Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.     

Role of nitric oxide in beta3-adrenoceptor activation on basal tone of internal anal sphincter

Effects of activation of beta3-adrenoceptor (beta3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective beta3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a beta3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l-N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS [1-[2-(trifluoromethylphenyl)imidazole]] had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by beta3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by beta3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

Inhibition of glycogen synthase kinase-3beta is sufficient for airway smooth muscle hypertrophy

We examined the role of glycogen synthase kinase-3beta (GSK-3beta) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3beta, each of which inhibit GSK-3beta activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-beta, a proasthmatic cytokine. GSK-3beta inhibition increased mRNA expression of alpha-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon) attenuated LiCl- and SB216763-induced protein synthesis and expression of alpha-actin and SM22, indicating that eIF2B is required for GSK-3beta-mediated airway smooth muscle hypertrophy. eIF2Bepsilon siRNA also blocked CT-1- but not TGF-beta-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3beta-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3beta, blocked protein synthesis and alpha-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-beta. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased alpha-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3beta phosphorylation. These data suggest that inhibition of the GSK-3beta/eIF2Bepsilon translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-beta-induced hypertrophy does not depend on GSK-3beta/eIF2B signaling.     

PGE2 amplifies the effects of IL-1beta on vascular smooth muscle cell de-differentiation: a consequence of the versatility of PGE2 receptors 3 due to the emerging expression of adenylyl cyclase 8

Transition of vascular smooth muscle cells from a contractile/quiescent to a secretory/proliferative phenotype is one of the critical steps in atherosclerosis and is instigated by pro-inflammatory cytokines released from macrophages that have infiltrated into the vascular wall. In most inflammatory diseases, cell activation induced by these compounds leads to a massive production of type E2 prostaglandin (PGE2) which often takes over and even potentiates the pro-inflammatory cytokine-related effects. To evaluate PGE2 incidence on atheroma plaque development, we investigated whether and how this compound could enhance the dedifferentiation of smooth muscle cells initially induced by interleukin-1beta (IL-1beta). To address this issue, we took advantage of vascular smooth muscle cells in primary culture and tracked two markers: PLA2 secretion and alpha-actin filament disorganization. In such a context, we found that PGE2 synergizes with IL-1beta to further enhance the phenotype transition of smooth muscle cells, through cAMP-protein kinase A. As indicated by pharmacological studies, the full PGE2-dependent potentiation of IL-1beta induced PLA2 secretion is associated with a change of regulation exerted by the subtypes 3 G(i)-coupled PGE2 receptors toward adenylyl cyclase(s) activated by the subtype 4 G(s)-linked PGE2 receptor. Whereas on contractile cells, stimulated subtypes 3 inhibit type 4-dependent PLA2 secretion, this negative regulation is switched to positive on IL-1beta-treated cells. Using real time PCR, pharmacological tools and small interfering RNA (siRNA), we demonstrated that the different integration of PGE2 signals depends on the upregulation of calcium/calmodulin stimulable adenylyl cyclase 8.     

Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3 beta phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3 beta phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3beta antibody. Endogenous IGF-I stimulated GSK-3 beta phosphorylation and inhibited GSK-3 beta activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3 beta phosphorylation and the resulting GSK-3 beta inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced beta-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3 beta inhibitor. Endogenous IGF-I inhibited beta-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3 beta activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

The mechanism for the activation of latent TGF-beta during co-culture of endothelial cells and smooth muscle cells: cell-type specific targeting of latent TGF-beta to smooth muscle cells

Transforming growth factor-beta (TGF-beta) is secreted in a latent form and activated during co-culture of endothelial cells and smooth muscle cells. Plasmin located on the surface of endothelial cells is required for the activation of latent TGF-beta (LTGF-beta) during co-culture, and the targeting of LTGF-beta to the cellular surface is requisite for its activation. In the present study, the cellular targeting of LTGF- beta was examined. We detected the specific binding of 125I-large LTGF- beta 1 isolated from human platelets to smooth muscle cells but not to endothelial cells. A mAb against the latency-associated peptide (LAP) of large LTGF-beta 1 complex, which blocked the binding of 125I-large LTGF-beta 1 to smooth muscle cells, inhibited the activation of LTGF- beta during co-culture. The binding of 125I-large LTGF-beta 1 could not be competed either by mannose-6-phosphate (300 microM) or by the synthetic peptide Arg-Gly-Asp-Ser (300 micrograms/ml). These results indicate that the targeting of LTGF-beta to smooth muscle cells is required for the activation of LTGF-beta during co-culture of endothelial cells and smooth muscle cells. The targeting of LTGF-beta to smooth muscle cells is mediated by LAP, and the domain of LAP responsible for the targeting to smooth muscle cells may not be related to mannose-6-phosphate or an Arg-Gly-Asp sequence, both of which have been previously proposed as candidates for the cellular binding domains within LAP.     

Interleukin-1 beta induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta.

The effect of interleukin-1 beta on the production of non-prostanoid vasoactive factors by cultured rat aortic smooth muscle cells was investigated. Under bioassay conditions, the perfusate from a column of confluent cells grown on beads and treated with interleukin-1 beta (1 ng/ml for 18 to 24 hr) abolished the contraction of a canine coronary ring without endothelium contracted by phenylephrine (1 microM), while the perfusate from control cells had no effect. The relaxing activity of the perfusate was observed when transit times were increased from 1 sec to 5 min. Nitro L-arginine (100 microM) reversed the relaxations and L-arginine stereoselectively restored the relaxations. Interleukin-1 beta (1 ng/ml) evoked a time-dependent accumulation of cyclic GMP but not cyclic AMP in cultured smooth muscle cells. The transfer of fresh or stored (-70 degrees C) conditioned culture medium from interleukin-1 beta-treated cells but not from control cells, to cultured smooth muscle cells stimulated the production of cyclic GMP. These observations demonstrate that interleukin-1 beta induces the production of transferable factor which relaxes vascular smooth muscle and stimulates the production of cyclic GMP.     

The heparin binding domain of vitronectin is the region that is required to enhance insulin-like growth factor-I signaling

We have shown that vitronectin (Vn) binding to a cysteine loop sequence within the extracellular domain of the beta3-subunit (amino acids 177-184) of alphaVbeta3 is required for the positive effects of Vn on IGF-I signaling. When Vn binding to this sequence is blocked, IGF-I signaling in smooth muscle cells is impaired. Because this binding site is distinct from the site on beta3 to which the Arg-Gly-Asp sequence of extracellular matrix ligands bind (amino acids 107-171), we hypothesized that the region of Vn that binds to the cysteine loop on beta3 is distinct from the region that contains the Arg-Gly-Asp sequence. The results presented in this study demonstrate that this heparin binding domain (HBD) is the region of Vn that binds to the cysteine loop region of beta3 and that this region is sufficient to mediate the positive effects of Vn on IGF-I signaling. We provide evidence that binding of the HBD of Vn to alphaVbeta3 has direct effects on the activation state of beta3 as measured by beta3 phosphorylation. The increase in beta3 phosphorylation associated with exposure of cells to this HBD is associated with enhanced phosphorylation of the adaptor protein Src homology 2 domain-containing transforming protein C and enhanced activation MAPK, a downstream mediator of IGF-I signaling. We conclude that the interaction of the HBD of Vn binding to the cysteine loop sequence of beta3 is necessary and sufficient for the positive effects of Vn on IGF-I-mediated effects in smooth muscle cells.     

IL-1beta inhibits intestinal smooth muscle proliferation in an organ culture system: involvement of COX-2 and iNOS induction in muscularis resident macrophages

Intestinal inflammation causes hyperplasia of smooth muscle that leads to thickening of the smooth muscle layer, resulting in dysmotility. IL-1beta is a proinflammatory cytokine that plays a central role in intestinal inflammation. In this study, to evaluate the effect of IL-1beta on proliferation of ileal smooth muscle cells in vivo, we utilized an organ culture system. When rat ileal smooth muscle tissue was cultured under serum-free conditions for 3 days, most smooth muscle cells maintained their arrangement and kept their contractile phenotype. When 10% FBS was added, an increased number of smooth muscle cells per unit area was observed. Moreover, immunohistochemical staining for PCNA demonstrated that FBS induced proliferation of smooth muscle cells. IL-1beta inhibited the proliferative effect of FBS. Furthermore, IL-1beta upregulated inducible nitric oxide (NO) synthase and cyclooxygenase-2 mRNA and protein and thus stimulated NO and PGE(2) productions. Moreover, exogenously applied NO and PGE(2) inhibited the increase of bromodeoxyuridine-positive cells stimulated with FBS. Immunostaining revealed that the majority of cyclooxygenase-2 and inducible NO synthase was located in the dense network of macrophages resident in the muscularis, which were immunoreactive to ED2. Based on these findings, IL-1beta acts as an anti-proliferative mediator, which acts indirectly through the production of PGE(2) and NO from resident macrophage within ileal smooth muscle tissue.     

Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad

A pseudomonad has been isolated from sewage, which can utilize 3-chlorobenzoic acid as a sole carbon source. In cells grown on benzoate the enzymes of the -ketoadipic acid pathway are present. Considerable enzymic activities for chlorinated substrates were found in benzoate grown cells only for the oxygenation of 3-chlorobenzoate and the dehydrogenation of 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. 3-Chlorobenzoate grown cells show additional high activities for the turnover of 3- and 4-chlorocatechols and chloromuconic acids.Abbreviations Used DHB (-)-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (derived from the trivial name, dihydrodihydroxybenzoate) - 3- and 5-Cl-DHB correspondingly 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B4 and inhibited by inhibitors of arachidonic acid oxidation

The mechanism of agonist-induced desensitization of the beta adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both alpha and beta adrenergic receptors and nicotinic receptors. beta receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37 degrees but not at 4 degrees, induced a dose dependent internalization of the beta adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. beta adrenergic receptor internalization was very specific: the alpha adrenergic agonist Phenylefrine had almost no effect on beta receptor levels, while Isoproterenol treatment had no effect on the number of alpha adrenergic or nicotinic receptors expressed at the cell surface of these cells. beta adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

GABAA receptors are expressed and facilitate relaxation in airway smooth muscle

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)) channels and metabotropic (GABA(B)) receptors. GABA(A) channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although GABA(A) channels and GABA(B) receptors have been functionally identified on peripheral nerves in the lung, GABA(A) channels have never been identified on ASM itself. We detected the mRNA encoding of the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, delta-, gamma(1-3)-, pi-, and theta-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the GABA(A) alpha(4)-, alpha(5)-, beta(3)-, and gamma(2)-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The GABA(A) beta(3)-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific GABA(A) channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the GABA(A)-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional GABA(A) channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease.