Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment?

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.

Signal transduction mechanisms involved in in vitro ram sperm capacitation

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 ± 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 ± 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium ([Formula: see text]) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in [Formula: see text] was unaffected, but the initial response and overall time to [Formula: see text] peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.