bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Chronic Administration of The GABAB Receptor Agonist Baclofen on Spontaneous And Cocaine‐Induced Behavior in Rats.
Authors: CARMO GAIL PEREIRA DO , KING GEORGE
Published: June 2021

There is increasing evidence that the GABAB receptor agonist baclofen may have potential as a therapeutic agent in cocaine abuse. Research demonstrates that baclofen effectively reduces the stimulant and reinforcing effects of cocaine. However, the effects of chronic baclofen exposure on cocaine‐induced behaviors have not been systematically examined.

OBJECTIVE: The present study evaluated the behavioral responses to cocaine challenge following continuous baclofen administration.

METHODS: Sprague‐Dawley rats were pretreated with baclofen (0, 0.31, 0.625, 1.25 mg/kg/day) continuously for 14 days, and withdrawn for 7 days. Body weights of all subjects were monitored during withdrawal. Following withdrawal, subjects received a cocaine (0, 7.5, 15 mg/kg) challenge, and locomotor activity and rearing were recorded for 60 min.

RESULTS: The highest dose of baclofen (1.25 mg/kg/day) increased spontaneous locomotion during withdrawal. However, baclofen did not alter body weights or rearing, suggesting that the effects of baclofen were not due to non‐specific increases in behavior. Additionally, the 1.25 mg/kg/day baclofen dose significantly decreased locomotor activity and rearing induced by 15 mg/kg cocaine.

CONCLUSIONS: The current findings provide further evidence that chronic baclofen treatment produces enduring alterations in spontaneous and cocaine‐induced locomotor activity.
Nitrous oxide (N2O)‐induced nitric oxide (NO)‐dependent release of β‐endorphin (β‐EP) from the arcuate nucleus to stimulate opioid receptors in the periaqueductal gray (PAG) to cause antinociception in the rat
Authors: Ohgami Yusuke , Chung Eunhee , Quock Raymond M
Published: June 2021

N2O stimulates an NO‐dependent release of endogenous opioid peptides to induced antinociception in rodents (Emmanouil & Quock, Anesth. Prog. 54:9–18, 2007). The present study was carried out to determine whether the arcuate nucleus was the brain site of action of N2O. Male Sprague Dawley rats, 250–300 g, were stereotaxically implanted with microdialysis probes in the arcuate nucleus. Exposure to 70% N2O increased dialysate levels of NOx as well as β‐EP, compared to levels in fractions collected under room air. Pretreatment of rats with the NOS‐inhibitor L‐NAME prior to N2O exposure antagonized the increases in both NO metabolites and β‐EP. In other rats that were stereotaxically implanted with intracerebral microinjection cannulae, pretreatment with naltrexone in the periaqueductal gray (PAG) but not the arcuate nucleus antagonized N2O‐induced antinociception in the rat hot plate test. These findings indicate a functional link between increased NO activity and neuronal release of β‐EP in response to N2O and support our hypothesis that β‐EP released from the arcuate nucleus is carried to the PAG to cause antinociception. (Supported by State of Washington Initiative Measure No. 171.)
The FAAH inhibitor URB‐597 ameliorates cannabinoid withdrawal in mice
Authors: Schlosburg Joel Evan , LA Carlson Brittany , Lichtman Aron H
Published: June 2021

Administration of Δ9‐tetrahydrocannabinol (THC) has been demonstrated to ameliorate the severity of withdrawal symptoms in rodents and humans. The purpose of the current investigation was to determine if upregulation of the endogenous cannabinoid anandamide would reduce the physical withdrawal signs in a mouse model of cannabinoid dependence. By blocking the primary enzyme responsible for anandamide degradation, fatty acid amide hydrolase (FAAH), the influence of elevated anandamide levels was examined on somatic signs of withdrawal in THC‐dependent mice. While FAAH (−/−) mice showed similar withdrawal profiles to normal mice, acute administration of the FAAH inhibitor URB‐597 significantly diminished the severity of withdrawal symptoms in wild type mice, but not in FAAH (−/−) mice. Unlike THC, repeated injections of high doses of URB‐597 failed to lead to cannabinoid dependence. These findings suggest that FAAH inhibitors lack dependence liability, but FAAH may prove a promising therapeutic target to treat cannabis dependence.

Research supported by: PO1DA017259, RO1DA15197, RO1DA015683
Modulation of the Reinforcing and Discriminative Stimulus Effects of Cocaine by the Dopamine D3 Receptor Partial Agonist CJB 090 [N‐(4‐(4‐(2,3‐Dichlorophenyl)piperazin‐4‐(pyridine‐2‐yl)benzamide] in Squirrel Monkeys
Authors: Achat‐Mendes Cindy , Platt Donna M. , Spealman Roger D. , Newman Amy H.
Published: June 2021

Dopamine D3 receptor mechanisms have been implicated repeatedly in the behavioral effects of cocaine. This study evaluated the effects of the D3 receptor partial agonist CJB 090 in monkeys trained to: 1) discriminate cocaine from vehicle, 2) self‐administer cocaine on a second order fixed‐interval, fixed‐ratio schedule of i.v. drug injection or 3) self‐administer food on a comparable second order schedule of food delivery. In cocaine discrimination studies, CJB 090 did not substitute for cocaine, but significantly attenuated cocaine's discriminative stimulus effects when administered as a pretreatment. CJB 090 also significantly attenuated the partial cocaine‐like stimulus effects of the D3 receptor agonist PD128907, but enhanced the partial cocaine‐like stimulus effects of the D2 receptor agonist sumanirole. In self‐administration studies, CJB 090 did not attenuate responding maintained by i.v. injections of cocaine at a dose that reduced responding maintained by food. In a separate group of monkeys, observational tests revealed that CJB 090 did not induce scratching or biting (species‐typical effects of D2/3 receptor agonists) or catalepsy (typical effect of D2/3 receptor antagonists). The results provide little evidence that CJB 090 reduced cocaine's reinforcing effects. However they do suggest that CJB 090, acting primarily via a D3 receptor mechanism, antagonized the discriminative stimulus effects of cocaine at a dose that did not induce adverse observable effects.

Supported by DA011054, NIDA‐IRP, RR00168. We thank J. Cao for CJB 090 synthesis.
Effects of an acute social stressor on brain glucose utilization and cocaine self‐administration in socially housed monkeys
Authors: Gould Robert Warren , Czoty Paul W , Hanlon Colleen A , Wesley Michael J , Gill Kathryn E , Miller Mack D , Porrino Linda J , Nader Michael A
Published: June 2021

In monkeys, neurobiological differences related to social rafnk have been identified that are hypothesized to arise from chronic exposure to stress and have been shown to affect cocaine reinforcement. This study examined the effects of acute exposure to a social stressor on regional rates of cerebral glucose metabolism (CMRglc) and cocaine self‐administration in 4 dominant and 4 subordinate male cynomolgus monkeys. CMRglc was assessed using [18F]fluorodeoxyglucose (FDG) and positron emission tomography (PET). Each monkey received FDG prior to placement in his home cage or a novel “intruder” cage which was surrounded by 4 unknown group‐housed monkeys for 40 min prior to a PET scan. At another time, exposure to each condition was followed by a self‐administration session in which cocaine (0.003–0.1 mg/kg) and food were concurrently available. In the FDG studies, CMRglc was reduced in motor, visual and somatosensory cortical areas and increased in anterior cingulate and basal ganglia in the intruder condition relative to control conditions; in addition, there were other changes related to social rank. Preliminary results show that acting as an intruder increased cocaine choice only in subordinate monkeys. These results document distinct patterns of neurobiological activity occurring after acute social stress that may enhance the reinforcing strength of cocaine in vulnerable individuals. DA10584, DA06634.
Modulation of of l‐Aromatic Amino Acid Decarboxylase by 3, 4‐dihydroxyphenylalanine in the MPTP mouse model.
Authors: King Jennifer Margaret , Charlton Clivel G.
Published: June 2021

Parkinson's disease (PD) is a neurological disorder resulting from a deficiency of dopamine (DA) in the neostriatum due to degeneration of dopaminergic neurons in the substantia nigra. The major therapeutic drug for PD is L‐3,4‐dihydroxyphenylalanine (L‐DOPA), which is the precursor of DA. However, long‐term treatment of L‐DOPA induces side effects including dyskinesias, wearing‐off and on‐off phenomenon. Previous studies in our lab led to investigation of the metabolic events of L‐DOPA treatment, including the role of L‐dopa on L‐aromatic amino acid decarboxylase (LAAD), the enzyme that catalyzes the formation of DA from L‐DOPA. In this project we prepare 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) PD disease mouse model and treat them with L‐dopa at 100 mg/kg. We studied the locomotor activity of the mice and performed Western blot assay for LAAD. The results showed that the sub‐chronic administration of L‐DOPA decreased LAAD expression in the striatum of the MPTP‐PD mouse model, but increased it in the cortex. L‐DOPA generates high amounts of DA, therefore, the data suggest that DA causes a feedback or allosteric downregulation of striatal LAAD. This down‐regulation of LAAD may contribute to the wearing‐off phenomenon and may show that the key side effects of L‐dopa may be due to the disturbance of the homeostasis of the catecholamine enzymes by the overload of L‐DOPA.
Simvastatin Protects Neurons from Cytotoxicity by Up‐regulating Bcl‐2 mRNA and Protein: The Role of Endothelin‐1 on Bcl‐2 Expression
Authors: Wood Wellington Gibson , Butterick Tammy , Johnson‐Anuna Leslie , Eckert Gunter , Igbavboa Urule , Muller Walter
Published: June 2021

There is great interest albeit some controversy in the use of statins for the prevention or treatment of neurodegenerative diseases such as ischemic stroke, Alzheimer's disease and AIDS dementia. We have recently reported (JPET, 2005, 312,786–793; Neurobiol Dis, 2007, 25, 438–445) the novel findings that simvastatin in vivo stimulated brain gene and protein expression of Bcl‐2 and ET‐1 which have been shown to play pivotal roles in pro‐survival pathways. In in vitro studies (J Neurochem, 2007, 101, 77–86) we found that simvastatin prevented neuronal apoptosis which was mevalonate/cholesterol independent and that the neuroprotective effects of simvastatin were eliminated by suppression of Bcl‐2 expression. There is some evidence that ET‐1 may increase Bcl‐2 abundance. To determine if simvastatin induced ET‐1 influences Bcl‐2 regulation we examined the temporal levels of gene and protein expression of ET‐1 following drug treatment in primary mouse cortical neurons and SH‐SY5Y human neuroblastoma cells (5Y). Primary neurons incubated with simvastatin showed a significant increase in ET‐1 mRNA preceding the elevation of Bcl‐2 gene expression and protein levels. There was also a significant increase in ET‐1 protein in the conditioned media and lysate of mouse neurons. Exogenous ET‐1 incubated with 5Y cells significantly increased Bcl‐2 protein. The ET‐1 receptor antagonists BQ‐123 and BQ 788 inhibited the stimulatory effects of simvastatin on Bcl‐2 protein levels. These findings provide new insight into the potential use of statins which may have efficacy in neurodegenerative diseases in which programmed cell death has been implicated. Supported in part by NIH grants AG‐18357, AG‐23524 and the Department of Veterans Affairs.
Glucocorticoid receptor interacting protein‐1 (GRIP‐1) is a novel transcriptional co‐activator for IRF‐1‐inducible genes
Authors: Damera Gautam , Gu Su , Kassel Olivier , Amrani Yassine , Tliba Omar
Published: June 2021

IFN Regulatory Factor‐1 (IRF‐1) is an important transcription factor involved in the regulation of IFNs and TNFα‐inducible genes. GR‐interacting protein 1 (GRIP‐1) plays a critical role in the transcriptional regulation of glucocorticoid receptor (GR). Recent evidence showed that GRIP‐1 interacts and modulates IRF‐3‐dependent gene expression. We here examined whether manipulating the levels of such co‐factor modulates the transcriptional regulation of IRF‐1‐inducible genes. Airway smooth muscle (ASM) cells were co‐transfected with 2 μg constitutively active (CA)‐GRIP‐1 (kindly provided by Dr. Rogatsky) or pcDNA3 (used as a negative control) vectors and 2 μg of IRF‐1 reporter construct. After 48 h, cells were either left untreated or treated with TNFα (10 ng/ml) and IFNγ(1000 UI/ml) for 6 h. In parallel experiments, cells were co‐transfected with 2 μg CA‐GRα, 2 μg of CA‐GRIP‐1 or pcDNA3 vectors and 2 μg of IRF‐1 reporter construct. After 48 h, cells were treated as described above. We found that CA‐GRIP‐1 by itself dramatically enhanced IRF‐1 reporter activity in both basal and cytokine‐treated cells. Interestingly, overexpression of GRα inhibited IRF‐1 reporter activity in pcDNA3 transfected cells, an inhibition that was completely prevented in GRIP‐1‐overexpressing cells. Further, chromatin‐immunoprecipitation assay revealed a marked enrichment of GRIP‐1 on IRF‐1 DNA binding sites of CD38 promoter in cells‐treated with TNFα and IFNγ. Together, these data expand the role of GRIP‐1 from co‐activator restricted to nuclear receptors to include other transcription factors such as IRF‐1.
Influence of Pumpkin Seed Oil Supplementation on Cardiovascular and Histological Outcomes in Female Non‐ovariectomized and Ovariectomized Rats
Authors: Lyttle Kerise Alecia , Gossell‐Williams Maxine D , Clarke Teran , Gardner Michael , Simon Oswald
Published: June 2021

Pumpkin (Cucurbita sp.) seed oil (PSO) is a rich source of phytoestrogens, consequently the aim of this study was to examine the effect of PSO supplementation on lipid profile, systolic and diastolic blood pressure and histological parameters in non‐ovariectomized and ovariectomized rats. 22 female Sprague‐Dawley rats divided into non‐ovariectomized rats for corn oil supplementation (Control CO; n=6), non‐ovariectomized rats for PSO supplementation (Control PSO; n=5), ovariectomized for corn oil supplementation (OVX/CO; n=6) and ovariectomized for PSO supplementation (OVX/PSO; n=5) were treated orally 5 days per week for 12 weeks with corn oil 40mg/kg or PSO 40 mg/kg. Control PSO had lower TC, LDL −C and trigylcerides and higher HDL‐C than the Control CO (P<0.001). OVX/CO had higher TC, LDL −C and trigylcerides and lower HDL‐C than the Control CO (P <0.001), these changes were prevented in the OVX/PSO rats (P<0.001). Control PSO and OVX/PSO groups had lower systolic and diastolic blood pressure weekly readings. Histological analysis demonstrated that the uterus and vagina of the OVX/PSO group was protected from atrophy associated with lack of estrogen. We conclude that PSO supplementation prevents changes in plasma lipids and blood pressure that occur with inadequate estrogen availability.
Estrogen Dependent Response to Nicotine in Substantia Nigra and Ventral Tegmental Area of OVX female rats
Authors: Sabban Esther L , Serova Lidia I
Published: June 2021

The incidence of Parkinson's disease is higher in men than women and nicotine (Nic) reduces the risk. To study the interaction between Nic and estrogen (E), OVX rats were treated for 16 days with estradiol benzoate (EB) or vehicle and received Nic injections on days 13–16 (twice daily). Tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA levels were determined by qRT‐PCR in VTA and SN. The VTA of OVX rats was especially susceptible to the lowest dose of Nic. In contrast, with EB treatment, only the highest dose of Nic was effective. In SN, both doses of Nic did not change TH mRNA levels in OVX rats with or without EB injections, but EB treatment increased sensitivity of GTPCH gene to Nic. To determine whether E can modulate Nic triggered transcriptional regulation of TH and GTPCH genes, dopaminergic MN9D cells were co‐transfected with pGTPCH/Luc and expression vector for ERa or ERβ. The results revealed that Nic and E elevated GTPCH promoter activity with both ERa and ERβ. The response was reduced with Nic and E combined. The findings provide insight for specificity of Nic regulation of catecholamine and tetrahydrobiopterin biosynthesis in DA neurons in gender specific fashion.
Gender differences in 5‐HT induced hypotension
Authors: Davis Robert Patrick , Diaz Jessica Lynn , Watts Stephanie Wengert
Published: June 2021

Recent studies have shown that 5‐hydroxytryptamine; (5‐HT) has a hypotensive effect in male DOCA‐salt hypertensive rats. Studies have also shown that males have higher blood pressure when compared with age matched pre‐menopausal women. We hypothesized that the hypotensive effects of 5‐HT infusion would result in a greater blood pressure fall in female rats when compared with males. Serotonin and vehicle were administered via osmotic pump (25ug/kg/min) for seven days to male and female normotensive rats. Arterial blood pressure was monitored via radiotelemetry. After twenty‐four hours, female pressure fell to 79 ± 2.14 mmHg, while male pressure fell to 97 ± 2.67 mmHg.

Animals were sacrificed seven days later, and intravascular 5‐HT measurements were made. Female rats had greater intravascular arterial and venous 5‐HT concentrations than males. 5‐HT concentrations were greatest in female veins (Jugular 56.502 ± 6.25 ng/mg protein) compared to arteries (Carotid 10.818 ± 1.34 ng/mg protein). These results suggest that 5‐HT has a greater effect on the female cardiovascular system when compared with the male. Finally, this study is the first to show greater concentrations of 5‐HT in female veins when compared with males.
Inhibitory phosphorylation of TASK‐1 is associated with atrial fibrillation
Authors: Harleton Erin , Besana Alessandra , Argenziano Michael , Robinson Richard B. , Feinmark Steven J.
Published: June 2021

Activation of the platelet‐activating factor (PAF) receptor leads to inhibition of TASK‐1, a two pore‐domain potassium channel. PAF‐induced TASK‐1 inhibition requires the activity of PKCε and causes repolarization abnormalities in isolated murine ventricular myocytes. We have previously shown that TASK‐1 current is absent from atrial myocytes derived from humans suffering from atrial fibrillation (AF). We now show that AF‐associated loss of TASK‐1 current occurs in spite of significantly higher levels of channel protein expression in AF atrium as compared to atrial tissue from patients in normal sinus rhythm, suggesting regulation by post‐translational modification. TASK‐1 current was restored in atrial myocytes by PP2A treatment, showing that inhibition in AF was phosphorylation‐dependent. We have made a construct of human TASK‐1 carboxy‐terminus fused to glutathione‐S‐transferase and made site‐directed mutants of potential PKC sites. Kinase reactions of these constructs with PKCε identified the phosphorylation site as T383, a result that was confirmed by LC‐MS/MS analysis. Similar mutations in the full length channel abrogated the ability of PAF to inhibit TASK‐1 current in whole cell patch clamp studies. These data provide an exciting new marker, and possible therapeutic target, for AF, the most common human arrhythmia.

Supported by NIH grants 5R01HL070105‐04, 5TL1RR024158‐02 and Servier
Gq‐coupled Receptor signaling – A kinetic analysis in living cells
Authors: Hoffmann Carsten , Hein Peter , Zabel Ulrike , Ziegler Nicole , Berlot Cathrine , Lohse Martin J , Bünemann Moritz
Published: June 2021

The activation of G protein‐coupled receptors (GPCRs) is the initial step in the signaling cascade which is triggered by binding of an agonist to a GPCR. Here we report the kinetics of the initial steps in the signaling cascade of a Gq‐coupled receptor. We employed the M3‐subtype of the muscarinic acetylcholine receptor (M3‐AChR) as a model receptor to study Gq‐signaling. Using a fluorescence resonance energy transfer (FRET) approach based on the FlAsH‐tetracysteine tag technology in combination with CFP we generated a M3‐receptor construct that reports conformational changes during receptor activation by changes in the FRET‐signal. Acetylcholine or carbachol both rapidly activated receptors on a millisecond timescale. FRET based detection of M3‐AChR – Gq‐Protein interaction revealed kinetics that closely resembled receptor activation kinetics. Furthermore G‐Protein activation was measured using YFP‐ tagged Gαq and an N‐terminally tagged CFP‐γ2 subunit of the heterotrimeric G‐protein. Activation of the Gq‐Protein was significantly slower than receptor activation and undistinguishable for acetylcholine or carbachol.
The Design And Use of Covalently Bound Agonists To Probe Kappa‐Opioid Receptors
Authors: Yan Feng , Bikbulatov Ruslan , Zjawiony Jordan , Roth Bryan
Published: June 2021

Salvinorin A, the most potent naturally occuring hallucinogen, has gained great attention since the Kappa‐Opioid Receptor (KOR) was identified as its principal molecular target (Roth et al, PNAS 2002). Previously, extensive efforts were made to understand how salvinorin A binds to and activates KOR. Our goal here was to design a series of ligands capable of covalently binding to KOR to further explore the ligand‐receptor interactions at atomic level. From substituted cysteine accessibility methods (SCAM) we knew that C315(7.38) is water accessible and highly reactive to methanethiosulfonate (MTS) reagents, therefore C315(7.38) could provide an anchoring point for proximity‐accelerated chemical coupling reaction. We designed a series of covalent binding ligands of variable size and electrophilic properties. Thus far, two compounds–RB‐48 and RB‐64–were the most promising candidates after extensive molecular pharmacology examinations. They showed high affinity, high efficacy and irreversible binding properties. We are currently using these ligands along with mass spectrometry to identify the anchoring residue(s). This study will facilitate our goals to elucidate the structure and function of KOR, a family A GPCR, which is involved in analgesia, pain, mood and reward. To our knowledge, this is the first design and application of site‐directed chemical coupling ligands to probe KOR.
The C‐terminal PDZ binding motifs in hMT1 and hMT2 melatonin receptors are required for membrane localization, but not for melatonin‐induced receptor desensitization
Authors: Zhang Yahong , Chen Lin , Masana Monica I. , Dubocovich Margarita L.
Published: June 2021

The melatonin receptors hMT1 and hMT2 are differentially desensitized by melatonin (MLT) which contributes to temporally regulated MLT signaling. At physiological concentrations (~500 pM), MLT functionally desensitizes and internalizes hMT2 receptors. hMT1 Receptors desensitize but do not internalize by supraphysiological concentration of MLT. The present study examined the PDZ binding motifs at carboxyl termini of hMT1 (VDSV) and hMT2 (ADAL) in regulating receptor membrane expression in HEK cells. Deletion of VDSV and ADAL motifs reduced 2‐[I125]‐iodomelatonin (100 pM) binding to the receptors (fmol/mg protein: 1112 ± 154 vs 19 ± 2.6 for hMT1; 694 ± 152 vs 2.1 ± 1.3 for hMT2), and diminished their membrane localization assessed by confocal microscopy. Interestingly, their differential desensitization by MLT were preserved in hMT1 PDZ and hMT2 PDZ. MLT reduced 2‐[I125]‐iodomelatonin binding in cells expressing hMT2 PDZ (2.1± 1.3 vs 0.3 ± 0.3 fmole/mg protein), but not hMT1 PDZ. This is consistent with previous results that switching hMT1 and hMT2 carboxyl termini does not alter their characteristic desensitization/internalization responses to MLT. In conclusion, C‐terminal PDZ binding motif is required for hMT1 and hMT2 membrane expression and that the molecular determinant(s) for their differential desensitization by MLT is not localized at carboxyl terminus. Supported by MH 42922 to MLD.
Peripheral nerve injury induces DOR trafficking to cell surface in dorsal horn neurons
Authors: Holdridge Sarah Victoria , Cahill Catherine Marie
Published: June 2021

Neuropathic (NP) pain remains a significant challenge for clinicians as it is typically refractory to traditional analgesics. However, research in ours and other laboratories increasingly suggests a therapeutic role of delta opioid receptor (DOR) agonists in treating NP pain. Spinal administration of a selective DOR agonist, attenuated nerve injury‐induced allodynia and hyperalgesia with enhanced antinociceptive effects in NP rats compared with controls. This functional enhancement was not due to increased DOR biosynthesis, as DOR protein did not change. In the present study, we examined DOR subcellular localization by electron microscopy immunohistochemistry to determine whether NP injury produces a redistribution of DORs from internal stores to the cell surface. Quantification of immunopositive dendrites in the dorsal horn revealed a bilateral increase in plasma membrane‐associated DORs within lamina V neurons of NP rats compared to shams. The recruitment of DORs to neuronal plasma membranes may represent a compensatory mechanism by which neurons may sustain an inhibitory tone during chronic pain states, and in turn, indicate a viable drug target. (CIHR, J.P. Bickell Foundation, CFI/OIT, CRC)
Complex interactions of sibutramine with α1D‐adrenoceptors
Authors: Pupo Andre Sampaio , Nojimoto Fernanda Diehl , Akinaga Juliana , Lima Vanessa
Published: June 2021

This study investigates the interactions of sibutramine, a hypophagic drug non‐selective inhibitor of norepinephrine (NE) and 5‐HT uptake, with the α1‐ARs subtypes of the rat vas deferens (α1A), spleen (α1B) and aorta (α1D) and with human α1‐ARs subtypes expressed in HEK 293 cells. Sibutramine potentiated the contractions induced by NE in the vas deferens, consistent with an indirect sympathomimetic action. In the rat spleen, sibutramine (up to 100 μM) was unable to affect the contractions induced by NE, suggesting it is inactive at α1B‐ARs. In rat aorta sibutramine 3 to 100 μM competitively antagonized the contractions induced by NE (Schild slope = 0.95 ± 0.05; pA2 = 5.5 ± 0.1) indicating it interacts with α1D‐ARs. Sibutramine inhibited the specific binding of [3H]‐prazosin to membranes from HEK cells expressing α1A‐ARs and α1B‐ARs with very low affinities (pKi ≈ 3.5). Low concentrations of sibutramine (30 nM to 1 μM) partially inhibited (≈50%) the specific binding of [3H]‐prazosin to membranes from HEK cells expressing α1D‐ARs. In dissociation experiments, a low concentration of sibutramine (0.3 μM) increased by 5‐fold the dissociation of [3H]‐prazosin from membranes of HEK cells expressing α1D‐ARs whereas sibutramine 10 μM delayed it by 3‐fold. Although presenting complex behavior in radioligand binding assays, sibutramine interacts selectively with native and recombinant α1D‐ARs.
Dopamine receptor interacting proteins: unraveling the receptor signalplex
Authors: Hazelwood Lisa A , Free R Benjamin , Cabrera David M , Sibley David R
Published: June 2021

Dopamine receptors (DARs) are known to exist as constituents of a large macromolecular complex of interacting proteins, termed the signalplex. These interacting proteins may be transmembrane or cytosolic, and can play a variety of roles in receptor trafficking, function or desensitization. To identify interacting partners, we have employed a co‐immunoprecipitation assay for D1 and D2 DARs from both mouse brain and transfected cell lines. When coupled with mass spectrometry (MS) sequencing, these experiments have allowed detection and subsequent peptide‐matching to more than 50 proteins within a non‐redundant protein database. Among the novel interacting proteins identified through these experiments were various subunits of the Na+,K+‐ATPase (NKA) and the regulatory protein sorting nexin 25 (SNX25). Studies have revealed multiple isoforms of SNX25 that influence DAR expression and function, possibly via sorting or internalization of the receptors. Experiments with the NKA have shown that the DARs and NKA can reciprocally regulate one another, providing a control mechanism for both receptor signaling and cellular ion balance. These data offer insights into the cellular components of the DAR signalplex, and indicate that interacting proteins can influence the DARs at various stages of receptor processing and function. LAH and RBF contributed equally to this work.

Support Contributed By: NINDS/NIH and the NIGMS PRAT Program (LAH)
Peripheral Delta Opioid Receptors (DOR) Require Priming for Functional Competence In Vivo
Authors: Rowan Matt P , Ruparel Nikita B. , Patwardhan Amol M. , Berg Kelly A. , Hargreaves Kenneth M. , Clarke William P.
Published: June 2021

Opioid receptors expressed on peripheral sensory neurons in culture are inactive for inhibitory signaling under basal conditions, but become competent to inhibit neuropeptide release and adenylyl cyclase activity after brief pre‐treatment (priming) with bradykinin (BK), arachidonic acid, or direct activators of protein kinase C (PKC). Opioid receptors can also function as oligomers, but in vivo data on priming and oligomers is limited. Here we evaluated whether peripheral DOR require priming in vivo to inhibit PGE2‐induced thermal hyperalgesia in the rat. Results indicate that the BK priming effect was PKC‐dependent, and the antihyperalgesic response to the DOR agonist, DPDPE, was blocked by the antagonist, naltrindole. Administration of the DOR‐kappa opioid receptor (KOR) heterodimer‐selective agonist, 6′GNTI, also produced BK priming‐dependent reversal of thermal hyperalgesia. This study supports in vitro findings that peripheral opioid receptors are inactive under basal conditions and require priming via inflammatory mediators, such as BK, for functional competence in vivo, and suggests a role for peripheral DOR‐KOR hetero‐oligomers in vivo. Supported in part by P01 DA016719 and THCEB/ARP.
Ligand mediated protein‐protein interaction between the angiotensin receptor type AT1 and the human NHE6 isoform
Authors: Hetrick Mary Michelle , Mandavia Chirag , Gavini Nara , Pulakat Lakshmi
Published: June 2021

The Renin Angiotensin System (RAS) plays a vital role in the regulation of blood pressure and fluid homeostasis. RAS is regulated through the hormone Angiotensin II (Ang II), a powerful vasoconstrictor that serves as a regulator of hypertension and other cardiovascular diseases. Involvement of Ang II in the regulation of sodium levels by activation of the Na+/H+ Exchangers (NHE) has been demonstrated in many tissues. Of the seven known NHE isoforms, only two, NHE1 and NHE6, exist in heart tissue. The activities of the NHE6 protein remain unclear. To analyze the conditions leading to activation of the NHE6 protein in mammalian cells, the human MCF‐7 cell line was used. The Heameagglutinin‐tagged NHE6 plasmid was introduced to the MCF‐7 cells via transient transfection. The transfected cells were exposed to Ang II and results implicated that the presence of Ang II was cardinal for strong interaction between AT1 and NHE6. It was ascertained that MCF‐7 co‐transfected with AT2 and Heameagglutinin‐tagged NHE6, also required the presence of Ang II for activation. Further investigation revealed that tyrosine and threonine phosphorylation of NHE6 occurred when AT1 was activated by ligand binding. In summary, our results insinuate Ang II activates NHE6 through the AT1 receptor and therefore may play a role in the development of the pathophysiological conditions leading to the onset of cardiac ischemia.
Sodium‐induced allosteric changes in D2 dopamine receptor local dynamics modulate ligand selectivity
Authors: Schetz John A. , Ericksen Spencer S. , Cummings David F. , Weinstein Harel
Published: June 2021

The basis of allostery in GPCRs and its implications for ligand recognition remain a mystery. While determining the source of large differences in measured ligand affinity reported for a mutant D2 dopamine receptor, we uncovered a unique and significant allosteric response to the physiological modulator sodium. This response is characterized by a sodium‐dependent enhanced affinity for ligands of a specific structural class having either agonist or antagonist functional properties. To explore possible mechanisms for our empirically‐derived results, the influence of sodium on D2 receptor dynamics was examined by computational analysis of the molecular normal modes of motion. This result revealed divergence in the component motions of the D2 receptor with and without sodium bound to the putative sodium binding regions. One particularly sensitive mode involved the concerted motions of the extracellular portions of transmembrane‐spanning domains, which when computed in the presence of sodium appeared to disrupt a favorable pi‐stacking interaction between TM2 and TM3 freeing an aromatic residue in the receptor model for favorable interactions with aromatic portions of the ligands increasing their affinity. This demonstrates how a sodium‐dependent change in dynamic properties can yield the experimentally observed allosteric phenomenon.

Supported R01 MH063162 (JAS), P01 DA012923 and K05 DA00060 (HW).
AP‐1 is activated by relaxin family peptides in cells expressing the relaxin family peptide receptor 3 (RXFP3)
Authors: van der Westhuizen Emma T , Sexton Patrick M , Summers Roger J
Published: June 2021

Relaxin‐3 is a neuropeptide involved in appetite regulation and stress behaviour in rats. RXFP3 is the cognate receptor for relaxin‐3 that is coupled to cAMP inhibition and ERK1/2 activation in CHO‐K1 and HEK293 cell lines recombinantly expressing the receptor. Competition for [125I]‐INSL5/H3 relaxin chimera binding is observed with H3 relaxin and the B‐chain of H3 relaxin, but not with any other relaxin family peptides. We identified that H3 relaxin but also H2 relaxin and porcine relaxin activate transcription from AP‐1 elements in CHO and HEK cells transiently expressing human RXFP3 receptors, measured using a reporter gene. H2 relaxin was more potent at activating transcription than either H3 relaxin or porcine relaxin. Experiments with inhibitors of p38 MAPK (RWJ67657, 10 μM), MEK1/2 (PD98059, 20μM) and JNK (SP600125, 10μ M) showed that AP‐1 activation involved p38 MAPK in CHO‐K1 cells stimulated with H3 relaxin, H2 relaxin or porcine relaxin. In HEK293 cells, AP‐1 activation in response to H3 relaxin or porcine relaxin involved p38 MAPK, MEK1/2 and JNK, but, when stimulated with H2 relaxin, it involved only a MEK1/2 pathway. We propose that different relaxin family peptides interact at different sites on RXFP3, to activate distinct signalling pathways.

This research was supported by National Health and Medical Research Council Project Grants 436713 and 454375
Role of exchange protein activated by cAMP (Epac1) in A2B adenosine receptor (A2BAR) regulation of vascular cell proliferation
Authors: Olah Mark , Sheth Sandeep , Fang Ying
Published: June 2021

A2B adenosine receptor activation in human umbilical vein endothelial cells (HUVEC) stimulates extracellular‐signal regulated kinase (ERK1/2) via a pathway that requires activation of Epac1. In that ERK1/2 stimulation is associated with cell proliferation, this study examined the hypothesis that Epac1 has a role in HUVEC proliferation. In HUVEC, 10 μM NECA induced cell proliferation as assessed by [3H]thymidine incorporation and this response was completely blocked by the MEK inhibitor PD98059. Retrovirus‐mediated overexpression of Epac1 increased basal HUVEC proliferation by ~75% and slightly but significantly enhanced NECA‐induced proliferation. The enhanced proliferation mediated by Epac1 required guanine nucleotide exchange factor (GEF) activity as a GEF domain‐deleted construct had no effect on basal HUVEC proliferation. To further examine the role of Epac1 in cAMP‐regulated cell proliferation, A7r5 vascular smooth muscle cells were examined. In A7r5 cells, NECA and forskolin inhibited ERK1/2 activity and decreased cell proliferation. Overexpression of Epac1 in A7r5 cells had no effect on basal proliferation and NECA produced a similar inhibition of proliferation in control and Epac1‐overexpressing A7r5 cells. These data indicate that Epac1 has a role in HUVEC proliferation, however, solely overexpression of Epac1 in A7r5 cells does not convert A2BAR activation to a stimulatory event.
Edema formation is reduced by hydrogen sulfide inhibitors in perfused rat lung
Authors: Russell Michael J , Austin Tucker , Barr Zac , Kepner Star , Savage Kristy , Russ Roy D
Published: June 2021

Edema is a significant post‐surgery complication in transplanted lungs and may be attributed in part to ischemia‐reperfusion injury. Endogenous hydrogen sulfide (H2S) may mediate beneficial effects in the heart, but effects in the lung are unknown. In the present study we investigated the role of H2S on ischemia‐reperfusion induced edema formation in rat lungs exposed to 90 min warm ischemia followed by 30 min of ventilation and perfusion. Lungs were perfused in situ using a peristaltic pump at constant flow to produce physiological (19–24 mmHg) pressure waves at 37°C. Ventilation was initiated at 3 cm H2O positive end‐expiratory pressure (PEEP), and lungs were inflated to 10 cm H2O PEEP and not ventilated during warm ischemia. After a final 30 min of ventilation and reperfusion, isolated right lobes were blotted dry, weighed, and dried in an oven for 24 hr at 80°C. Exogenous hydrogen sulfide (Na2S, 200 μM) and inhibition of the H2S producing enzyme cystathionine β‐synthase with amino oxyacetic acid did not affect edema formation, whereas inhibition of the H2S producing enzyme cystathionine γ‐lyase (CSE) with propargylglycine and inhibition of pyridoxyl‐5‐phosphate with hydroxylamine significantly reduced edema formation. These findings suggest that H2S may facilitate post‐ischemic edema formation in rat lungs via the CSE pathway. Supported by the MedCen Foundation of the Medical Center of Central Georgia.
A Novel fusogenic lipid vesicle (FLV) based targeted delivery system displays compstatin on cell membranes and effectively reduces complement deposition in vitro
Authors: Todnem Nathan D , Goga Ledia , Pushpakumar Sathnur , Cramer Daniel , Perez‐Abadia Gustavo , Barker John H , Maldonado Claudio
Published: June 2021

Excessive complement (C) activation due to ischemia/reperfusion following organ transplantation has been associated with graft dysfunction. Several anti complement inhibitors have been used in the past to reduce this damage however their systemic administration can leave patients vulnerable to infection. To develop a targeted therapy we created a novel method to rapidly display complement inhibitors on cell membranes by using fusogenic lipid vesicles (FLVs) containing nickel (Ni) tethers. Compstatin a C3 convertase blocker was modified with a hexahistidine (His) domain (C‐His) to bind to these tethers. The purpose of this study was to determine whether the display of C‐His was effective in reducing complement deposition in vitro. We hypothesized that cells decorated with C‐His would reduce C3b deposition after C activation. Studies were conducted in human fibroblasts and SKOV‐3 cells. Ni tethering on cell membranes was optimized by altering FLV formulation, concentration and incubation time. C‐His bioactivity was tested using hemolytic assay. Trastuzimab and human serum was used for C activation, and C3b deposition was measured using FACS analysis. We found that C‐His successfully coupled to Ni tethers on SKOV‐3 cells and reduced C3b deposition. We conclude that membrane display of C‐His is effective in reducing C deposition and that this therapy has potential in transplantation.
Estradiol inhibits vascular hyperpermeability following hemorrhagic shock
Authors: Hunter Felicia A , Tharakan Binu , Childs Ed W
Published: June 2021

Recent studies from our laboratory showed the involvement of mitochondria mediated ‘intrinsic’ apoptotic signaling in vascular hyperpermeability following hemorrhagic shock (HS). We hypothesized that 17ß‐estradiol, which shows anti‐apoptotic properties, would attenuate HS‐induced vascular hyperpermeability. The objective of this study was to determine if 17ß‐Estradiol would inhibit hyperpermeability in rat mesenteric post capillary venules in vivo and protect mitochondrial transmembrane potential (MTP), following HS. In rats, HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg. To study vascular permeability, the rats were injected intravenously with FITC‐albumin (50 mg/kg) and the FITC‐albumin flux was measured by determining the changes in integrated optical intensity obtained intra and extra vascularly using intravital microscopy. Changes in MTP were determined using the fluorescent dye JC‐1. HS induced hyperpermeability (p<0.05). 17ß‐Estradiol (2 mg/kg body weight) attenuated HS‐induced hyperpermeability (p<0.05). 17ß‐Estradiol pre‐treatment prevented HS‐induced decrease in MTP (p<0.05). These findings demonstrate that 17ß‐estradiol maintains vascular endothelial barrier functions following HS. Its protective effects may be mediated through the inhibition of ‘intrinsic’ apoptotic signaling.
Inhibition of Glycan Shedding from the Endothelial Glycocalyx and Adhesion of Leukocytes by Doxycycline in Post‐capillary Venules
Authors: Lipowsky Herbert H , Sah Rachna A , Lescanic Ann
Published: June 2021

Previous studies have demonstrated that shedding of glycans from the endothelial cell (EC) glycocalyx and leukocyte (WBC)‐EC adhesion in response to the chemoattractant fMLP could be attenuated by doxycycline (Doxy) through the inhibition of matrixmetalloproteases (MMPs). Given that Doxy is a strong calcium chelator, the present studies were undertaken to determine if the calcium chelator EDTA could produce a similar effect. To this end, WBC‐EC adhesion and adhesion of lectin (BS‐1) coated microspheres (0.1 um diameter) in post‐capillary venules of mesentery (rat) were studied during superfusion of the tissue with Ringers solution plus EDTA in the presence or absence of fMLP. In the absence of fMLP, with 1 mM EDTA sequestration of microspheres steadily increased with time, and with 3 mM sequestration reached levels 50–100% greater than without EDTA. In contrast, with 0.5 uM Doxy alone, microsphere adhesion remained constant under control conditions. Calcium chelation at levels (1 mM EDTA) that inhibited fMLP induced WBC adhesion to the same extent as Doxy (0.5 uM) caused a disruption of the venular permeability barrier that resulted in increased microsphere sequestration in the venular wall. Thus, the inhibition of microsphere shedding and WBC adhesion in response to fMLP by doxycycline appear to be caused by inhibition of MMPs in contrast to calcium chelation. Supported by R01 HL39286.
Prolyl Hydroxylase Inhibition Attenuates Hypoxia‐Induced Microvascular Inflammation Via iNOS Upregulation
Authors: Parker Tuley R. , Gonzalez Norberto C , Holloway Naomi B , Blanco V Gustavo , Casillan Alfred J , West Cameron E , Michael Moncure , Thomas James H , Wood John G
Published: June 2021

Acute systemic hypoxia (Hx) promotes leukocyte adherence in post‐capillary venules of rats. When animals are chronically hypoxic, the initial microvascular inflammation resolves in part due to upregulation of inducible nitric oxide synthase (iNOS). Inhibition of prolyl hydroxylase (PHD) has been shown to increase hypoxia‐inducible factor‐1 (HIF‐1) levels, which can increase iNOS expression. Objective: This study evaluated whether inhibition of PHD attenuated Hx‐induced microvascular inflammation, and if upregulation of iNOS was involved. Methods: Intravital microscopy was used to measure leukocyte adherence in mesenteric venules of anesthetized rats. The PHD inhibitor EDHB was administered to rats 24 hrs prior to experiments. Results: Hx caused a rapid increase in leukocyte adherence in vehicle‐ but not in EDHB‐treated rats. Superfusion of the mesentery with the iNOS inhibitor L‐NIL caused a small but significant increase in adherent leukocytes during Hx in EDHB‐ but not vehicle‐treated rats. Conclusion: These results show that inhibition of PHD reduces Hx‐induced microvascular inflammation in part through iNOS upregulation, and are consistent with a HIF‐1‐dependent mechanism.
Evaluating criteria for intact capillaries in Rana pipiens using hydraulic conductivity (Lp), rolling and sticking white blood cells (WBC), and an index of systemic inflammation
Authors: Williams Donna A. , Wipke‐Tevis Deidre D. , Flood Mary H.
Published: June 2021

WBC rolling/sticking along capillary walls have been used for decades to identify inflammation and exclude microvessels from studies of basal capillary function in vivo. For microcirculatory preparations a broader view of animal health state also is required, but often remains undefined. We measured activation of WBC obtained from a peripheral blood sample as an index of systemic inflammation in Rana pipiens and then measured Lp on individual capillaries always devoid of rolling/sticking WBC. We hypothesized that capillary Lp following our standard protocol (abrupt change in shear stress (Δτ)) would be higher in frogs with activated WBC (oxidative burst) compared to control. Frogs (Rana pipiens, N=38) were pithed and mesentery was exteriorized and superfused with frog Ringer's (14–16°C). True capillaries were cannulated at 10 cm H2O, perfused with 10 mg·ml−1BSA/Ringer's, stimulated with Δτ, and Lp assessed at 30 cm H2O (modified Landis technique). Mean±SE Lp for control capillaries (no activated WBC; within 95% confidence intervals (CI) of Δτ/Lp response curve) was 2.7±0.3 × 10−7 cm·s−1·cm H2O−1 (n=15). In frogs with activated WBC, Lp was higher (11.4±1.6 × 10−7, n=9; P<0.0001) compared to control. A subset of frogs with no activated WBC displayed Lp values outside CI that averaged 8.9±1.1 × 10−7 (n=12), were higher than control, and did not differ from the activated WBC group. These data suggest that capillary rolling/sticking WBC may not provide comprehensive information concerning inflammation of intact capillaries in Rana pipiens. Systemic activated WBC accounted for half of the identified outliers. Supported by RO1 HL63125.
Hypertrophic remodeling of diabetic mesenteric resistance arteries is attenuated by Tempol and apocynin
Authors: Souza Flavia M , Katz Paige S , Lord Kevin C , Potter Barry , Vassallo Dalton , Lucchesi Pamela A
Published: June 2021

The effects of antioxidants on Type 2 diabetes‐induced remodeling of mesenteric resistance arteries are not well known. This study identified structural changes found in small mesenteric resistance arteries in the diabetic (db/db) mouse model. Effects of a membrane permeable free radical scavenger (Tempol, 1 mM) and the NAD(P)H oxidase inhibitor (apocynin, 5 mM) on blood pressure (MAP, measured by telemetry), glucose levels (Accu‐Chek) and vascular structure (pressure myography) were assessed in 16 wk db/db and Db/db (control) mice with or without 4 wks of treatment. MAP was similar between Db/db and db/db mice and unaffected by drug treatment. Fasting blood glucose levels were higher in db/db mice (Db/db 115±10 vs. db/db 505±28 mg/dl, p<0.001). The lumen diameter (Db/db 107±7.5; db/db 131±5 microns, p〈0.05 at 75 mm Hg) and the media cross‐sectional area were increased in db/db compared to Db/db, suggesting hypertrophic outward remodeling. Treatment of db/db mice with Tempol reduced the growth index by 45% and apocynin by 56% compared to untreated db/db. Oxidative stress, measured by electron spin resonance with CMH as spin trap, was increased in db/db and was reversed by Tempol or apocynin. These data suggest that oxidative stress plays an important role in diabetes‐induced hypertrophic vascular remodeling of resistance arterioles. Supported by COBRE P20RR18766 and HL056046 (PAL) and CAPES (DV).