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     

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     

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     

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     

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     

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     

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     

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     

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     

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     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

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     

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     

Na(+) pump alpha 2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice

The relative expression of ₁ - and ₂-Na⁺/K⁺-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the ₁-isoform serves a "housekeeping" role, whereas the ₂-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the ₁- or the ₂-Na⁺/K⁺-ATPase isoform was ablated were utilized. Both of these knockouts, and , are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in -aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; -aortas had intermediate values. In contrast, neonatal -aorta was similar to WT. Western blot analysis indicated a population of 70% ₁- and 30% ₂-isoforms in the WT. Thus in terms of the total Na⁺/K⁺-ATPase protein, the -aorta (at 70%) would be similar to the -aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual -isoforms of the Na⁺/K⁺-ATPase differ functionally and that the ₂-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the ₂-isoform is distributed differently than the ₁-isoform. Importantly, these isoforms do not localize to the same regions. sodium; potassium; ATPase; contraction; transgenic     

Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells

Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. ₁-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate G_q/11 signaling pathways that subsequently increase intracellular Ca²⁺ concentration ([Ca²⁺]_ic). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca²⁺]_ic. Avian tendon cells were analyzed for mRNA expression of ₁-adrenoceptors by RT-PCR. Avian tendons expressed the _1A- and _1B-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca²⁺-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca²⁺]_ic. KMD-3213, an _1A-adrenoceptor antagonist, significantly reduced the Ca²⁺ response. Other adrenoceptor antagonists had no effect on the Ca²⁺ response. The absence of extracellular Ca²⁺ also significantly reduced the response to NE, indicating that Ca²⁺ influx contributed to the rise in [Ca²⁺]_ic. This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca²⁺ response is coupled to the _1A-adrenoceptor subtype. -adrenoceptors; fibroblasts; catecholamines; tenocytes     

Activation of PLC-delta1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca²⁺ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G_i/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen²,D-Pen⁵]enkephalin, and A₁ agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca²⁺ influx and was not observed in the absence of extracellular Ca²⁺, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca²⁺ channels. Treatment of the cells with a G_q/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike G_i/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G₁₃ minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca²⁺ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca²⁺ release or Ca²⁺ influx via voltage-gated Ca²⁺ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by G_i/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca²⁺ influx via store-operated Ca²⁺ channels. phospholipase C; G protein     

Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence

The ₂-adrenergic receptor (₂-AR) and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both ₂-AR and BK_Ca channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the ₂-AR and the BK_Ca channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of ₂-AR colocalizing with the BK_Ca channel. Coimmunoprecipitation studies indicate that BK_Ca and ₂-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK_Ca channel blocker, paxilline, significantly antagonized the relaxant effect of the ₂-AR agonist ritodrine. These novel findings provide an insight into the coupling between the ₂-AR and BK_Ca channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. ₂-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction     

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin _IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on _IIb3, we examined _IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with _IIb3 in resting platelets and that 120 dyn/cm² shear stress leads to their disassociation from _IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the ₃ tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to _IIb3. Syk's disassociation from ₃ is inhibited when VWF binding to either GpIb-IX-V or _IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to _IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing _IIb3 with ₃ truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates _IIb3 function and suggest that shear-induced _IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the ₃-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction     

Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6

Ca⁺-induced Ca²⁺ release (CICR) in the heart involves local Ca²⁺ signaling between sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) ₁-subunit (1C_YM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing 1C_YM and RyR2 were vesiculated and exhibited spontaneous Ca²⁺ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with 1C_YM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca²⁺ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca²⁺ transients that required extracellular Ca²⁺. Depolarization-induced Ca²⁺ release in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing 1C_YM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca²⁺ current densities. In addition, the gain of CICR in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local _1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca²⁺ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca²⁺ release and the incidence of spontaneous Ca²⁺ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6. heart failure; dihydropyridine receptor; excitation-contraction coupling     

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming _1A-subunits of human P/Q-type Ca²⁺ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human _1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of _1A-WT with each _1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba²⁺ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of _1A-mutants increased, suggesting the presence of an _1A-mutant-specific suppression effect. When we coexpressed _1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of _1A-WT and mutant using a splice variant of _1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-_1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects. channelopathy; voltage clamp; Xenopus oocytes; cerebellum; splice variants