abolished by CBX. However, another adipokine, adiponectin significantly blocked pannexin‐1 channel activity to 29.9±4.4% of control, a level similar to the effects of CBX and PBNC. Correspondingly, visfatin significantly increased ATP release from podocytes by almost 2 folds, which was prevented by CBX, PBNC or adiponectin. These results suggest that PANX1 functions as an anion channel in podocytes to control ATP release, which is regulated by adipokines.

Support or Funding Information

This study is supported by NIH grants DK54927, DK102539, HL057244, HL075316, and HL122937.
Effects of phosphodefective FFAR4 C‐terminal mutants on COX‐2 expression in macrophages
Authors: Cheshmehkani Ameneh , Moniri Nader H.
Published: November 2023

Macrophages have critical roles in the initiating, maintaining, and resolving inflammation, and mediate this response partially through expression of cyclooxygenase‐2 (COX‐2), which generates prostaglandins (PG). Free‐fatty acid receptor‐4 (FFAR4), a G protein‐coupled receptor that was previously termed GPR120, has been shown to play important roles in regulating macrophage function to produce robust anti‐inflammatory effects. In this regard, agonism of FFAR4 with docosahexaenoic acid (DHA) has previously been shown to reduce expression of COX‐2 and subsequent PGE2 synthesis. Our previous work, as well as that of others, has shown that FFAR4 signaling is regulated by phosphorylation of the receptor on its C‐terminal tail and that mutation of these phosphorylation sites diminishes the receptors ability to interact with β‐arrestin partner proteins. The goal of the current study was to assess the role of the C‐terminal tail of FFAR4 in regulating COX2 expression and PGE2 synthesis. Here, we have created C‐terminal truncated mutants of both the short (FFAR4‐S) and long (FFAR4‐L) isoforms of FFAR4 and stably expressed them in murine RAW264.7 macrophages. Our results show that stable expression of wild‐type (WT) FFAR4‐S in RAW cells significantly reduces COX2 expression, while expression of the C‐terminal truncated mutant of FFAR4‐S does not affect COX2 expression compared to control RAW macrophages. Meanwhile, stable expression of WT‐FFAR4‐L, which has previously been shown to be intrinsically biased solely towards β‐arrestin signaling, also resulted in reduced COX‐2 expression, while expression of the truncated FFAR4‐L mutant reversed this effect. These results suggest that C‐terminal phosphorylation and subsequent β‐arrestin recruitment are integral in FFAR4‐mediated alteration of COX2 expression in macrophages.

Support or Funding Information

Supported by NIH/NIDDK grant DK098730 to N.H.M.
Apelin‐13 inhibits lipid accumulation in human hepatocytes
Authors: Park Soojin , Huang Jin , Im Dongsoon
Published: November 2023

Background

Apelin is an adipokine and acts through a G protein‐coupled receptor named APJ. Its protective function on lipid accumulation in adipocytes has been proven. However, functions of apelin in hepatocytes have not been investigated.

METHODS

In order to assess functional role of apelin and APJ in hepatocytes, we used an in vitro model of liver X receptor‐mediated hepatocellular steatosis. In human hepatoma cells, APJ expression was confirmed.

RESULTS

A specific liver X receptor activator induced lipid accumulation and apelin‐13 inhibited the lipid accumulation. Apelin‐13‐induced inhibition was ameliorated by treatment of F13A, a specific APJ antagonist. Furthermore, silencing of APJ by siRNA transfection also inhibited apelin‐13 action. Liver X receptor activator induced expression of SREBP‐1c, a key transcription factor for lipogenesis. Apelin‐13 inhibited the SREBP‐1c induction and the inhibition was ameliorated by F13A in the hepatocytes. Treatment of pertussis toxin, LY294002, U0126, and SB202190 blocked apelin‐13‐induced inhibition of lipid accumulation, suggesting involvement of Gi/o proteins, PI‐3‐kinase, and MAPK kinases.

CONCLUSION

Therefore, the present data suggest that apelin may function to protect lipid accumulation in the liver.
Effects of Pentadecapeptide BPC 157 on Experimental Rat Model of Dry Eye
Authors: Kralj Tamara , Kokot Antonio , Kasnik Katarina , Drmic Domagoj , Zlatar Mirna , Seiwerth Sven , Sikiric Predrag
Published: November 2023

Aim

We hypothesize the stable gastric pentadecapeptide BPC 157 as a therapy of dry eye syndrome in rats. BPC 157 already cures ocular tissues. BPC 157 eye drops (ug‐ or ng‐regimen) rescued total debridement of corneal epithelium (Coll Antropol 2005;29(1):321–5), successfully closed perforating corneal incisions in rats (Exp Eye Res 2015;136:9–15), counteracted particular NO‐specific parallel miotic effect of L‐NAME and L‐arginine and atropine‐induced mydriasis in rats and guinea pigs (Eur J Pharmacol 2016;771:211–9). In addition, BPC 157 counteracts various ulcerative lesions in skin and gastrointestinal tract, and BPC 157/NO‐relationship is established in various experimental models and species (Curr Pharm Des 2014;20(7):1126–35), providing that it would counteract the damaging effects of lacrimal gland extirpation, and dry eye syndrome. Dry eye syndrome is a relatively common disease with multifactorial causes, which may cause simptoms variating from discomfort to blindness. This study aimed to evaluate the effect of BPC 157 on the rat dry eye model.

Methods

We used male Albino Wistar rats, 200g, randomly divided. After application of Ketmine‐HCl 50–60 mg/kg + Xylazine‐HCl 5–10 mg/kg, Tetrakain 0,5% drops, left extraorbital lacrimal gland was isolated from surrounding tissues and surgically removed. Rats received stable gastric pentadecapeptide BPC 157 (Diagen, Slovenia) topically (5 drops (2 μg/ml)) or an equivolume of destilled water (controls). Corneal defects were analyzed with microcamera observation using blue light after 0.5% fluorescein eye drop instillation. These examinations were performed in both treated and control eyes at baseline and on weeks 1, 2 and 4.

Results

Pentadecapeptide BPC 157 administered topically significantly reduced clinical signs of dry eye by decreasing corneal defect in all treated groups (Figure 1), while in control groups corneal defect persisted (Figure 2)

Conclusion

BPC 157 was originally an anti‐ulcer peptide used in trials for ulcerative colitis and now is in trials for the treatment of multiple sclerosis (Curr Pharm Des 2014;20(7):1126–35). Now, we demonstrated that pentadecapeptide BPC 157 counteracts dry eye lesions in rats.

Support or Funding Information

Univerity of Zagreb, Zagreb, Croatia (Grant number BM099)