Protein Kinase A Inhibits Lysophosphatidic Acid-Induced Migration of Airway Smooth Muscle Cells

ABSTRACT Lysophosphatidic acid (LPA) is a bioactive phospholipid that is released from activated platelets and affects contractile properties of airway smooth muscle cells. However, possible roles of LPA on cell migration, one of the initial events of airway remodeling, are not clarified. This study aimed to examine the effects of LPA on migration and actin fiber formation in bovine tracheal smooth muscle cells (BTSMCs). Random and oriented cell migrations were examined with wound assay and Boyden chamber assay, respectively. Cytosolic actin fibers were stained with rhodaminephalloidin. Membrane translocation of RhoA, a hallmark of RhoA activation, was assessed by Western blotting. LPA augmented the migration of BTSMCs from wounded confluent monolayer but did not accelerate the chemotactic migration toward LPA. LPA also induced a transient actin reorganization and RhoA activation. Dense actin fibers were observed mainly in the wound edge but not in migrated cells, thereby suggesting the role of actin reorganization in the initiation of cell migration. LPA-induced actin fiber formation was blocked by Y27632 [R-(ϩ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide], an inhibitor of Rho kinase. Effects of LPA on migration and actin fiber formation were also inhibited by cAMP-elevating agents, i.e., dibutyryl cAMP, forskolin, isoproterenol, and theophylline. KT5720 (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], a protein kinase A (PKA) inhibitor, reversed the inhibitory actins of cAMP on LPA-induced responses. These results indicate that LPA induces cAMP/PKAsensitive, RhoA-mediated random migration of BTSMCs. Regulation of this mechanism would be beneficial for the control of airway remodeling. Proliferation and hypertrophy of airway smooth muscle cells cause airway remodeling Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that is generated in serum This work was supported in part by a grant-in-aid from the Japan Society for the Promotion of Science. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org

Overexpression of c-Met/hepatocyte growth factor receptors in human prostatic adenocarcinoma.

Hepatocyte growth factor (HGF) and c-met proto-oncogene product (c-Met) have varied biological functions in different tissues and have been implicated in mitogenic, motogenic and morphogenic responses in both organ regeneration and carcinogenesis. Some studies have suggested that the overexpression of c-Met and epidermal growth factor receptor (EGFR) are associated with growth advantage, while transforming growth factor-beta receptor II (TGF beta R II) is associated with growth disadvantage of human prostatic adenocarcinoma. However, it is unclear if the expression of c-Met correlates with the expression of EGFR and TGF beta R II, and with the proliferative status of human prostatic adenocarcinoma. Using immunohistochemical staining with anti-c-Met (C-12), anti-EGFR (NCL-EGFR) and anti-TGF beta R II (L-21) antibodies, we determined the frequency of expression of c-MET, EGFR, and TGF beta R II respectively in a series of 134 radical prostatectomy specimens. We evaluated the relationship between the expression of these receptors and clinicopathological characteristics. Overall, c-Met immunostaining was detected in 54 of 134 (40.3%) cases, EGFR in 45 (33.6%) and TGF beta R II in 64 (48.4%). The overexpression of c-Met was significantly more common in poorly differentiated (P &#60; 0.0001) and in the diffusely infiltrated specimens (P &#60; 0.0005). In contrast, TGF beta R II was significantly overexpressed in the well differentiated specimens (P &#60; 0.0001) and associated negatively with c-Met (P &#60; 0.0001). Overall, these data suggest that c-Met/HGF receptor and TGF beta R II overexpression may be involved in the differentiation of human prostatic adenocarcinoma, c-Met with de-differentiation and TGF beta R II with differentiation.</p

Multiple noncoding exons 1 of nuclear receptors NR4A family (nerve growth factor-induced clone B, Nur-related factor 1 and neuron-derived orphan receptor 1) and NR5A1 (steroidogenic factor 1) in human cardiovascular and adrenal tissues

金沢大学医薬保健研究域医学系Objective: Nuclear receptors are involved in a wide variety of functions, including aldosteronogenesis. Nuclear receptor families NR4A [nerve growth factor-induced clone B (NGFIB), Nur-related factor 1 (NURR1) and neuron-derived orphan receptor 1 (NOR1)] and NR2F [chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TFI), COUP-TFII and NR2F6) activate, whereas NR5A1 [steroidogenic factor 1 (SF1)] represses CYP11B2 (aldosterone synthase) gene transcription. The present study was undertaken to elucidate the mechanism of differential regulation of nuclear receptors between cardiovascular and adrenal tissues. Methods: We collected tissues of artery (n = 9), cardiomyopathy muscle (n = 9), heart muscle (noncardiomyopathy) (n = 6), adrenal gland (n = 9) and aldosterone-producing adenoma (APA) (n = 9). 5′-rapid amplification of cDNA ends (RACE) identified transcription start sites. Multiplex reverse-transcription PCR (RT-PCR) determined use of alternative noncoding exons 1 (ANEs). Results: In adrenocortical H295R cells, angiotensin II, KCl or cAMP, all stimulated CYP11B2 transcription and NR4A was upregulated, whereas NR2F and NR5A1 were downregulated. 5′-RACE and RT-PCR revealed four ANEs of NGFIB (NR4A1), three of NURR1 (NR4A2), two of NOR1 (NR4A3) and two of SF1 (NR5A1) in cardiovascular and adrenal tissues. Quantitative multiplex RT-PCR showed NR4A and NR5A1 differentially employed multiple ANEs in a tissue-specific manner. The use of ANEs of NGFIB and NURR1 was significantly different between APA and artery. Changes in use of ANEs of NGFIB and NOR1 were observed between cardiomyopathy and noncardiomyopathy. The NR4A mRNA levels in artery were high compared with cardiac and adrenal tissues, whereas the NR5A1 mRNA level in adrenal tissues was extremely high compared with cardiovascular tissues. Conclusion: NR4A and NR5A1 genes are complex in terms of alternative promoter use. The use of ANEs may be associated with the pathophysiology of the heart and adrenal gland. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Regulation of TRP channels: a voltage-lipid connection

TRIP (transient receptor potential) channels respond to a plethora of stimuli in a fine-tuned manner. We show here that both membrane potential and the level of PI (phosphatidylinositol) phosphates are efficient regulators of TRIP channel gating. Recent work has shown that this regulation applies to several members of the TRPV (TRP vanilloid) subfamily (TRPV1 and TRPV5) and the TRPM (TRP melastatin) subfamily (TRPM4/TRPM5/TRPM7/TRPM8), whereas regulation of members of the TRPC subfamily is still disputed. The mechanism whereby PIP2 (PI 4,5-bisphosphate) acts on TRPM4, a Ca2+- and voltage-activated channel, is shown in detail in this paper: (i) PIP2 may bind directly to the channel, (ii) PIP2 induces sensitization to activation by Ca2+, and (iii) PIP2 shifts the voltage dependence towards negative and physiologically more meaningful potentials. A PIP2-binding pocket seems to comprise a part of the TRIP domain and especially pleckstrin homology domains in the C-terminus.status: publishe

Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole

Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3- cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was ∼200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects. Copyright © 2008 Society for Neuroscience.This work was supported by grants from the Human Frontiers Science Program (RGP32/2004), the Belgian Federal Government (IUAP P5/05), the Research Foundation-Flanders (G.0172.03; G.0565.07), the Research Council of the KU Leuven (GOA 2004/07; EF/95/010), the Spanish Minsitry of Education (SAF2004-01011), the Generalitat Valenciana Predoctoral Fellowship Program (CTBPRB/2003/151), and the Fundación Marcelino Botín.Peer Reviewe

Direct Effect of Remifentanil and Glycine Contained in Ultiva® on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses.

BACKGROUND:Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord. METHODS:We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli. RESULTS:Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine. CONCLUSIONS:We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal and intravenous administrations

Antinociceptive effect of selective G protein-gated inwardly rectifying K+ channel agonist ML297 in the rat spinal cord.

The G protein-gated inwardly rectifying K+ (GIRK) channels play important signaling roles in the central and peripheral nervous systems. However, the role of GIRK channel activation in pain signaling remains unknown mainly due to the lack of potent and selective GIRK channel activators until recently. The present study was designed to determine the effects and mechanisms of ML297, a selective GIRK1/2 activator, on nociception in the spinal cord by using behavioral studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons. Rats were prepared for chronic lumber catheterization and intrathecal administration of ML297. The nociceptive flexion reflex was tested using an analgesy-meter, and the influence on motor performance was assessed using an accelerating rotarod. We also investigated pre- and post-synaptic actions of ML297 in spinal cord preparations by whole-cell patch-clamp recordings. Intrathecal administration of ML297 increased the mechanical nociceptive threshold without impairing motor function. In voltage-clamp mode of patch-clamp recordings, bath application of ML297 induced outward currents in a dose-dependent manner. The ML297-induced currents demonstrated specific equilibrium potential like other families of potassium channels. At high concentration, ML297 depressed miniature excitatory postsynaptic currents (mEPSCs) but not their amplitude. The ML297-induced outward currents and suppression of mEPSCs were not inhibited by naloxone, a μ-opioid receptor antagonist. These results demonstrated that intrathecal ML297 showed the antinociceptive effect, which was mediated through direct activation of pre- and post-synaptic GIRK channels. Selective GIRK channel activation is a promising strategy for the development of new agents against chronic pain and opioid tolerance