Coupling of store-operated calcium entry to vasoconstriction is acid-sensing ion channel 1a dependent in pulmonary but not mesenteric arteries

Figures

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  Fig 1. L-type Ca2+ channels contribute to ET-1 induced vasoconstriction in mesenteric but not pulmonary arteries. Representative traces (top panels) and summary data showing vasoconstriction (percent baseline inner diameter; middle panels) and changes in fura-2 ratio (F340/F380; bottom panels) in response to endothelin-1 (ET-1; 10−11–10−7 or 10−11–10−8) in the presence or absence of diltiazem (50 μM) in small pulmonary (A; left panels) or mesenteric (B; right panels) arteries. n = 4–6 per group, values are means ± SEM. *p ≤ 0.05 vs. vehicle; ** p < 0.01; analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test.

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  Fig 2. Orai1 and ASIC1a are expressed in pulmonary and mesenteric arteries and VSMC. Representative PCR gels (A) showing expression of Orai1 (357 bp) and ASIC1a (305 bp) in intact pulmonary arteries (PA), mesenteric arteries (MA), and brain tissue (positive control). All lanes were loaded with 5 μL cDNA. β-actin (244 bp) was used as a loading control for intact arteries. Representative western blots (B left) showing protein expression of Orai1 and ASIC1 (~100 kDa and 60 kDa) in intact PA, MA, and brain tissue (positive control). Coomassie blue staining shows equal protein loading between samples (B; right). Each experiment was replicated 3 times.

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  Fig 3. Store-operated Ca2+ entry is coupled to vasoconstriction in pulmonary but not mesenteric arteries. Representative traces and summary data of SOCE (A & B) and SOCE-induced vasoconstriction (C & D) in pulmonary and mesenteric resistance arteries. All studies were done in the presence of diltiazem (50μM) and cyclopiazonic acid (10μM). n = 7–10 animals/group. ***p ≤ 0.001 or **** p ≤ 0.0001 vs. pulmonary; analyzed by unpaired t-test.

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  Fig 4. Store-operated Ca2+ entry is dependent on ASIC1a in pulmonary but not mesenteric arteries. SOCE-induced changes in fura-2 ratio (Δ F340/F380; top panels) and associated vasoconstriction (% baseline; bottom panels) in small pulmonary and mesenteric arteries, respectively. Studies were conducted in the presence or absence of PcTX1 (20 nM) or AnCoA4 (20 μM). Vehicle data are the same as Fig 3. n = 5–10 animals/group. **p ≤ 0.01 vs. vehicle; *** p ≤ 0.001 vs. vehicle; analyzed by one-way ANOVA and individual groups compared with the Tukey’s multiple comparison test.

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  Fig 5. Store depletion increases the interaction between Orai1 and STIM1 in VSMCs. Representative images of Orai1/STIM1 interaction in pulmonary (A) and mesenteric (D) VSMCs under basal conditions (BL; left) or following store-depletion (SD; right). Summary data showing the average number of puncta per cell (B and E) and puncta size (C and F) in pulmonary (B-C) and mesenteric (E-F) VSMCs. The nuclei are labeled with SYTOX (green), actin is labeled with Alexa Fluor 647 Phalloidin (blue), and puncta formation (white). N = 7–10; each data point represents an individual well taken from 1–3 experiments (cell cultures generated from different animals and ran on different days). *P ≤ 0.05 vs. baseline.

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  Fig 6. Store depletion increases the interaction between ASIC1a and STIM1 in pulmonary, but not in mesenteric VSMCs. Representative images of ASIC1a/STIM1 interaction in pulmonary (A) and mesenteric (D) VSMCs under basal conditions (BL; left) and following store-depletion (SD; right). Summary data showing the average number of puncta per cell (B and E) and puncta size (C and F) in pulmonary (B-C) and mesenteric (E-F) VSMCs. The nuclei are labeled with SYTOX (green), actin is labeled with Alexa Fluor 647 Phalloidin (blue), and puncta formation (white). n = 6–7 wells; each data point represents an individual well taken from 1–3 experiments (cell cultures generated from different animals and ran on different days). *P < 0.05 vs. baseline; **P < 0.01 vs. baseline; *** p < 0.001 vs. baseline; analyzed by unpaired t-test, except for C which was analyzed using Mann-Whitney test.

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  Fig 7. Vasoconstrictor and arterial wall [Ca2+]i responses to ET-1 are attenuated in response to ASIC1a inhibition and augmented in response to Orai1 inhibition in pulmonary arteries. Vasoconstriction (percent baseline inner diameter; top panels) and changes in fura-2 ratio (F340/F380; bottom panels) in response to endothelin-1 (ET-1; 10−11–10−7 or 10−11–10−8) in the presence or absence of PcTX1 (20 nM) or AnCoA4 (20 μM) in small pulmonary (A) and mesenteric (B) arteries. N = 4–7 per group, values are means ± SEM. *p ≤ 0.05 vs. vehicle; ** p < 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; analyzed by two-way anova followed by Dunnett’s multiple comparisons test.