Shear stress-induced NO production is dependent on ATP autocrine signaling and capacitative calcium entry

Abstract

Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production.

Keywords: ATP; capacitative calcium entry; endothelial cells; nitric oxide; shear stress.

Figure 1

Degradation of endogenous ATP attenuates shear stress-induced NO production. A-D. Sample traces of the NO response before (solid line) and after (dotted line) treatment with 1U/mL of apyrase. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change (at 50s). Sample responses from a step change to A. 1 dyn/cm² B. 6 dyn/cm² C. 10 dyn/cm². D. 20 dyn/cm². E. Within an experiment, apyrase showed an inhibition of the shear stress-induced NO response; however, due to the variability between experiments, we normalized the responses for an untreated step change to 10 dyn/cm² within each experiment. Comparisons of the Δ[NO] response before and after treatment with apyrase. Δ[NO] between untreated and apryase treated cells were found to be statistically significant for step changes to 6, 10 and 20 dyn/cm² but not to 1 dyn/cm². Δ[NO] for apyrase treated cells averaged 57% for 6 dyn/cm², 77% for 10 dyn/cm² and 80% for 20 dyn/cm² of the untreated responses. (Mean and SEM, one-tailed t-test, *p<0.05, n=4)

Figure 1

Degradation of endogenous ATP attenuates shear stress-induced NO production. A-D. Sample traces of the NO response before (solid line) and after (dotted line) treatment with 1U/mL of apyrase. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change (at 50s). Sample responses from a step change to A. 1 dyn/cm² B. 6 dyn/cm² C. 10 dyn/cm². D. 20 dyn/cm². E. Within an experiment, apyrase showed an inhibition of the shear stress-induced NO response; however, due to the variability between experiments, we normalized the responses for an untreated step change to 10 dyn/cm² within each experiment. Comparisons of the Δ[NO] response before and after treatment with apyrase. Δ[NO] between untreated and apryase treated cells were found to be statistically significant for step changes to 6, 10 and 20 dyn/cm² but not to 1 dyn/cm². Δ[NO] for apyrase treated cells averaged 57% for 6 dyn/cm², 77% for 10 dyn/cm² and 80% for 20 dyn/cm² of the untreated responses. (Mean and SEM, one-tailed t-test, *p<0.05, n=4)

Figure 1

Degradation of endogenous ATP attenuates shear stress-induced NO production. A-D. Sample traces of the NO response before (solid line) and after (dotted line) treatment with 1U/mL of apyrase. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change (at 50s). Sample responses from a step change to A. 1 dyn/cm² B. 6 dyn/cm² C. 10 dyn/cm². D. 20 dyn/cm². E. Within an experiment, apyrase showed an inhibition of the shear stress-induced NO response; however, due to the variability between experiments, we normalized the responses for an untreated step change to 10 dyn/cm² within each experiment. Comparisons of the Δ[NO] response before and after treatment with apyrase. Δ[NO] between untreated and apryase treated cells were found to be statistically significant for step changes to 6, 10 and 20 dyn/cm² but not to 1 dyn/cm². Δ[NO] for apyrase treated cells averaged 57% for 6 dyn/cm², 77% for 10 dyn/cm² and 80% for 20 dyn/cm² of the untreated responses. (Mean and SEM, one-tailed t-test, *p<0.05, n=4)

Figure 1

Degradation of endogenous ATP attenuates shear stress-induced NO production. A-D. Sample traces of the NO response before (solid line) and after (dotted line) treatment with 1U/mL of apyrase. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change (at 50s). Sample responses from a step change to A. 1 dyn/cm² B. 6 dyn/cm² C. 10 dyn/cm². D. 20 dyn/cm². E. Within an experiment, apyrase showed an inhibition of the shear stress-induced NO response; however, due to the variability between experiments, we normalized the responses for an untreated step change to 10 dyn/cm² within each experiment. Comparisons of the Δ[NO] response before and after treatment with apyrase. Δ[NO] between untreated and apryase treated cells were found to be statistically significant for step changes to 6, 10 and 20 dyn/cm² but not to 1 dyn/cm². Δ[NO] for apyrase treated cells averaged 57% for 6 dyn/cm², 77% for 10 dyn/cm² and 80% for 20 dyn/cm² of the untreated responses. (Mean and SEM, one-tailed t-test, *p<0.05, n=4)

Figure 1

Degradation of endogenous ATP attenuates shear stress-induced NO production. A-D. Sample traces of the NO response before (solid line) and after (dotted line) treatment with 1U/mL of apyrase. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change (at 50s). Sample responses from a step change to A. 1 dyn/cm² B. 6 dyn/cm² C. 10 dyn/cm². D. 20 dyn/cm². E. Within an experiment, apyrase showed an inhibition of the shear stress-induced NO response; however, due to the variability between experiments, we normalized the responses for an untreated step change to 10 dyn/cm² within each experiment. Comparisons of the Δ[NO] response before and after treatment with apyrase. Δ[NO] between untreated and apryase treated cells were found to be statistically significant for step changes to 6, 10 and 20 dyn/cm² but not to 1 dyn/cm². Δ[NO] for apyrase treated cells averaged 57% for 6 dyn/cm², 77% for 10 dyn/cm² and 80% for 20 dyn/cm² of the untreated responses. (Mean and SEM, one-tailed t-test, *p<0.05, n=4)

Figure 2

Apyrase attenuates shear stress-induced eNOS phosphorylation. Bar graph comparing shear stress-induced eNOS phosphorylation after 3 min in untreated and apyrase treated cells. Phosphorylation increased in untreated cells after 3 mins and was attenuated when endogenous ATP was degraded using apyrase as compared to the no flow condition. Responses are normalized by the untreated response (Mean and SEM were plotted, *p<0.05 one-tailed t-test n=5 each condition).

Figure 3

Inhibition of purinergic receptors attenuates shear stress-induced NO production. Purinergic receptors were blocked with using the non-specific blocker suramin. Cells were incubated with 200 μM of suramin for 10 min prior to insertion into the chamber. Each membrane was exposed to a series of 4 step changes from 0.1 to 10 dyn/cm². A. Sample traces of the response for untreated (solid line) and suramin (dotted line) treated cells. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change occurring at 50s. B. Bar graph comparing the Δ[NO] response to shear stress in untreated and suramin treated cells. The Δ[NO] response in cells treated with suramin averaged 40% of the untreated responses and were statistically significant. (Mean and SEM were plotted, one-tailed t-test. n=3 each, *p<0.05).

Figure 3

Inhibition of purinergic receptors attenuates shear stress-induced NO production. Purinergic receptors were blocked with using the non-specific blocker suramin. Cells were incubated with 200 μM of suramin for 10 min prior to insertion into the chamber. Each membrane was exposed to a series of 4 step changes from 0.1 to 10 dyn/cm². A. Sample traces of the response for untreated (solid line) and suramin (dotted line) treated cells. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change occurring at 50s. B. Bar graph comparing the Δ[NO] response to shear stress in untreated and suramin treated cells. The Δ[NO] response in cells treated with suramin averaged 40% of the untreated responses and were statistically significant. (Mean and SEM were plotted, one-tailed t-test. n=3 each, *p<0.05).

Figure 4

Inhibition of store-operated channels (SOC) attenuates shear stress-induced NO production. SOCs were inhibited using the blocker SKF-96365 (SKF). Cells were treated with 50 μM of SKF for 10 min prior to insertion into the chamber. Each membrane was exposed to a series of 5 step changes of 0.1 to 10 dyn/cm². A. Sample traces of the response for untreated (solid line) and SKF treated (dotted line) cells. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change occurring at 50s. B. Bar graph representing the Δ[NO] response to shear stress in untreated and SKF treated cells. The Δ[NO] response for SKF treated cells averaged 60% of the untreated responses and were found to be statistically significant (Mean and SEM, one-tailed t-test. n=3 each, *p<0.05).

Figure 4

Inhibition of store-operated channels (SOC) attenuates shear stress-induced NO production. SOCs were inhibited using the blocker SKF-96365 (SKF). Cells were treated with 50 μM of SKF for 10 min prior to insertion into the chamber. Each membrane was exposed to a series of 5 step changes of 0.1 to 10 dyn/cm². A. Sample traces of the response for untreated (solid line) and SKF treated (dotted line) cells. The steady-state concentration was offset to zero in order to show the individual NO response due to the step change occurring at 50s. B. Bar graph representing the Δ[NO] response to shear stress in untreated and SKF treated cells. The Δ[NO] response for SKF treated cells averaged 60% of the untreated responses and were found to be statistically significant (Mean and SEM, one-tailed t-test. n=3 each, *p<0.05).

Figure 5

ATP induced eNOS phosphorylation is dependent on increases in intracellular calcium. Cells were simulated with 100 μM ATP with calcium. Prior to stimulation, cells were treated with 30 μM of BAPTA for 30 min. Cells were harvested before stimulation (t=0) and at time points 1, 3, 5, and 10 min after stimulation. All peNOS/eNOS ratios were normalized by t=0. Treatment with BAPTA abolished the eNOS phosphorylation response. (p<0.05 *; p<0.01 ** one-tailed t-test, n=4 each)

Figure 6

ATP induced eNOS phosphorylation is dependent on the calcium influx from the extracellular space. Cells were simulated with 100 μM ATP in the presence (ATP w CA) or absence of extracellular calcium (ATP wo Ca). Cells were harvested before stimulation (t=0) and at time points 1, 3, 5, and 10 min after stimulation. All peNOS/eNOS ratios were normalized by t=0. eNOS phosphorylation remained elevated after 1 min, which was not seen in the absence of extraceullular calcium. After the initial response to stimulation in the absence of calcium, PBS with calcium was added (t=10) which caused an increase in eNOS phosphorylation that was not observed when PBS without calcium was added. (p<0.05 *,#; p<0.01 **, ## two-tailed t-test; ATP wo Ca⁺² n=5, ATP w Ca⁺² n=4, addition of PBS with Ca⁺² n=3, No Ca n=3)

Figure 7

Inhibition of ATP-induced CCE by SKF-96365. Prior to the experiment, cells were incubated with PBS with Ca⁺² or 50 μM of SOC inhibitor SKF-96365 for 10 min. A. Representative traces of the calcium response in untreated (solid line) and SKF (dotted line) treated cells to 500 μL of 100 μM ATP with calcium. B. Bar graph representing average responses for each condition at the peak response, 60s and 100s. SKF reduced transient and sustained calcium response, which was statically significant from untreated and SKF treated cells. Average responses represent multiple coverslips (cs) and total cell count among coverslips (n) Untreated: #cs= 8, n=512; SKF: #cs=5, n=298. (Mean and SEM, Two-tailed t-test p<0.0001)

Figure 7

Inhibition of ATP-induced CCE by SKF-96365. Prior to the experiment, cells were incubated with PBS with Ca⁺² or 50 μM of SOC inhibitor SKF-96365 for 10 min. A. Representative traces of the calcium response in untreated (solid line) and SKF (dotted line) treated cells to 500 μL of 100 μM ATP with calcium. B. Bar graph representing average responses for each condition at the peak response, 60s and 100s. SKF reduced transient and sustained calcium response, which was statically significant from untreated and SKF treated cells. Average responses represent multiple coverslips (cs) and total cell count among coverslips (n) Untreated: #cs= 8, n=512; SKF: #cs=5, n=298. (Mean and SEM, Two-tailed t-test p<0.0001)

Figure 8

ATP induced eNOS phosphorylation is dependent on CCE. Cells were simulated with 100 μM ATP with calcium. Prior to stimulation, cells were treated with 50 μM of SKF-96365 for 10 min. Cells were harvested before stimulation (t=0) and at time points 1, 3, 5, and 10 min after stimulation. All peNOS/eNOS ratios were normalized by t=0. SKF-96365 treated cells (SKF) show an attenuated eNOS phosphorylation at 1 and 3 min, which was statistically significant. (p<0.05 * one-tailed t-test, ATP w Ca⁺² n=4, SKF n=5)

Figure 9

Inhibition of PKC attenuates the ATP stimulated sustained calcium response. Prior to stimulation with 500 μL of 100 μM ATP with calcium, cells were incubated with 30 μM of the general PKC inhibitor cheler for 2 min. A. Representative traces of the calcium response in untreated (solid line) and cheler (dotted line) treated cells to 500 μL of 100 μM ATP with calcium. B. Bar graph representing average responses for each condition at the peak response, 60(s) and 100(s). Cheler treated cells showed attenuation of the peak and sustained calcium response. Average responses represent multiple coverslips (cs) and total cell count among coverslips (n) Untreated: #cs= 3, n=120, Cheler: #cs=4, n=160, two-tailed t-test ***p<0.0001.

Figure 9

Inhibition of PKC attenuates the ATP stimulated sustained calcium response. Prior to stimulation with 500 μL of 100 μM ATP with calcium, cells were incubated with 30 μM of the general PKC inhibitor cheler for 2 min. A. Representative traces of the calcium response in untreated (solid line) and cheler (dotted line) treated cells to 500 μL of 100 μM ATP with calcium. B. Bar graph representing average responses for each condition at the peak response, 60(s) and 100(s). Cheler treated cells showed attenuation of the peak and sustained calcium response. Average responses represent multiple coverslips (cs) and total cell count among coverslips (n) Untreated: #cs= 3, n=120, Cheler: #cs=4, n=160, two-tailed t-test ***p<0.0001.

Figure 10

Inhibition of PKC attenuates ATP induced phosphorylation of eNOS. Prior to stimulation with 100 μM ATP with calcium, cells were treated with 30 μM of cheler for 2 min prior to stimulation. Cells were harvested before stimulation (t=0) and at time points 1, 3, 5, and 10 min after stimulation. All peNOS/eNOS ratios were normalized by t=0. Cheler treated cells showed an attenuated agonist stimulated phosphorylation of eNOS, which was statistically significant at 1 and 3 min (p<0.05 * one-tailed t-test, ATP w Ca⁺² n=4, Cheler n=5).

Figure 11

Proposed pathway for the mechanism of shear stress-induced NO. Flow causes the release of ATP in nM-μM levels, which activate purnergic receptors causing the release of IP₃ and DAG. IP₃ then causes the ER to deplete intracellular calcium stores. Depletion of ER calcium stores leads to the activation of store-operated channels (SOCs) (known as capacitative calcium entry (CCE)), which is PKC-dependent. CCE then activates calcium-dependent calmodulin (Ca⁺²-CaM), which leads to phosphorylation of eNOS and production of NO.