PKA-dependent Mechanism Research Articles

Abstract B071: Mesothelial cell motility and contractility promote ovarian cancer metastatic potential through a PKA-dependent mechanism

Abstract Ovarian cancer is the leading cause of death among gynecological cancers, and metastasis to the peritoneum occurs in over 60% of patients. To form these metastatic implants, disseminated cancer cells attach and invade through the mesothelial barrier that lines the peritoneal organs in the abdominal cavity. A better understanding of cancer-mesothelial interactions will lead to identification of mesothelial-specific prognostic markers and the discovery of therapeutic targets to limit ovarian cancer metastatic spread. The goal of our studies is to dissect how mesothelial cell contractility and barrier integrity impact the metastatic potential of high-grade serous ovarian cancer. We developed an imaging-based live cell assay that assesses the rate at which the ovarian cancer spheroids transmigrate across mesothelial monolayers (we termed this process “clearance”) and preconditioned the mesothelial monolayer with different pharmacologic agents. We discovered that treatment with forskolin, a cAMP activator, significantly reduced the number of clearing spheroids compared to the control untreated condition. Inhibition of protein kinase A (PKA) in mesothelial cells that were treated with forskolin reversed the anti-metastatic effects and restored clearance rates to baseline levels. Next, we analyzed mesothelial cell-cell junctions, cytoskeletal organization, traction forces and cell motility patterns. Compared to the control condition, forskolin treated mesothelial cells exhibited higher expression levels of ZO-1 and β-catenin junctions, as well as reduction in actin stress fibers. Consistent with these results, we found that forskolin reduced traction forces and mesothelial cell migration was assessed through a wound healing assay. To evaluate the impact of increasing mesothelial contractility, we treated mesothelial cells with calyculin A, a potent phosphatase inhibitor that elevated expression of phospho-myosin to promote traction forces and increase spheroid clearance rates. Transcriptomic and proteomic studies of mesothelial monolayers treated with forskolin revealed a role for the c-Jun pathway. c-Jun is known to regulate cell migration and response to environmental stress, and thus presents a promising candidate for enhancing mesothelial barrier function and protecting against ovarian cancer spheroid invasion. Our findings suggest that normalizing mesothelial cell contractility via a PKA-dependent mechanism enhances barrier integrity and limits the invasive potential of ovarian cancer spheroids. A better understanding of cancer-mesothelial paracrine signaling and environmental stressors in the peritoneal cavity that lead to mesothelial barrier dysfunction hold promise for the discovery of mesothelial-targeted therapies to reduce ovarian cancer metastatic dissemination. Citation Format: Dorota Jazwinska, Youngbin Cho, Ioannis Zervantonakis. Mesothelial cell motility and contractility promote ovarian cancer metastatic potential through a PKA-dependent mechanism [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B071.

Influence of the PHY domain on the ms-photoconversion dynamics of a knotless phytochrome

The ability of some knotless phytochromes to photoconvert without the PHY domain allows evaluation of the distinct effect of the PHY domain on their photodynamics. Here, we compare the ms dynamics of the single GAF domain (g1) and the GAF-PHY (g1g2) construct of the knotless phytochrome All2699 from cyanobacterium Nostoc punctiforme. While the spectral signatures and occurrence of the intermediates are mostly unchanged by the domain composition, the presence of the PHY domain slows down the early forward and reverse dynamics involving chromophore and protein binding pocket relaxation. We assign this effect to a more restricted binding pocket imprinted by the PHY domain. The photoproduct formation is also slowed down by the presence of the PHY domain but to a lesser extent than the early dynamics. This indicates a rate limiting step within the GAF and not the PHY domain. We further identify a pH dependence of the biphasic photoproduct formation hinting towards a pKa dependent tuning mechanism. Our findings add to the understanding of the role of the individual domains in the photocycle dynamics and provide a basis for engineering of phytochromes towards biotechnological applications.Graphical abstract

Sex Differences in Protein Kinase A Signaling of the Latent Postoperative Pain Sensitization That Is Masked by Kappa Opioid Receptors in the Spinal Cord

Latent sensitization (LS) of pain engages pronociceptive signaling pathways in the dorsal horn that include NMDA receptor (NMDAR)→adenylyl cyclase-1 (AC1)→protein kinase A (PKA), and exchange proteins directly activated by cyclic AMP (Epacs). To determine whether these pathways operate similarly between males and females or are under the inhibitory control of spinal κ opioid receptors (KOR), we allowed hyperalgesia to resolve after plantar incision and then blocked KOR with intrathecal administration of LY2456302, which reinstated hyperalgesia and facilitated touch-evoked immunoreactivity of phosphorylated extracellular signal-regulated kinase (pERK) in neurons (NeuN) but not astrocytes (GFAPs) nor microglia (Iba1). LY2456302 reinstated hyperalgesia even when administered 13 months later, indicating that chronic postoperative pain vulnerability persists for over a year in a latent state of remission. In both sexes, intrathecal MK-801 (an NMDAR competitive antagonist) prevented LY2456302-evoked reinstatement of hyperalgesia as did AC1 gene deletion or the AC1 inhibitor NB001. NB001 also prevented stimulus-evoked pERK. In both sexes, the Epac inhibitor ESI-09 prevented reinstatement, whereas the Epac activator 8-CPT reinstated hyperalgesia. By contrast, the PKA inhibitor H89 prevented reinstatement only in male mice, whereas the PKA activator 6-bnz-cAMP itself evoked reinstatement at all doses tested (3–30 nmol, i.t.). In neither sex did incision change gene expression of KOR, GluN1, PKA, or Epac1 in dorsal horn. We conclude that sustained KOR signaling inhibits spinal PKA-dependent mechanisms that drive postoperative LS in a sex-dependent manner. Our findings support the development of AC1, PKA, and Epac inhibitors toward a new pharmacotherapy for chronic postoperative pain.SIGNIFICANCE STATEMENTBecause of neural mechanisms that are not well understood, men and women respond differently to treatments for chronic pain. We report that surgical incision recruits a pronociceptive latent pain sensitization that persisted for over a year and was kept in check by the sustained analgesic activity of κ opioid receptors. NMDAR→AC1→cAMP→Epac signaling pathways in the dorsal horn of the spinal cord maintain latent sensitization in both males and females; however, only males recruit a PKA-dependent mechanism. This work presents a novel male-specific mechanism for the promotion of chronic postoperative pain.

Eicosapentaenoic acid membrane incorporation stimulates ABCA1-mediated cholesterol efflux from human THP-1 macrophages

A high intake in polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) (C20:5 n-3), is cardioprotective. Dietary PUFAs incorporate into membrane phospholipids, which may modify the function of membrane proteins. We investigated the consequences of the membrane incorporation of several PUFAs on the key antiatherogenic ABCA1-mediated cholesterol efflux pathway. Human THP-1 macrophages were incubated with EPA, arachidonic acid (AA) (C20:4 n-6) or docosahexaenoic acid (DHA) (C22:6 n-3) for a long time to mimic a chronic exposure. EPA 70 μM, but not AA 50 μM or DHA 15 μM, increased ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 28% without altering aqueous diffusion. No variation in ABCA1 expression or localization was observed after EPA treatment. EPA incorporation did not affect the phenotype of THP-1 macrophages. The membrane phospholipids composition of EPA cells displayed higher levels of both EPA and its elongation product docosapentaenoic acid, which was associated with drastic lower levels of AA. Treatment by EPA increased the ATPase activity of the transporter, likely through a PKA-dependent mechanism. Eicosanoids were not involved in the stimulated ABCA1-mediated cholesterol efflux from EPA-enriched macrophages. In addition, EPA supplementation increased the apo AI binding capacity from macrophages by 38%. Moreover, the increased apo AI binding in EPA-enriched macrophages can be competed. In conclusion, EPA membrane incorporation increased ABCA1 functionality in cholesterol-normal human THP-1 macrophages, likely through a combination of different mechanisms. This beneficial in vitro effect may partly contribute to the cardioprotective effect of a diet enriched with EPA highlighted by several recent clinical trials.

The Glucagon-Like Peptide 1 Receptor Agonist Liraglutide Stimulates Mechanistic Target of Rapamycin (mTOR) Signaling via PKA And Akt

Glucagon-like peptide 1 receptor (GLP-1R) agonists enhance glucose-stimulated insulin secretion and act on several regions of the brain to reduce food intake and body weight, making the GLP-1R a major therapeutic target for the treatment of type 2 diabetes and obesity. Surprisingly, little is known about the signaling mechanisms mediating the food intake-lowering effects of GLP-1R agonists. We have previously shown that inhibiting the mechanistic Target of Rapamycin (mTOR) in the ventromedial hypothalamus blocks anorexia induced by GLP-1R activation in this brain nucleus (1). Therefore, the goal of the present studies is to elucidate the mechanisms by which GLP-1R activation stimulates mTOR signaling. To accomplish this, we treated Chinese Hamster Ovary cells stably expressing the human GLP-1R with the GLP-1R agonist liraglutide (Lira) in combination with inhibitors of various signaling molecules. Since PKA is a canonical target of GLP-1R signaling, and PKA phosphorylates mTOR and its regulating protein Raptor following β-adrenergic stimulation (2), we used the PKA inhibitors H89 and KT 5720 to examine whether PKA is required for the stimulation of mTOR activity by Lira. We expressed myc-tagged mTOR or Raptor in GLP-1R stably expressing CHO cells, treated them with Lira, immunoprecipitated myc-mTOR or myc-Raptor, and immunoblotted for the PKA substrate RRXS/T motif. We found that Lira significantly increased PKA-substrate motif phosphorylation of myc-Raptor but not myc-mTOR, and this was blocked by pre-treatment with H89. Lira also failed to stimulate phosphorylation of a Ser791Ala Raptor mutant that cannot be phosphorylated by PKA (2). To test whether Akt, a well-known regulator of mTOR activity, contributes to the activation of mTOR signaling by Lira, we pre-treated GLP-1R stably expressing CHO cells with either of the Akt inhibitors Akt-i 1/2 and MK-2206 followed by treatment with Lira or forskolin (Fsk), a cAMP inducer and PKA activator. Pre-treatment with either Akt-i 1/2 or MK-2206 blocked mTOR activation by both Lira and Fsk. This suggests that the contribution of Akt to Lira-induced mTOR activation is likely downstream of cAMP production. Taken together, our results suggest a novel two-pronged, PKA-dependent mechanism for the stimulation of mTOR signaling following GLP-1R activation – directly via phosphorylation of Raptor and indirectly via stimulation of Akt. Future studies will assess the respective contributions and temporal dynamics of each of these pathways. Reference: (1) Burmeister et al., Am J Physiol Endocrinol Metab. 2017 Aug;313: E651–E662. (2) Liu et al., J Clin Invest. 2016;126(5):1704-1716.

Secreted PDZD2 exerts an insulinotropic effect on INS-1E cells by a PKA-dependent mechanism

Secreted PDZD2 (sPDZD2) is a signaling molecule generated upon proteolytic processing of the multi-PDZ-containing protein PDZD2. Previous analysis of gene-trap mice deficient in the synthesis of full-length PDZD2, but not the secreted form, revealed a role of PDZD2 in the regulation of glucose-stimulated insulin secretion. Here, using the pancreatic INS-1E β cells as in vitro model, we showed that depletion of PDZD2/sPDZD2 by RNA interference suppressed the expression of β-cell genes Ins1, Glut2 and MafA whereas treatment with recombinant sPDZD2 rescued the suppressive effect. Similar to GLP-1, sPDZD2 stimulated intracellular cAMP levels, activated β-cell gene expression in a PKA-dependent manner and induced the phosphorylation and nuclear localization of PDX1. Depletion of PDX1 inhibited the sPDZD2 insulinotropic effect, which could also be demonstrated in mouse islets. In summary, our findings are consistent with sPDZD2 serving a signaling function in regulating β-cell gene expression.

Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism.

Clinical studies have shown that microduplications at 7q36.3, containing VIPR2, confer significant risk for schizophrenia and autism spectrum disorder (ASD). VIPR2 gene encodes the VPAC2 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP-induced cAMP responsiveness, but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We have previously found that repeated administration of a selective VPAC2 receptor agonist Ro25-1553 in the mouse during early postnatal development caused synaptic alterations in the prefrontal cortex and sensorimotor gating deficits. In this study, we aimed to clarify the effects of VPAC2 receptor activation on neurite outgrowth in cultured primary mouse cortical neurons. Ro25-1553 and VIP caused reductions in total numbers and lengths of both neuronal dendrites and axons, while PACAP38 facilitated elongation of dendrites, but not axons. These effects of Ro25-1553 and VIP were blocked by a VPAC2 receptor antagonist PG99-465 and abolished in VPAC2 receptor-deficient mice. Additionally, Ro25-1553-induced decreases in axon and dendritic outgrowth in wild-type mice were blocked by a protein kinase A (PKA) inhibitor H89, but not by a PKC inhibitor GF109203X or a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor U0126. PACAP38- induced facilitation of dendritic outgrowth was blocked by U0126. These results suggest that activation of the VPAC2 receptor impairs neurite outgrowth and decreases branching of cortical neurons by a PKA-dependent mechanism. These findings also imply that the VIPR2-linkage to mental health disorders may be due in part to deficits in neuronal maturation induced by VPAC2 receptor overactivation.

Spinal protein kinase A and phosphorylated extracellular signal-regulated kinase signaling are involved in the antinociceptive effect of phytohormone abscisic acid in rats.

The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

GPER and ERα mediate estradiol enhancement of mitochondrial function in inflamed adipocytes through a PKA dependent mechanism

Obesity is associated with inflammation, dysregulated adipokine secretion, and disrupted adipose tissue mitochondrial function. Estradiol (E2) has been previously reported to increase mitochondrial function and biogenesis in several cell lines, but neither the type of oestrogen receptor (ERα, ERβ and GPER) involved nor the mechanism whereby such effects are exerted have been fully described. Considering the anti-inflammatory activity of E2 as well as its effects in enhancing mitochondrial biogenesis, the aim of this study was to investigate the contribution of ERα, ERβ, and GPER signaling to the E2-mediated enhancement of adipocyte mitochondrial function in a pro-inflammatory situation.3T3-L1 cells were treated for 24 h with ER agonists (PPT, DPN, and G1) and antagonists (MPP, PHTPP, and G15) in the presence or absence of interleukin 6 (IL6), as a pro-inflammatory stimulus. Inflammation, mitochondrial function and biogenesis markers were analyzed. To confirm the involvement of the PKA pathway, cells were treated with a GPER agonist, a PKA inhibitor, and IL6. Mitochondrial function markers were analyzed.Our results showed that activation of ERα and GPER, but not ERβ, was able to counteract the proinflammatory effects of IL6 treatment, as well as mitochondrial biogenesis and function indicators. Inhibition of PKA prevented the E2- and G1-associated increase in mitochondrial function markers.In conclusion E2 prevents IL6 induced inflammation in adipocytes and promotes mitochondrial function through the combined activation of both GPER and ERα. These findings expand our understanding of ER interactions under inflammatory conditions in female rodent white adipose tissue.

Curcumin affects gene expression and reactive oxygen species via a PKA dependent mechanism in Dictyostelium discoideum.

Botanicals are widely used as dietary supplements and for the prevention and treatment of disease. Despite a long history of use, there is generally little evidence supporting the efficacy and safety of these preparations. Curcumin has been used to treat a myriad of human diseases and is widely advertised and marketed for its ability to improve health, but there is no clear understanding how curcumin interacts with cells and affects cell physiology. D. discoideum is a simple eukaryotic lead system that allows both tractable genetic and biochemical studies. The studies reported here show novel effects of curcumin on cell proliferation and physiology, and a pleiotropic effect on gene transcription. Transcriptome analysis showed that the effect is two-phased with an early transient effect on the transcription of approximately 5% of the genome, and demonstrates that cells respond to curcumin through a variety of previously unknown molecular pathways. This is followed by later unique transcriptional changes and a protein kinase A dependent decrease in catalase A and three superoxide dismutase enzymes. Although this results in an increase in reactive oxygen species (ROS; superoxide and H2O2), the effects of curcumin on transcription do not appear to be the direct result of oxidation. This study opens the door to future explorations of the effect of curcumin on cell physiology.

RGS19 upregulates Nm23-H1/2 metastasis suppressors by transcriptional activation via the cAMP/PKA/CREB pathway.

The Nm23 metastasis suppressor family is involved in physiological and pathological processes including tumorigenesis and metastasis. Although the inverse correlation of Nm23 level with tumor metastasis potential has been widely observed, the mechanisms that regulate the expression of Nm23 remain poorly understood. Our previous studies have revealed that Nm23-H1/2 isoforms are upregulated by RGS19, a regulator of G protein signaling (RGS) protein which accelerates the termination of Gi signals. Here, we examined the ability of RGS19 to stimulate transcriptional regulation of Nm23 by screening a panel of luciferase reporter genes. Transient and stable overexpression of RGS19 upregulated the Nm23-H1/2 protein levels and activated several transcription factors including CREB, AP-1 and SRE in HEK293 cells. Interestingly, agents that increase the intracellular cAMP level and the phosphorylation of CREB (e.g., adrenergic receptor agonist, forskolin, and cAMP analogues) upregulated the expression of Nm23-H1/2 in HEK293 cells and several cancer cell lines including A549, HeLa, MDA-MB-231, and MDA-MB-435s cells. Conversely, inhibition of protein kinase A (PKA) by H-89 suppressed the phosphorylation of CREB and reduced the expression of Nm23-H1/2. Furthermore, activation of PKA attenuated cancer cell migration in wound healing and transwell assays. Collectively, these results revealed a PKA-dependent mechanism for controlling Nm23-H1/2 expression.

Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

BackgroundParathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear.Material/MethodsIn HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed.ResultsThe FRET analysis found that PTH(1–34), [G1,R19]PTH(1–34) (GR(1–34), and [G1,R19]PTH(1–28) (GR(1–28) were all activated by PKC. The PKC activation ability of GR(1–28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1–34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1–28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1–28) or GR(1–34), and the difference was blunted by Go6983. PTH(1–34), GR(1–28), and GR(1–34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC.ConclusionsPLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1–28); the other was PKA-independent and associated with PTH(29–34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism.

1α,25-Dihydroxyvitamin D3 (1,25-D3) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D3 on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D3 stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active de novo protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D3 did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D3 stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice.

Background:Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood.Objectives:The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice.Methods:Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease.Results:BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases.Conclusions:Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa.Citation:Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238–245; http://dx.doi.org/10.1289/EHP378

TPA promotes cortical neuron survival via mTOR-dependent mechanisms.

Tissue plasminogen activator (tPA) is a thrombolytic agent commonly used in the treatment of ischemic stroke. While the thrombolytic effects of tPA have been well established, the impact of this blood-brain barrier (BBB) crossing drug on neurons is not known. Given the widespread use of tPA in the clinical setting and the strict therapeutic window established for effective use of the drug, we examined the molecular mechanisms mediating the impact of tPA on postnatal cortical neurons isolated from the mouse brain. Dissociated postnatal primary cortical neurons were treated with tPA and the effects on neuron survival were evaluated. Pharmacological inhibitors of several signaling pathways previously implicated in neuroprotection (mTOR, JAK/STAT, MAPK and PKA-dependent mechanisms) were used to pinpoint the mechanistic effectors of tPA on neuron survival in vitro. We report here that tPA treatment results in a time-dependent neuroprotective effect on postnatal cortical neurons that relies predominantly on Janus kinase (JAK) and mammalian target of rapamycin (mTOR) signaling mechanisms. Taken together, these data suggest that tPA promotes neuroprotection in a temporally-regulated manner and that both JAK and mTOR signaling effectors are critical mediators of this neuroprotective effect. The results suggest the possibility of targeting these defined mechanisms to potentially expand the therapeutic window for tPA.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 μM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 μM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 μM). 8MM-IBMX (10 μM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Phosphodiesterase Type-1 Regulates Transient BK Currents and Contractility of Human Urinary Bladder Smooth Muscle

Large conductance Ca2+-activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human UBSM to determine the mechanism by which PDE1 regulates human urinary bladder function. A combined electrophysiological, functional, and pharmacological approach was applied using human UBSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly-isolated human UBSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)-induced contractions in human UBSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 μM) increased TBKC activity in human UBSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 μM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 μM). 8MM-IBMX (10 μM) hyperpolarized the UBSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human UBSM isolated strips.The results reveal that pharmacological inhibition of PDE1 attenuates human UBSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human UBSM. Overall, our data provide mechanistic evidence that pharmacological inhibition of PDE1 can be a useful therapeutic approach for the treatment of overactive bladder syndrome by attenuating UBSM excitability and contractility. These investigations are an important step in validating PDE1 as a potential novel therapeutic target for overactive bladder.Supported by NIH R01DK106964 and R01DK084284 to Georgi V. Petkov.

The vasodilator papaverine stimulates L-type Ca2+ current in rat tail artery myocytes via a PKA-dependent mechanism

Papaverine is an opium alkaloid, primarily used as an antispasmodic drug and as a cerebral and coronary vasodilator. Its phosphodiesterase inhibitory activity promotes increase of cAMP levels mainly in the cytosol. As cAMP is known to modulate L-type Ca2+ channel activity, here we tested the proposition that papaverine could affect vascular channel function. An in-depth analysis of the effect of papaverine on Ba2+ or Ca2+ current through L-type Ca2+ channel [IBa(L) or ICa(L)], performed in rat tail artery myocytes using either the whole-cell or the perforated patch-clamp method, was accompanied by a functional study on rat aorta rings. Papaverine increased current amplitude under both the perforated or whole-cell configuration. Stimulation of the current by papaverine was concentration-, Vh-, frequency-, and charge carrier-dependent, and fully reverted by drug washout. The PKA inhibitor H89, but not the PKG inhibitor Rp-8-Br-cGMPS, antagonised papaverine- as well as IBMX- (another phosphodiesterase inhibitor) induced IBa(L) stimulation. In cells pre-treated with IBMX, application of papaverine failed to increase current amplitude. Papaverine sped up the inactivation kinetics of IBa(L), though only at concentrations ≥30μM, and shifted the voltage dependence of the inactivation curve to more negative potentials. In rings, the vasorelaxing activity of papaverine was enhanced by previous treatment with nifedipine. In conclusion, papaverine stimulates vascular L-type Ca2+ channel via a PKA-dependent mechanism, thus antagonising its main vasodilating activity.

Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms

Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A2A receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

Ruscogenin suppresses mouse neutrophil activation: Involvement of protein kinase A pathway

Ruscogenin, a natural steroidal sapogenin, presents in both food and medicinal plants. It has been found to exert significant anti-inflammatory activities. Considering that activation of neutrophil is a key feature of inflammatory diseases, this study was performed to investigate the inhibitory effect of ruscogenin and its underlying mechanisms responsible for neutrophil activation. Ruscogenin displayed potent antioxidative effects against Formyl-Met-Leu-Phe (FMLP)-induced extra- and intracellular superoxide generation in mouse bone marrow neutrophils, with IC50 values of 1.07±0.32μM and 1.77±0.46μM, respectively. Phorbol myristate acetate (PMA)-elicited extra- and intracellular superoxide generation were also suppressed by ruscogenin, with IC50 values of 1.56±0.46μM and 1.29±0.49μM, respectively. However, ruscogenin showed weak inhibition in NaF-induced response. Inhibition of superoxide generation was mediated neither by a superoxide-scavenging ability nor by a cytotoxic effect. Furthermore, ruscogenin inhibited the membrane translocation of p47phox and p67phox. It reduced FMLP-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and p21-activated kinase (PAK). The cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were increased by ruscogenin. Moreover, ruscogenin inhibited phosphorylation of protein kinase B (Akt), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). In addition, the inhibitory effects of ruscogenin on superoxide production and the phosphorylation of Akt, p38MAPK, and ERK1/2 were reversed by PKA inhibitor (H89), suggesting a PKA-dependent mechanism. In summary, our data suggest that ruscogenin inhibits activation of neutrophil through cPLA2, PAK, Akt, MAPKs, cAMP, and PKA signaling pathways. Increased PKA activity is associated with suppression of the phosphorylation of Akt, p38MAPK, and ERK1/2 pathways.