Figure 6.

Activation of HASMCs by the OR2AG1-specific agonist amyl butyrate led to an influx of Ca²⁺ dependent on cAMP production and opening of CNG channels. (A) Amyl butyrate (300 μM) was applied for 30 s in Ringer's solution with 2 mM extracellular Ca²⁺ or in a Ca²⁺-free Ringer's solution with 5 mM EGTA. Under Ca²⁺ free conditions, the cytosolic Ca²⁺ increase was completely abolished (N = 3). (B) Amyl butyrate (300 μM) was applied for 30 s after a 5 min incubation with the adenylyl cyclase inhibitor SQ22536 (200 μM). Ca²⁺ influx was significantly reduced after incubation with SQ22536 (N = 3). (C) A 2 min incubation with MDL12330A (50 μM), an adenylyl cyclase inhibitor, and a following co-application with amyl butyrate (300 μM, duration: 30 s) significantly inhibited amyl the butyrate-induced Ca²⁺ increase (N = 4). (D) The CNG channel inhibitor L-cis-diltiazem (100 μM) was co-applied with amyl butyrate (300 μM) for 30 s and significantly reduced the Ca²⁺ influx induced by amyl butyrate. The inhibitory effect of L-cis-diltiazem was reversible after a wash-out (N = 5). Bars of all experiments indicate the stimulus duration. All error bars represent the ±SEM of at three to five independent experiments. Significance was tested with an unpaired two-sample Student's t-test or a Mann-Whitney U test. ^*p < 0.05, ^**p < 0.01.