However, their role in the adherence of T cells to asthmatic ASM remains to be investigated. the molecular mechanisms that modulate BHR in asthma (reviewed in (15)). Open in a separate window Physique 1 Excitation-contraction coupling in airway easy muscleEffects of pro-inflammatory cytokines and 2-adrenergic receptor agonists on excitation-contraction coupling in ASM cells. Contractile agonists activate receptors that influence intracellular signaling, affecting calcium homeostasis and sensitization as well as the function and expression of GPCRs and CD38. Inflammatory cytokines bind to receptors and modulate calcium homeostasis by increasing expression of CD38 and increasing Ca2+ release from the sarcoplasmic reticulum. Inflammatory cytokines such as IL-13, IL-1 and TNF- also increase Rho kinase activity to modulate the calcium sensitization pathways. 2-adrenergic receptor agonists regulate calcium homeostasis and calcium sensitization by inhibiting RhoA activation, Ca2+ release from the sarcoplasmic reticulum, and actin-myosin crossbridging. cADPR, cyclic ADP ribose; CaM, calmodulin; DAG, diacylglycerol; GEF, guanine exchange factor; GPCR, G-proteinCcoupled receptor; IP3, inositol tri-phosphate; MLC, myosin light chain; MLCK, myosin light Temanogrel chain kinase; PIP2, phophatylinositol 4,5 biphosphate; PLC, phospholipase C; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; 2AR, 2-adrenergic receptor. The level of intracellular calcium regulates, in part, ASM shortening. Activation of an ASM cell by an agonist induces a rapid rise in [Ca2+]i, associated with the release of intracellular calcium stores, to a peak level roughly tenfold higher than the resting level (100 nM to greater than 1 M with maximum agonist stimulation). Following this peak, calcium levels fall but remain elevated provided that the excitatory stimulus remains present. The elevation in [Ca2+]i activates the calcium/calmodulin-sensitive myosin light chain kinase (MLCK), leading to phosphorylation of the regulatory myosin light chain (MLC20) at Serine 19. Phosphorylation of this residue by myosin ATPase activity initiates crossbridge cycling between myosin and actin. ATP binding, hydrolysis and ADP release continue as long as MLC20 is usually phosphorylated; dephosphorylation by the MLC phosphatase terminates crossbridge cycling and relaxes easy muscle (reviewed in (16)). Considering the central role of Ca2+ in regulating ASM contractile function, investigators postulate that alterations in Ca2+-regulatory mechanisms likely impair ASM contractility. Studies using cultured human tracheal or bronchial easy muscle cells, as models of ASM responsiveness, convincingly exhibited that Gq-protein coupled receptors (GPCR)-associated signaling in ASM can be modulated by a variety of inflammatory stimuli. Cytokines, such as TNF-, augment agonist-induced ASM contractility by enhancing, in a non-specific manner, agonist-evoked Ca2+ transients (to bradykinin, carbachol) (15). The hypothesis that changes in GPCR-associated Ca2+ signaling represent an important mechanism underlying the development of BHR has also been supported by other studies. Tao and colleagues showed that ASM cells derived from hyperresponsive inbred rats have an augmented bradykinin-induced Ca2+ response when compared to ASM cells derived from normoresponsive rats (17). Deshpande and colleagues exhibited that in addition to TNF-, other cytokines including IL-1 and, in to a lesser degree, IFN augments Ca2+ responses induced by carbachol, bradykinin and thrombin (18). In a similar manner, IL-13, a Th2 type important mediator Temanogrel in allergic asthma (19), also non-specifically increased Ca2+ responses to agonists (20C23). Microarray technology used to study the modulation of gene expression of ASM by IL-13 revealed a diversity of potential molecular mechanisms influencing ASM responsiveness, including changes in cytoskeletal proteins, receptors or calcium regulators (24). Together, these data show that pro-asthmatic cytokines, in a nonspecific manner, enhance GPCR-associated Ca2+ responses in ASM, a mechanism likely to affect ASM contractility. Reports in C3H/HeJ, Balb/C and A/J mice revealed that differences in ASM contractility among species may not require changes in GPCR agonist-induced Ca2+ responses but rather involve changes in the Ca2+ sensitivity of the contractile apparatus (25). A possible mechanism involves the small monomeric G protein Rho that can augment ASM contractility by increasing levels of MLC phosphorylation via the Rho-activated kinase (ROCK) dependent suppression of MLC phosphatase (26, 27). Both RhoA and ROCK are activated by a variety of stimuli associated with the development of BHR including cytokines (28C31), sphingolipids (32C34), mechanical stress (35) and isoprostane (36). The RhoA/Rho kinase pathway regulates the expression of serum response factor-dependent easy muscle specific genes in canine ASM cells (37), a mechanism that identifies the importance of the Rho-kinase pathway in maintaining a contractile phenotype recently described in bovine ASM tissues (38). Rho pathways modulate diverse.Studies conducted using intact tissue, as opposed to cells from patients with asthma, have revealed increased Temanogrel immunohistochemical detection of a marker of proliferation C proliferative cell nuclear antigen, indicating that evidence for increased proliferation of ASM is not merely a phenomenon of cell culture (5, 126). Table 2 Modulation of human airway smooth muscle proliferation# could contribute to unopposed growth (129). It was originally thought that the structural changes in asthmatic airways that constitute airway remodeling developed as a result of a persistent inflammatory stimulus. enhancing ASM contraction and/or altering ASM relaxation (see Physique 1). Understanding the mechanisms by which inflammatory mediators modulate ASM contractile reactivity may offer new insight into the molecular mechanisms that modulate BHR in asthma (reviewed in (15)). Open in a separate window Physique 1 Excitation-contraction coupling in airway easy muscleEffects of pro-inflammatory cytokines and 2-adrenergic receptor agonists on excitation-contraction coupling in ASM cells. Contractile agonists activate receptors that influence intracellular signaling, affecting calcium homeostasis and sensitization as well as the function and expression of GPCRs and CD38. Inflammatory cytokines bind to receptors and modulate calcium homeostasis by increasing expression of CD38 and increasing Ca2+ release from the sarcoplasmic reticulum. Inflammatory cytokines such as IL-13, IL-1 and TNF- also increase Rho kinase activity to modulate the calcium sensitization pathways. 2-adrenergic receptor agonists regulate calcium homeostasis and calcium sensitization by inhibiting RhoA activation, Ca2+ release from the sarcoplasmic reticulum, and actin-myosin crossbridging. cADPR, cyclic ADP ribose; CaM, calmodulin; DAG, diacylglycerol; GEF, guanine exchange factor; GPCR, G-proteinCcoupled receptor; IP3, inositol tri-phosphate; MLC, myosin light chain; MLCK, myosin light chain kinase; PIP2, phophatylinositol 4,5 biphosphate; PLC, phospholipase C; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; 2AR, 2-adrenergic receptor. The level of intracellular calcium regulates, in part, ASM shortening. Activation of an ASM cell by an agonist induces a rapid rise in [Ca2+]i, associated with the release of intracellular calcium stores, to a peak level roughly tenfold higher than the resting level (100 nM to greater than 1 M with maximum agonist stimulation). Following this peak, calcium levels fall but remain elevated provided that the excitatory stimulus remains present. The elevation in [Ca2+]i activates the calcium/calmodulin-sensitive myosin light chain kinase (MLCK), leading to phosphorylation of the regulatory myosin light chain (MLC20) at Serine FLJ16239 19. Phosphorylation of this residue by myosin ATPase activity initiates crossbridge cycling between myosin and actin. ATP binding, hydrolysis and ADP release continue as long as MLC20 is phosphorylated; dephosphorylation by the MLC phosphatase terminates crossbridge cycling and relaxes smooth muscle (reviewed in (16)). Considering the central role of Ca2+ in regulating ASM contractile function, investigators postulate that alterations in Ca2+-regulatory mechanisms likely impair ASM contractility. Studies using cultured human tracheal or bronchial smooth muscle cells, as models of ASM responsiveness, convincingly demonstrated that Gq-protein coupled receptors (GPCR)-associated signaling in ASM can be modulated by a variety of inflammatory stimuli. Cytokines, such as TNF-, augment Temanogrel agonist-induced ASM contractility by enhancing, in a non-specific manner, agonist-evoked Ca2+ transients (to bradykinin, carbachol) (15). The hypothesis that changes in GPCR-associated Ca2+ signaling represent an important mechanism underlying the development of BHR has also been supported by other studies. Tao and colleagues showed that ASM cells derived from hyperresponsive inbred rats have an augmented bradykinin-induced Ca2+ response when compared to ASM cells derived from normoresponsive rats (17). Deshpande and colleagues demonstrated that in addition to TNF-, other cytokines including IL-1 and, in to a lesser degree, IFN augments Ca2+ responses induced by carbachol, bradykinin and thrombin (18). In a similar manner, IL-13, a Th2 type important mediator in allergic asthma (19), also non-specifically increased Ca2+ responses to agonists (20C23). Microarray technology used to study the modulation of gene expression of ASM by IL-13 revealed a diversity of potential molecular mechanisms influencing ASM responsiveness, including changes in cytoskeletal proteins, receptors or calcium regulators (24). Together, these data show that pro-asthmatic cytokines, in a nonspecific manner, enhance GPCR-associated Ca2+ responses in ASM, a mechanism likely to affect ASM contractility. Reports in C3H/HeJ, Balb/C and A/J mice revealed that differences in ASM contractility among species may not require changes in GPCR agonist-induced Temanogrel Ca2+ responses but rather involve changes in the Ca2+ sensitivity of the contractile apparatus (25). A possible mechanism involves the small monomeric G protein Rho that can augment ASM contractility by increasing levels of MLC phosphorylation via the Rho-activated kinase (ROCK) dependent suppression of MLC phosphatase (26, 27). Both RhoA and ROCK are activated by a variety of stimuli associated with the development of BHR including cytokines (28C31), sphingolipids (32C34), mechanical stress (35) and isoprostane (36). The RhoA/Rho kinase pathway regulates the expression of serum response factor-dependent smooth muscle specific genes in canine ASM cells (37), a mechanism that identifies the importance of the Rho-kinase pathway in maintaining a contractile phenotype recently described in bovine ASM tissues (38). Rho pathways modulate diverse cellular responses in ASM cells including the regulation of Ca2+ influx (39) and cell proliferation (40). Possibly, abnormal RhoA activity and/or expression will dramatically alter ASM contractility not only via the Ca2+ sensitization but also through the increased expression of Rho-dependent contractile proteins. A report using the Y-27632 inhibitor.