Type 1 inositol (1,4,5)-trisphosphate receptor activates ryanodine receptor 1 to mediate calcium spark signaling in adult mammalian skeletal muscle.

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Regulation of Macrophage Polarization and Wound Healing.

BACKGROUND: Macrophages (Mφs) participate in wound healing by coordinating inflammatory and angiogenic processes. Mφs respond to environmental cues by adopting either "classically" activated (M1) proinflammatory or "alternatively" activated (M2a, M2b, M2c, M2d) wound healing phenotypes. THE PROBLEM: Mφ polarization is essential for wound healing and aberrations in this process are linked to several pathologies. It is important to elucidate molecular mechanisms underlying Mφ polarization. BASIC/CLINICAL SCIENCE ADVANCES: Mφs are categorized as proinflammatory (M1) or anti-inflammatory/wound healing (M2). M1 Mφs are observed in initial tissue damage responses, are induced by exogenous pathogen-associated molecular patterns or endogenous damage-associated molecular patterns, and exhibit increased phagocytosis and pro-inflammatory cytokine production, facilitating innate immunity and wound debridement. M2 Mφs predominate later in repair, express vascular endothelial growth factor, transforming growth factor beta, and interleukin 10 (IL-10), are activated by varied stimuli, assist in the resolution of inflammation, and promote tissue formation and remodeling. Recent work has characterized a novel "M2d" phenotype resulting from adenosine-dependent "switching" of M1 Mφs that exhibits a pattern of marker expression that is distinct from canonical IL-4/IL-13-dependent M2a Mφs. Recent studies have demonstrated important roles for specific transcriptional elements in M1 and M2a Mφ polarization, notably members of the interferon regulatory factor family interferon regulatory factor 5 (IRF5) and IRF4, respectively. The role of these IRFs in M2d polarization and wound healing remains to be determined. CLINICAL CARE RELEVANCE: Knowledge of microenvironmental signals and molecular mechanisms that mediate Mφ polarization should permit their manipulation to regulate important physiological processes and resolve pathological conditions. CONCLUSION: Proper Mφ polarization is essential to effective wound healing, and distinct phenotypes, such as the angiogenic M2d Mφ, may be of critical importance to this process. The IRF5 transcription factor has been shown to play a key role in M1 Mφ activation and the Jumonji domain containing-3-IRF4 pathway has been implicated in M2 Mφ activation.

Sites of protein kinase A activation of the human ClC-2 Cl(-) channel.

Human ClC-2 Cl(-) (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pH(o). Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pH(o) and to examine the relationship between PKA and low pH(o) activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pH(o) were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pH(o) activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pH(o) 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pH(o) 6 only RGET was needed for PKA activation of hClC-2. Low pH(o) activation of hClC-2 Cl(-) channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pH(o) 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pH(o) 7.4. RGET, but not RRAT, was sufficient for activation at pH(o) 6.0. However, in the RGET691(D) mutant, there was PKA activation at pH(o) 6.0.