Airway smooth muscle relaxation results from a reduction in the frequency of Ca(2+ )oscillations induced by a cAMP-mediated inhibition of the IP(3 )receptor

BACKGROUND: It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca(2+ )oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca(2+ )oscillations. METHODS: The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca(2+ )signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy. RESULTS: All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca(2+ )oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca(2+ )oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca(2+ )oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca(2+ )stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca(2+ )available to stimulate contraction because an elevation of intracellular Ca(2+ )concentration induced by exposure to a Ca(2+ )ionophore (ionomycin) or by photolysis of caged-Ca(2+ )did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca(2+)-activated K(+ )channels, which would be expected to increase Ca(2+ )influx and contraction. By contrast, the photolysis of caged-IP(3 )in the presence of agonist, to further elevate the intracellular IP(3 )concentration, reversed the slowing of the frequency of the Ca(2+ )oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP(3)R to IP(3 )was reduced by FSK and this was supported by the reduced ability of IP(3 )to release Ca(2+ )in SMCs in the presence of FSK. CONCLUSION: These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca(2+ )oscillation frequency by reducing internal Ca(2+ )release through IP(3 )receptors

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7

Airway smooth muscle as a target of asthma therapy: history and new directions

Ultimately, asthma is a disease characterized by constriction of airway smooth muscle (ASM). The earliest approach to the treatment of asthma comprised the use of xanthines and anti-cholinergics with the later introduction of anti-histamines and anti-leukotrienes. Agents directed at ion channels on the smooth muscle membrane (Ca(2+ )channel blockers, K(+ )channel openers) have been tried and found to be ineffective. Functional antagonists, which modulate intracellular signalling pathways within the smooth muscle (β-agonists and phosphodiesterase inhibitors), have been used for decades with success, but are not universally effective and patients continue to suffer with exacerbations of asthma using these drugs. During the past several decades, research energies have been directed into developing therapies to treat airway inflammation, but there have been no substantial advances in asthma therapies targeting the ASM. In this manuscript, excitation-contraction coupling in ASM is addressed, highlighting the current treatment of asthma while proposing several new directions that may prove helpful in the management of this disease