Troglitazone and pioglitazone interactions via PPAR-gamma-independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines

Troglitazone (Tro) and pioglitazone (Pio) activation of peroxisome proliferator-activated receptor (PPAR)- and PPAR--independent pathways was studied in cell lines derived from porcine renal tubules. PPAR--dependent activation of PPAR response element-driven luciferase gene expression was observed with Pio at 1 µM but not Tro at 1 µM. On the other hand, PPAR--independent P-ERK activation was observed with 5 µM Tro but not with Pio (5–20 µM). In addition, Pio (1–10 µM) increased metabolic acid production and activated AMP-activated protein kinase (AMPK) associated with decreased mitochondrial membrane potential, whereas Tro (1–20 µM) did not. These results are consistent with three pathways through which glitazones may act in effecting metabolic processes (ammoniagenesis and gluconeogenesis) as well as cellular growth: 1) PPAR--dependent and PPAR--independent pathways, 2) P-ERK activation, and 3) mitochondrial AMPK activation. The pathways influence cellular acidosis and glucose and glutamine metabolism in a manner favoring reduced plasma glucose in vivo. In addition, significant interactions can be demonstrated that enhance some physiological processes (ammoniagenesis) and suppress others (ligand-mediated PPAR- gene expression). Our findings provide a model both for understanding seemingly opposite biological effects and for enhancing therapeutic potency of these agents. peroxisome proliferator-activated receptor-; phospho-extracellular signal-regulated kinase; intracellular pH; Na⁺/H⁺ exchanger; AMP-activated protein kinase; mitochondria     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of G_s and G occurs and active G_s, measured as GTP-³⁵S bound, decreases. Because intracellular Ca²⁺ is elevated in Duchenne muscular dystrophy and G_s is known to activate the dihydropyridine receptor Ca²⁺ channel, whether laminin also altered intracellular Ca²⁺ was investigated. Laminin-1 decreases active (GTP-S-bound) G_s, and the Ca²⁺ channel is inhibited by laminin-1. The laminin ₁-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C₂C₁₂ myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain     

Obligatory role for phospholipase C-gamma(1) in villin-induced epithelial cell migration

While there is circumstantial evidence to suggest a requirement for phospholipase C-₁ (PLC-₁) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-₁ play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-₁. Inhibition of villin phosphorylation prevented villin-PLC-₁ complex formation as well as villin-induced cell migration. The absolute requirement for PLC-₁ in villin-induced cell migration was demonstrated by measuring cell motility in PLC-₁^–/– cells and by downregulation of endogenous PLC-₁. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-₁ at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration. fluorescence resonance energy transfer; actin     

Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IB kinase (IKK)/nuclear factor-B (NF-B) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of ₅₁ integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C- inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca²⁺ pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca²⁺]_i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC- activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC--PKC-IKK-NF-B signaling cascade. Another crucial factor, [Ca²⁺]_i increase, may at least be required to activate PKC needed for NF-B activation. nuclear factor-B; phosphatidylinositol 3-kinase; phospholipase C-; protein kinase C; intracellular Ca²⁺ concentration     

Functional analysis of Na+/K+-ATPase isoform distribution in rat ventricular myocytes

The Na⁺/K⁺-ATPase (NKA) is the main route for Na⁺ extrusion from cardiac myocytes. Different NKA -subunit isoforms are present in the heart. NKA-1 is predominant, although there is a variable amount of NKA-2 in adult ventricular myocytes of most species. It has been proposed that NKA-2 is localized mainly in T-tubules (TT), where it could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. However, there is controversy as to where NKA-1 vs. NKA-2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I_Pump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K_1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-1, K_1/2 = 141 ± 17 µM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, 63% of I_Pump generated by NKA-2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-2/NKA-1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-2 is 4.5 times higher in the T-tubules vs. ESL, whereas NKA-1 is almost uniformly distributed between the TT and ESL. T-tubules; Na⁺/K⁺ pump current; ouabain; external sarcolemma; detubulation     

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins

Heterotrimeric G_i proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G_i proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of G_i2 or G_i1/3 protein in G_i2-knockout (G_i2–/–) or G_i1/3-knockout (G_i1/3–/–) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from G_i2–/– mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from G_i1/3–/– mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B₂ (TxB₂) production was decreased in M harvested from G_i2–/– mice. Also, LPS-induced production of IL-10 and TxB₂ was decreased in M from G_i1/3–/– mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in G_i2–/– mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated G_i2–/– mice compared with WT mice. These data suggest that G_i proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. G_i protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung     

Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca²⁺-sensitive K⁺ (mtK_Ca) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK_Ca channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K⁺ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (_m). To test this hypothesis, we measured effects of NS-1619, a putative mtK_Ca channel opener, and valinomycin, a K⁺ ionophore, on mitochondrial respiration, _m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O₂·min^–1·mg protein^–1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O₂·min^–1·mg protein^–1, respectively, with the FADH₂-linked substrate succinate (+ rotenone); these effects were abolished by the mtK_Ca channel blocker paxilline. _m was not decreased by 10–30 µM NS-1619 with either substrate, but H₂O₂ release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H₂O₂ release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained _m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K⁺ influx during states 2 and 4 increases mitochondrial respiration while maintaining _m; this allows singlet electron uptake by O₂ and ROS generation. mitochondrial bioenergetics; heart mitochondria     

Mechanical loading and injury induce human myotubes to release neutrophil chemoattractants

The purpose of this study was to 1) test the hypothesis that skeletal muscle cells (myotubes) after mechanical loading and/or injury are a source of soluble factors that promote neutrophil chemotaxis and superoxide anion (O₂^–·) production and 2) determine whether mechanical loading and/or injury causes myotubes to release cytokines that are known to influence neutrophil responses [tumor necrosis factor- (TNF-), IL-8, and transforming growth factor-1 (TGF-1)]. Human myotubes were grown in culture and exposed to either a cyclic strain (0, 5, 10, 20, or 30% strain) or a scrape injury protocol. Protocols of 5, 10, and 20% strain did not cause injury, whereas 30% strain and scrape injury caused a modest and a high degree of injury, respectively. Conditioned media from strained myotubes promoted chemotaxis of human blood neutrophils and primed them for O₂^–· production in a manner that was dependent on a threshold of strain and independent from injury. Neutrophil chemotaxis, but not priming, progressively increased with higher magnitudes of strain. Conditioned media only from scrape-injured myotubes increased O₂^–· production from neutrophils. Concentrations of IL-8 and total TGF-1 in conditioned media were reduced by mechanical loading, whereas TNF- and active TGF-1 concentrations were unaffected. In conclusion, skeletal muscle cells after mechanical loading and injury are an important source of soluble factors that differentially influence neutrophil chemotaxis and the stages of neutrophil-derived reactive oxygen species production. Neutrophil responses elicited by mechanical loading, however, did not parallel changes in the release of IL-8, TGF-1, or TNF- from skeletal muscle cells. inflammation; cytokines; exercise; free radicals     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization     

Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca²⁺-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca²⁺ signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKII₂, a splice variant of the gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII₂ protein and a significant decrease in CaMKII protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII₂ in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII₂ activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKII₂ is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation. calmodulin kinase II; phenotype modulation     

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca²⁺-activated K⁺ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and ₁-subunits (BK-₁), or both - and ₄-subunits (BK-₄) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-₁ within a similar concentration range. Because methAEA could potentiate BK-, BK-₁, and BK-₄ with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-₁ did not change intracellular Ca²⁺ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB₁ antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB₂ agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener     

The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR ₇-subunit has a property of high Ca²⁺ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR₇ existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR ₅- and ₂-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR₇. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR₇ and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR₇, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR₇ has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR₇ with AC within plasma membranes. In addition, nAChR₇ may regulate the AC activity via Ca²⁺ within lipid rafts. cholesterol; PC-12 cells     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.

     

TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation

Expression of TNF-, a pleiotropic cytokine, is elevated during stroke and cerebral ischemia. TNF- regulates arterial diameter, although mechanisms mediating this effect are unclear. In the present study, we tested the hypothesis that TNF- regulates the diameter of resistance-sized (150-µm diameter) cerebral arteries by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. Laser-scanning confocal imaging revealed that TNF- increased Ca²⁺ spark and Ca²⁺ wave frequency but reduced global intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells of intact arteries. TNF- elevated reactive oxygen species (ROS) in smooth muscle cells of intact arteries, and this increase was prevented by apocynin or diphenyleneiodonium (DPI), both of which are NAD(P)H oxidase blockers, but was unaffected by inhibitors of other ROS-generating enzymes. In voltage-clamped (–40 mV) cells, TNF- increased the frequency and amplitude of Ca²⁺ spark-induced, large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel transients 1.7- and 1.4-fold, respectively. TNF--induced transient K_Ca current activation was reversed by apocynin or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a membrane-permeant antioxidant, and was prevented by intracellular dialysis of catalase. TNF- induced reversible and similar amplitude dilations in either endothelium-intact or endothelium-denuded pressurized (60 mmHg) cerebral arteries. MnTMPyP, thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase blocker that inhibits Ca²⁺ sparks, and iberiotoxin, a K_Ca channel blocker, reduced TNF--induced vasodilations to between 15 and 33% of control. In summary, our data indicate that TNF- activates NAD(P)H oxidase, resulting in an increase in intracellular H₂O₂ that stimulates Ca²⁺ sparks and transient K_Ca currents, leading to a reduction in global [Ca²⁺]_i, and vasodilation. cerebrovascular circulation; ryanodine-sensitive Ca²⁺ release channel; Ca²⁺-activated K⁺ channel; reactive oxygen species; vasodilation     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction