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The production of biologics that treat complex diseases, such as cancer, autoimmune, and infectious disease, requires careful monitoring and control of cell cultures. While bioprocess optimizations have dramatically improved production yields, a lack of analytical tools has made it challenging to identify accompanying intracellular improvements. Intracellular redox can diminish the growth and productivity of biologics-producing cells and adversely impact product quality profiles yet characterizing redox is challenging due to its complex and highly transient nature. In this study, we integrated a fluorescent thiol-based redox biosensor to monitor intracellular redox in one bisAb- and two monoclonal antibody-producing clonal cell lines in a 14-day fed-batch bioreactor. We characterized biosensor functionality using three fluorescence measurement techniques and determined sensor oxidation correlates with the intracellular ratio of reduced (GSH) and oxidized glutathione (GSSG), an important cellular antioxidant. Our fed-batch bioreactor studies showed that sensor expression minimally affected bioprocess outcomes, including growth, productivity, product quality attributes, or intracellular redox attributes, including mitochondrial reactive oxygen species and total cellular GSH levels in all cell lines tested. Biosensor measurements taken throughout the culture revealed that the intracellular environment in these cell lines became more reduced throughout the culture, with the exception of a high pH condition which became more oxidized. Our results demonstrate the potential of using biosensors to monitor intracellular changes in near-real-time with minimal process effects, thus potentially improving future bioprocess optimizations.

Pain signals are relayed to the brain via a nociceptive system, and in rare situations, this nociceptive system contains genetic variants that can limit pain response. Here we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and further if we can target this region by a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N knock-in mouse using CRISPR/Cas9, we discovered the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral response to chemical noxious stimuli and less hypersensitivity to nerve injury-induced pain, while leaving the response to noxious heat intact. Furthermore, blocking this K710 region in wild-type rodents by a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and rescued pain hypersensitivity induced by nerve injury back to baseline. These findings identify K710 TRPV1 as a discrete site crucial for the control of nociception and provides new insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

AimsE-cigarette aerosol containing aldehydes, including acetaldehyde, are metabolized by the enzyme aldehyde dehydrogenase 2 (ALDH2). However, little is known how aldehyde exposure from e-cigarettes, when coupled with an inactivating ALDH2 genetic variant, ALDH2*2 (present in 8% of the world population), affects cardiovascular oxidative stress. The aim of this study was to determine how e-cigarette aerosol exposure, when coupled with genetics, impacts cardiovascular oxidative stress in wild type ALDH2 and ALDH2*2 knock-in mice.Methods and ResultsUsing selective ion flow mass spectrometry, we determined that e-cigarette aerosol contains acetaldehyde that are 10-fold higher than formaldehyde or acrolein. Next, using wild type ALDH2 and ALDH2*2 knock-in rodents, we identified organ-specific differences in ALDH2 with the heart having ~1.5-fold less ALDH2 enzyme activity relative to the liver and lung. In isolated cardiac myocytes, acetaldehyde exposure (30 seconds, 0.1-1 μM) caused a 4-fold greater peak in calcium levels for ALDH2*2 relative to ALDH2 cardiomyocytes. ALDH2*2 cardiomyocytes exposed to acetaldehyde also demonstrated a 2-fold increase in ROS production and 2.5-fold increase in 4HNE protein adducts relative to ALDH2 cardiomyocytes. For intact rodents, ALDH2*2 knock-in mice exposed to e-cigarette aerosol had an increased heart rate beginning 5 days after exposure compared to wild type ALDH2 mice (775±30 bpm versus 679±33 bpm, respectively, *pConclusionsThe increased oxidative stress from e-cigarette aerosol aldehydes triggers the proinflammatory NF-κB pathway. As ALDH2 expression and activity is lower in the heart relative to the lung, the heart could be more susceptible to increases in cardiovascular oxidative stress from e-cigarette aerosol; particularly for those carrying an ALDH2*2 genetic variant which limits acetaldehyde metabolism.Graphical AbstractE-cigarette aerosol exposure triggers increases in ROS that lead to increased protein carbonylation, MDA production, and elevates phosphorylated NF-kB. This exposure to e-cigarette aerosol leads to increases in cardiovascular oxidative stress. For the ALDH2*2 variant, there is limited ability to metabolize the reactive aldehydes from e-cigarette aerosol and with increased levels of oxidative stress at baseline relative to wild type ALDH2, e-cigarette aerosol increased oxidative stress, protein carbonylation, and phosphorylation of NF-kB relative to wild type ALDH2 rodents.

Supplemental Digital Content is Available in the Text. ALDH2 activity influences endometriosis and its associated pain., Endometriosis affects ∼176 million women worldwide, yet on average, women experience pain ∼10 years from symptom onset before being properly diagnosed. Standard treatments (drugs or surgery) often fail to provide long-term pain relief. Elevated levels of reactive aldehydes such as 4-hydroxynonenal (4-HNE) have been implicated in the peritoneal fluid of women with endometriosis and upon accumulation, reactive aldehydes can form protein-adducts and/or generate pain. A key enzyme in detoxifying reactive aldehydes to less reactive forms is the mitochondrial enzyme aldehyde dehydrogenase-2 (ALDH2). Here, we tested the hypothesis that aberrant reactive aldehyde detoxification by ALDH2 underlies endometriosis and its associated pain. We determined, in the eutopic and ectopic endometrium of women with severe (stage IV) peritoneal endometriosis, that ALDH2 enzyme activity was decreased, which was associated with decreased ALDH2 expression and increased 4-HNE adduct formation compared to the eutopic endometrium of controls in the proliferative phase. Using a rodent model of endometriosis and an ALDH2*2 knock-in mouse with decreased ALDH2 activity, we determined that increasing ALDH2 activity with the enzyme activator Alda-1 could prevent endometriosis lesion development as well as alleviate pain-associated behaviors in proestrus. Overall, our findings suggest that targeting the ALDH2 enzyme in endometriosis may lead to better treatment strategies and in the proliferative phase, that increased 4-HNE adduct formation within the endometrium may serve as a less invasive diagnostic biomarker to reduce years of suffering in women.

The functional expression of transient receptor potential cation channel of the ankyrin-1 subtype (TRPA1) has recently been identified in adult mouse cardiac tissue where stimulation of this ion channel leads to increases in adult mouse ventricular cardiomyocyte (CM) contractile function via a Ca2+-Calmodulin-dependent kinase (CaMKII) pathway. However, the extent to which TRPA1 induces nitric oxide (NO) production in CMs, and whether this signaling cascade mediates physiological or pathophysiological events in cardiac tissue remains elusive. Freshly isolated CMs from wild-type (WT) or TRPA1 knockout (TRPA1-/-) mouse hearts were treated with AITC (100 µM) and prepared for immunoblot, NO detection or ischemia protocols. Our findings demonstrate that TRPA1 stimulation with AITC results in phosphorylation of protein kinase B (Akt) and endothelial NOS (eNOS) concomitantly with NO production in a concentration- and time-dependent manner. Additionally, we found that TRPA1 induced increases in CM [Ca2+]i and contractility occur independently of Akt and eNOS activation mechanisms. Further analysis revealed that the presence and activation of TRPA1 promotes CM survival and viability following ischemic insult via a mechanism partially dependent upon eNOS. Therefore, activation of the TRPA1/Akt/eNOS pathway attenuates ischemia-induced CM cell death.

Engineered Chinese hamster ovary (CHO) cells are the most widely utilized cell line for protein-based therapeutics production at industrial scales. Process development strategies which improve production capacity and quality are often implemented without an understanding of underlying intracellular changes. Intracellular redox conditions drive reactions in pathways critical to biologics production, including bioenergetic and biosynthetic pathways, necessitating methods to quantify redox-related changes. Advances in methods for analytical redox quantification presented here, including bioreactor probes, redox-targeted proteomics, genetically encoded redox-sensitive fluorescent proteins, and biochemical assays, are creating new opportunities to characterize the effects of redox in biologics production. Implementing these methods will lead to enhanced media formulations, improved bioprocess strategies, and new cell line engineering targets and ultimately develop redox into an optimizable bioprocess parameter to improve the yield and quality of these lifesaving medicines.

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Introduction: E-cigarette aerosol contains reactive aldehydes including acetaldehyde, formaldehyde, and acrolein when e-cigarette is heated. Approximately 560 million people worldwide cannot efficiently metabolize aldehydes present in e-cigarette aerosol, due to a genetic deficiency in aldehyde dehydrogenase 2 enzyme (ALDH2), known as ALDH2*2. Little is known how aldehyde exposure from e-cigarettes, coupled with genetic differences in aldehyde metabolism, affects cardiovascular oxidative stress both at a physiological and cellular level. Hypothesis: E-cigarette aerosol exposure will elevate heart rate and cellular oxidative stress more substantially in ALDH2*2 knock-in mice versus wild type ALDH2 mice. Methods: To measure aldehyde levels, e-cigarette Juul aerosols were collected and quantified by selective ion flow gas mass spectrometry. Further, age-matched male wild type and homozygous ALDH2*2 mice (8-10 weeks old, ~25g) were implanted with EKG telemeters. After surgical recovery, mice were paired by genotype (one wild type ALDH2 and one ALDH2*2 mice) and exposed to either Juul aerosol or room air 4 sessions per day for 10 days. For each session, 7 puffs/min were drawn for the first two minutes (a total of 14 puffs), and the whole body exposure to Juul aerosol lasted 7 minutes, continued with 23 minutes smoking-free intervals in each session. Mice EKG waveforms were recorded daily. After 10 days of exposure, heart homogenates were subjected to biochemical assays including lipid peroxidation, 4-HNE protein adduct formation, and protein carbonylation. Results: Quantification of reactive aldehyde levels in e-cigarettes revealed that Juul aerosol contained acetaldehyde (5.3±0.32 ppm), formaldehyde (0.20±0.02 ppm), and acrolein (0.09±0.01 ppm). When exposed to Juul aerosol, ALDH2*2 mice showed a maximal increase in heart rate unlike ALDH2 wild type mice (774.6±29.5 bpm versus 678.9±32.8 bpm respectively, * p Conclusions: These findings indicate e-cigarette aerosols contain reactive aldehydes, primarily acetaldehyde. A deficiency in reactive aldehyde metabolism by having an ALDH2*2 deficiency may contribute to increases in heart rate and oxidative stress within the cardiovascular system while smoking e-cigarettes.

Endometriosis affects ∼176 million women worldwide, yet on average, women experience pain ∼10 years from symptom onset before being properly diagnosed. Standard treatments (drugs or surgery) often fail to provide long-term pain relief. Elevated levels of reactive aldehydes such as 4-hydroxynonenal (4-HNE) have been implicated in the peritoneal fluid of women with endometriosis and upon accumulation, reactive aldehydes can form protein-adducts and/or generate pain. A key enzyme in detoxifying reactive aldehydes to less reactive forms, is the mitochondrial enzyme aldehyde dehydrogenase-2 (ALDH2). Here, we tested the hypothesis that aberrant reactive aldehyde detoxification by ALDH2, underlies endometriosis and its associated pain. We determined, in the eutopic and ectopic endometrium of women with severe (stage IV) peritoneal endometriosis, that ALDH2 enzyme activity was decreased, which was associated with decreased ALDH2 expression and increased 4-HNE adduct formation compared to the eutopic endometrium of controls in the proliferative phase. Using a rodent model of endometriosis and an ALDH2*2 knock-in mouse with decreased ALDH2 activity, we determined that increasing ALDH2 activity with the enzyme activator Alda-1 could prevent endometriosis lesion development as well as alleviate pain-associated behaviors in proestrus. Overall, our findings suggest that targeting the ALDH2 enzyme in endometriosis may lead to better treatment strategies and in the proliferative phase, that increased 4-HNE adduct formation within the endometrium may serve as a less invasive diagnostic biomarker to reduce years of suffering in women.One Sentence SummaryALDH2 activity influences endometriosis and its associated pain.

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is activated by a broad range of noxious stimuli. Cdk5, a member of the Cdk family, has recently been identified as a modulator of pain signaling pathways. In the current study, we investigated the extent to which Cdk5 modulates TRPA1 activity. Cdk5 inhibition was found to attenuate TRPA1 response to agonist in mouse DRG sensory neurons. Additionally, the presence of active Cdk5 was associated with increased TRPA1 phosphorylation in transfected HEK293 cells that was roscovitine-sensitive and absent in the mouse mutant S449A full-length channel. Immunopurified Cdk5 was observed to phosphorylate human TRPA1 peptide substrate at S448A in vitro. Our results point to a role for Cdk5 in modulating TRPA1 activity.

Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free CaThese novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca

We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via reactions with the amino acids: cysteine, histidine and lysine. Thus, we sought to determine if 4-HNE mediated post-translational modification of TRPV1 could account for dysfunctional TRPV1-mediated signaling observed in diabetes. Our initial studies demonstrate 4-HNE infusion decreases TRPV1-dependent coronary blood flow in C57BKS/J (WT) mice. Further, we found that TRPV1-dependent vasorelaxation was suppressed after 4-HNE treatment in isolated mouse coronary arterioles. Moreover, we demonstrate 4-HNE significantly inhibited TRPV1 currents and Ca2+ entry utilizing patch-clamp electrophysiology and calcium imaging respectively. Using molecular modeling, we identified potential pore cysteines residues that, when mutated, could restore TRPV1 function in the presence of 4-HNE. Specifically, complete rescue of capsaicin-mediated activation of TRPV1 was obtained following mutation of pore Cysteine 621. Finally, His tag pull-down of TRPV1 in HEK cells treated with 4-HNE demonstrated a significant increase in 4-HNE binding to TRPV1, which was reduced in the TRPV1 C621G mutant. Taken together these data suggest that 4-HNE decreases TRPV1-mediated responses, at both the in vivo and in vitro levels and this dysfunction can be rescued via mutation of the pore Cysteine 621. Our results show the first evidence of an amino acid specific modification of TRPV1 by 4-HNE suggesting this 4-HNE-dependent modification of TRPV1 may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Background and Aim: Birds, unlike mammals, are resistant to the spicy hot sensation when consuming chili peppers. The inability for birds to sense the hot sensation is accredited to genetic divergence in the transient receptor potential vanilloid 1 (TRPV1) when compared to mammals. Recently, we described how TRPV1 is important for regulating myocardial ischemia-reperfusion injury in rodents. Here, we tested our hypothesis that K710 is a crucial mediator of capsaicin-induced calcium influx in myocardial H9C2 cells. Methods and Results: Sequence alignment of the C-terminus TRP domain of TRPV1 among species was performed. Unlike mammals having K710 TRPV1, birds instead have N710 TRPV1 (Fig 1A). Site-directed mutagenesis of K710N was performed on wild type (WT) rTRPV1 with a pCMV6-entry vector. The empty vector, WT and K710N plasmids were transfected into H9C2 cells and the cellular localization of TRPV1 channel were detected by immunofluorescent staining and channel activity by capsaicin challenge with live cell calcium imaging with Fura-2AM. Interestingly, the K710N mutation caused the TRPV1 channel to be retained intracellularly and co-localized with PDI, an endoplasmic reticulum marker (Fig 1B, Bar=25μM). K701N cells treated with 1μM capsaicin showed a reduced response to capsaicin compared to wild type TRPV1 transfected cells (Fig 1C-E, 011±0.01 in WT vs . 0.03±0.01 in K710N, n=10, P Conclusions: Site-directed mutagenesis of K710N caused rTRPV1 to be insensitive to capsaicin and suggests a potential target for limiting calcium influx from myocardial ischemia-reperfusion injury which could be a novel protective strategy to prevent damage from a heart attack.

Aldehydes, which are present within the air as well as food and beverage sources, are highly reactive molecules that can be cytotoxic, mutagenic, and carcinogenic. To prevent harm from reactive aldehyde exposure, the enzyme aldehyde dehydrogenase 2 (ALDH2) metabolizes reactive aldehydes to a less toxic form. However, the genetic variant of ALDH2, ALDH2*2, significantly reduces the ability to metabolize reactive aldehydes in humans. Therefore, frequent environmental aldehyde exposure, coupled with inefficient aldehyde metabolism, could potentially lead to an increased health risk for diseases such as cancer or cardiovascular disease. Here, we discuss the environmental sources of reactive aldehydes and the potential health implications particularly for those with an ALDH2*2 genetic variant. We also suggest when considering the ALDH2*2 genetic variant the safety limits of reactive aldehyde exposure may have to be reevaluated. Moreover, the ALDH2*2 genetic variant can also be used as an example for how to implement precision medicine in the field of environmental health sciences.

Delayed graft function is an early complication following kidney transplantation with an unclear molecular mechanism. Here we determined whether impaired reactive aldehyde metabolism is associated with delayed graft function. Human kidney biopsies from grafts with delayed graft function were compared with grafts that did not develop delayed graft function by Ingenuity gene pathway analysis. A second series of grafts with delayed graft function (n=10) were compared to grafts that did not develop delayed graft function (n=10) by measuring reactive aldehyde metabolism, reactive aldehyde-induced protein adduct formation, and aldehyde dehydrogenase (ALDH) gene and protein expression. In the first series of kidney biopsies, several gene families known for metabolizing reactive aldehydes, such as aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), and glutathione-S transferase (GSTA), were upregulated in kidneys that did not develop delayed graft function versus those that did. In the second series of kidney grafts, we focused on measuring aldehyde-induced protein adducts and ALDH enzymatic activity. The reactive aldehyde metabolism by ALDH enzymes was reduced in kidneys with delayed graft function compared to those that did not (37 ± 12∗vs. 79 ± 5 μg/min/mg tissue,∗P<0.005, respectively). ALDH enzymatic activity was also negatively correlated with length of hospital stay after a kidney transplant. Together, our study identifies a reduced ALDH enzymatic activity with kidneys developing delayed graft function compared to those that did not. Measuring ALDH enzymatic activity and reactive aldehyde-induced protein adducts can potentially be further developed as a biomarker to assess for delayed graft function and recovery from a kidney transplant.

Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) and vanilloid subtype-1 (TRPV1) are structurally related, non-selective cation channels that show a high permeability to calcium. Previous studies indicate that TRP channels play a prominent role in the regulation of cardiovascular dynamics and homeostasis, but also contribute to the pathophysiology of many diseases and disorders within the cardiovascular system. However, no studies to date have identified the functional expression and/or intracellular localization of TRPA1 in primary adult mouse ventricular cardiomyocytes (CMs). Although TRPV1 has been implicated in the regulation of cardiac function, there is a paucity of information regarding functional expression and localization of TRPV1 in adult CMs. Our current studies demonstrate that TRPA1 and TRPV1 ion channels are co-expressed at the protein level in CMs and both channels are expressed throughout the endocardium, myocardium and epicardium. Moreover, immunocytochemical localization demonstrates that both channels predominantly colocalize at the Z-discs, costameres and intercalated discs. Furthermore, specific TRPA1 and TRPV1 agonists elicit dose-dependent, transient rises in intracellular free calcium concentration ([Ca2+]i) that are abolished in CMs obtained from TRPA1−/− and TRPV1−/− mice. Similarly, we observed a dose-dependent attenuation of the TRPA1 and TRPV1 agonist-induced increase in [Ca2+]i when WT CMs were pretreated with increasing concentrations of selective TRPA1 or TRPV1 channel antagonists. In summary, these findings demonstrate functional expression and the precise ultrastructural localization of TRPA1 and TRPV1 ion channels in freshly isolated mouse CMs. Crosstalk between TRPA1 and TRPV1 may be important in mediating cellular signaling events in cardiac muscle.

Background Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. Methods Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. Results Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. Conclusions TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.

WOS: 000384518200014, PubMed ID: 27586050, Essential oils were obtained by hydrodistillation of the umbels+seeds and stems of Ferula akitschkensis (FAEO(u/s) and FAEO(stm), respectively) and analyzed by gas chromatography and gas chromatography-mass spectrometry. Fifty-two compounds were identified in FASO(u/s); the primary components were sabinene, alpha-pinene, beta-pinene, terpinen-4-ol, eremophilene, and 2-himachalen-7-ol, whereas the primary components of FAEOstm were myristicin and geranylacetone. FAEO(u/s), beta-pinene, sabinene, gamma-terpinene, geranylacetone, isobornyl acetate, and (E)-2-nonenal stimulated [Ca2+](i) mobilization in human neutrophils, with the most potent being geranylacetone (EC50 = 7.6 +/- 1.9 mu M) and isobornyl acetate 6.4 +/- 1.7 (EC50 = 7.6 +/- 1.9 mu M). In addition, treatment of neutrophils with beta-pinene, sabinene, gamma-terpinene, geranylacetone, and isobornyl acetate desensitized the cells to N-formyl-Met-Leu-Phe (fMLF)- and interleukin-8 (IL-8)-induced [Ca2+](i) flux and inhibited fMLF-induced chemotaxis. The effects of beta-pinene, sabinene, gamma-terpinene, geranylacetone, and isobornyl acetate on neutrophil [Ca2+](i) flux were inhibited by transient receptor potential (TRP) channel blockers. Furthermore, the most potent compound, geranylacetone, activated Ca2+ influx in TRPV1-transfected HEK293 cells. In contrast, myristicin inhibited neutrophil [Ca2+](i) flux stimulated by fMLF and IL-8 and inhibited capsaicin-induced Ca2+ influx in TRPV1-transfected HEK293 cells. These findings, as well as pharmacophore modeling of TRP agonists, suggest that geranylacetone is a TRPV1 agonist, whereas myristicin is a TRPV1 antagonist. Thus, at least part of the medicinal properties of Ferula essential oils may be due to modulatory effects on TRP channels., National Institutes of Health IDeA Program COBRE Grant [GM110732]; Ministry of Education and Science, Kazakhstan [0504/GF3, 2117/GF4]; USDA National Institute of Food and Agriculture Hatch project [1009546]; Montana University System Research Initiative [51040-MUSRI2015-03]; Montana State University Agricultural Experiment Station, This research was supported in part by National Institutes of Health IDeA Program COBRE Grant GM110732; Grants 0504/GF3 and 2117/GF4 from the Ministry of Education and Science, Kazakhstan; a USDA National Institute of Food and Agriculture Hatch project 1009546; Montana University System Research Initiative 51040-MUSRI2015-03; and the Montana State University Agricultural Experiment Station.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr99 or Tyr138 of CaM was replaced with Phe, generating mutant CaM, Phe99-CaM, or Phe138-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe138-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM. Blocking phosphorylation of CaM at Tyr99 also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-Trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of ECmigration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr99 by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane. Fil: Chaudhuri, Pinaki. Cleveland Clinic; Estados Unidos Fil: Rosenbaum, Michael. Louis Stokes Cleveland Veterans Affairs Medical Center; Estados Unidos Fil: Sinharoy, Pritam. Kent State University; Estados Unidos Fil: Damron, Derek S.. Kent State University; Estados Unidos Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Graham, Linda. Cleveland Clinic; Estados Unidos

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.

BackgroundTransient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol is an intravenous anesthetic known to cause vasorelaxation. Our objectives were to examine the extent to which TRPA1 and/or TRPV1 ion channels mediate propofol-induced depressor responses in vivo and to delineate the signaling pathway(s) involved.MethodsMice were subjected to surgery under 1.5-2.5% sevoflurane gas with supplemental oxygen. After a stable baseline in mean arterial pressure (MAP) was achieved propofol (2.5, 5.0, 10.0 mg/kg/min) was administered to assess the hemodynamic actions of the intravenous anesthetic. The effect of nitric oxide synthase (NOS) inhibition with L-NAME and/or calcium-gated K+ channel (BKCa) inhibition with Penetrim A (Pen A), alone and in combination, on propofol-induced decreases in mean arterial pressure were assessed in control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-).ResultsPropofol decreased MAP in control mice and this effect was markedly attenuated in TRPA1-/- and TRPAV-/- mice but unaffected in TRPV1-/-mice. Moreover, pretreatment with L-NAME or Pen A attenuated the decrease in MAP in control and TRPV1-/- mice, and combined inhibition abolished the depressor response. In contrast, the markedly attenuated propofol-induced depressor response observed in TRPA1-/- and TRPAV-/- mice was unaffected by pre-treatment with Pen A or L-NAME when used either alone or in combination.ConclusionThese data demonstrate for the first time that propofol-induced depressor responses in vivo are predominantly mediated by TRPA1 ion channels with no involvement of TRPV1 ion channels and includes activation of both NOS and BKCa channels.