Reverse mode Na ⁺/Ca ²⁺ exchange mediated by STIM1 contributes to Ca ²⁺ influx in airway smooth muscle following agonist stimulation

Abstract

Background

Agonist stimulation of airway smooth muscle (ASM) results in IP ₃ mediated Ca ²⁺ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na ⁺ influx. This localised increase in Na ⁺ levels can potentially switch the Na ⁺/Ca ²⁺ exchanger into reverse mode and so result in a further influx of Ca ²⁺. The aim of this study was to characterise the expression and physiological function of the Na ⁺/Ca ²⁺ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca ²⁺ influx into these cells.

Methods

The expression profile of NCX (which encodes the Na ⁺/Ca ²⁺ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca ²⁺ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.

Results

NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na ⁺ loading and addition of extracellular Ca ²⁺ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca ²⁺ and induced relaxation of porcine peripheral airways.

Conclusions

Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca ²⁺ homeostasis and link store depletion via STIM1 directly with NCX activation.