Although allergic inflammation has a central function in the establishment and maintenance of airway hyperresponsiveness (AHR) in asthma, only 50% of patients with asthma are atopic (25
). Moreover, the degree of inflammation present in asthmatic lungs may not correlate with the degree of AHR (26
). Such findings suggest that intrinsic abnormalities in lung structural cells, including ASM, merit distinct consideration in severe or fatal disease. Unfortunately, only a few studies have addressed the excitation–contraction coupling and contractile function of ASM cells because of the difficulties in obtaining ASM cells in the setting of fatal asthma (death from asthma is relatively rare), and because of the lack of adequate techniques to address the contractile function of ASM cells in vitro
. Here we studied upstream, receptor-related events leading to cellular contraction (Ca2+
flux) and the expression of components of procontractile GPCR pathways in ASM cells from patients with and without asthma, in an attempt to uncover new or unique therapeutic targets.
Our experiments revealed differences in GPCR-evoked Ca2+
mobilization in asthmatic and nonasthmatic ASM cells. We found reduced peak Ca2+
concentrations after stimulation with histamine, but not bradykinin or thrombin, in asthmatic ASM cells compared with nonasthmatic ASM cells. Our results differ from those of Mahn and colleagues (27
), who found reduced responses of asthmatic ASM to bradykinin. They observed that the recovery of Ca2+
concentrations to baseline was delayed in asthmatic cells, which they attributed to depleted sarcoplasmic reticulum Ca2+
stores resulting from decreased SERCA concentrations. In contrast, we detected equivalent responses to the SERCA inhibitor thapsigargin and similar kinetics of Ca2+
recovery after the GPCR stimulation of healthy and asthmatic ASM cells. These results indicate that Ca2+
stores were comparable in our samples. Our study and that of Mahn and colleagues contain the key difference that Mahn and colleagues (27
) obtained bronchial specimens from subjects with mild to moderate asthma, whereas we studied ASM cells from bronchi obtained postmortem from patients who died of asthma.
We found that the total expression of receptor-stimulated signaling components such as Gαq and PLCβ1 was equivalent in our ASM samples, whereas amounts of bradykinin B2 and adenosine A2a receptor were significantly increased in asthmatic cells relative to control cells. We are unaware of any extensive comparison of GPCR expression in asthmatic and nonasthmatic ASM cells reported by others, and B2 receptor concentrations were not assessed in the study of Mahn and colleagues (27
). Cytokines, including IFN-γ and TGF-β, which are both increased in asthmatic airways (5
), up-regulate ASM GPCR expression in cultured ASM cells (cysteinyl leukotriene D1 and B2, respectively) (28
). Asthmatic ASM cells also express higher concentrations of C-C chemokine receptor 3, which may mediate migration and cytokine secretion (30
). Although the amounts of Gαq were increased in bronchial tissue from allergen-sensitized and challenged mice in a previous study, we observed no changes in Gαq expression in asthmatic human ASM cells compared with control cells. These findings are similar to those of McGraw and colleagues (31
), who demonstrated unchanged Gαq concentrations in Gαi2 inhibitory peptide transgenic mice, which demonstrate increased AHR relative to wild-type mice in the absence of allergic inflammation. Currently, the physiological significance of altered G-protein expression in these models has not been clearly established.
Because no obvious reductions in receptor, G-protein, or effector expression could account for the impaired histamine-evoked Ca2+
mobilization in asthmatic ASM cells, we analyzed the expression of other genes that could potentially regulate excitation–contraction pathways. Consistent with the proliferative phenotype of asthmatic cells observed by others (8
), the expression of cyclins and antiapoptosis factors (Akt, Elk1/4, and Bcl2) was substantially increased compared with that in control cells. In addition, asthmatic cells displayed a synthetic/secretory phenotype, as evidenced by the up-regulation of cytokines and matrix-modifying enzymes such as MMP9, VEGF, C-C chemokine ligand 2, and connective tissue growth factor, among others, as shown in previous studies (5
). We also detected increased expression of adenosine A2a and sphingosine-1–phosphate receptors in asthmatic ASM cells, which were reported by others to affect bronchial smooth muscle tone (33
), suggesting physiological relevance to asthma.
To sum up most important finding of this study, the expression of RGS5 mRNA and protein was significantly up-regulated in asthmatic ASM compared with nonasthmatic cells and lung tissue. Asthmatic ASM cells displayed reduced histamine responses relative to nonasthmatic ASM cells, but had comparable to responses to bradykinin and thrombin. These results suggest that RGS5 is a regulator of histamine-mediated contractile signaling in asthmatic ASM, but may not couple to bradykinin or PAR1 receptors in these cells. Our previous work demonstrated that short interfering (si)RNA-mediated RGS5 knockdown in healthy ASM cells augmented thrombin responses (19
). In a separate study, RGS5 specifically inhibited angiotensin II type 1A receptor–mediated but not muscarinic M3 receptor–mediated ERK activation in vascular smooth muscle cells. In contrast, Anger and colleagues found that RGS5 impaired carbachol-mediated IP3 formation and ERK activation in COS-7 cells expressing M3 receptors (36
). Collectively, these results suggest the receptor-selective, and perhaps cell type–specific, regulation of signaling by RGS5 (37
We also demonstrated that the overexpression of RGS5 inhibited PCLS contraction to carbachol. A current limitation of our PCLS transfection involved the nonselective overexpression/knockdown in the slices, and future studies using lentivirus driven by a smooth muscle–specific promoter will allow us to achieve selective expression in ASM. However, agonist-induced bronchoconstriction is visualized directly, which is mediated by ASM contraction. Although the contribution of factors secreted by epithelial cells or other cells cannot be formally excluded, the sections are aggressively washed immediately before the addition of an agonist, which induces a reduction in airway diameter within minutes. We showed that RGS5 overexpression blunts the Ca2+
signaling evoked by procontractile ligands, including carbachol (). In addition, because we observed previously that PCLS from Rgs5–/–
mice contracted significantly more to carbachol than did airways from WT mice (19
), these studies indicate that RGS5 is a physiologically relevant modulator of airway contraction.
We also determined previously that the prolonged exposure of ASM to β-adrenergic agonists down-regulates RGS5 expression (19
). Thus, we may be underestimating the extent of RGS5 up-regulation in ASM from patients with severe asthma who died of asthma, given that such patients were probably exposed to sustained high doses of β-agonist. What mechanisms might underlie the increased RGS5 quantities in asthmatic ASM? RGS5 is up-regulated in arterial smooth muscle in models of skin wound–healing and tumor angiogenesis (38
), suggesting that extracellular matrix–modifying enzymes and growth factors linked to airway remodeling in asthma also modulate RGS5 expression. Inflammatory cytokines (Th2-related, IL-1, and TNF-α) also directly influence GPCR responsiveness (39
). IL-13 increases the Ca2+
responsiveness of ASM to histamine, bradykinin, and acetylcholine (Ach), and the contraction of tracheas to Ach (40
). Although our preliminary work indicates that IL-13 does not affect RGS5 expression in ASM (data not shown), numerous cytokines and chemokines modify RGS protein transcription in other cell types (18
Finally, whether the up-regulation of RGS5 in asthmatic ASM is beneficial or maladaptive remains unclear. Recently, Li and colleagues demonstrated that transgenic mice expressing a cardiac-specific Rgs5
transgene were resistant to hypertrophic cardiomyopathy and fibrosis, whereas Rgs5–/–
mice were more sensitive to pressure overload–induced cardiomyopathy than were WT mice (41
). The up-regulation of RGS5 could be a compensatory event that protects ASM from chronic hyperstimulation in asthma. Future studies of bronchial contraction in mice with varying amounts of RGS5 expression in models of allergic and nonallergic pulmonary inflammation will be needed to address this and other issues more fully.