Airway smooth muscle (ASM) is subjected to changes in intracellular pH (pHi) due to physiological and pathological perturbations in acid/base balance, and such changes can exert a profound influence on ASM function. The overall purpose of this proposal is to determine the pathways of pHi regulation in ASM, the effect of alterations in pHi on ASM contractile function, and the cellular mechanisms by which pHi influences excitation- contraction coupling.
Specific Aim 1 is to define and characterize ion transport pathways involved in maintaining resting pHi in ASM and in mediating pHi recovery following an acid- and alkaline-load. The contribution of H+-transporting pathways (the Na-H exchanger, the H-K ATPase and the v-type H ATPase) and HCO3--transporting pathways (Cl-HCO3 transporters) will be assessed. This will be accomplished through spectrofluorimetric measurements of pHi in cultured ASM cells with BCECF. One ion transport pathway of particular interest is the H-K ATPase, since preliminary data suggests that selective inhibitors of this pathway cause relaxation of ASM.
Specific Aim 2 is identify a putative H-K ATPase in ASM by immunofluorescent staining with H-K ATPase antibodies, and by the measurement of H+ and K+ transport across the ASM cell membrane. It is hypothesized that ASM relaxation induced by H-K ATPase inhibitors is mediated by a change in pHi.
Specific Aim 3, therefore, is to determine the influence of pHi on ASM contractile function. This will be accomplished by examining the effect of physiological and pharmacological manipulations of pHi on tone and contractility of freshly isolated airways in vitro. It is hypothesized that an intracellular acidifications promotes ASM relaxation and alkalinization promotes contraction, and that these effects are mediated through an alterations in intracellular Ca2+ metabolism.
Specific Aim 4 is to investigate the relationship between pHi and intracellular Ca2+ concentration in ASM. This will be accomplished by the spectrofluorimetric measurement of Ca2+ in cultured ASM cells with Fura 2. Finally, it is hypothesized that ASM pHi, in addition to exerting an influence on muscle tone, may itself be influenced by receptor agonists which contract or relax ASM.
Specific Aim 5 is to determine the effect of receptor agonists on ASM pHi and to investigate the role that agonist- induced changes in pHi may play in excitation-contraction coupling.
