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. 2019 Jun 3;17(3):eAO4600.
doi: 10.31744/einstein_journal/2019AO4600.

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells

[Article in English, Portuguese]
Janyerson Dannys Pereira da Silva  1 , Gustavo Ballejo  1
Affiliations

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells

[Article in English, Portuguese]
Janyerson Dannys Pereira da Silva et al. Einstein (Sao Paulo). .

Abstract
in English, Portuguese

Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors.

Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings.

Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s-1) and thapsigargin (3.9±0.25mg.s-1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM).

Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

Objetivo:: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes.

Métodos:: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato.

Resultados:: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM).

Conclusão:: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

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Conflict of interest statement

Conflict of interest:

none.

Figures

Figure 1
Figure 1. Sustained endothelium-dependent relaxations elicited by ACh and Thap require extracellular calcium. Original recordings showing the effect of ACh (A), Thap (B) and GSK101 (C) in rat thoracic aortic rings pre-constricted with phenylephrine (PE). Effects were analyzed in endothelium-intact (+E), endothelium-denuded (-E) or when rat thoracic aortic rings with endothelium were incubated in nominally calcium free solution (Ca2+-free, prepared omitting CaCl2) during 40 minutes. Numbers below abbreviations indicate drug concentration in μM; scale bars, time in minute, (horizontal); and tension in g (vertical) variations. (D) and (E) Changes in tension (Δ) in percentage of PE-induced contraction produced by ACh, Thap and GSK101 in +E and −E aortic rings. (E) Effect of L-NNA (100μM) added on the plateau of relaxations caused by ACh, Thap and GSK101 or SNP (100nM). (F) Representative time-course of the relaxations produced by ACh (1μM), Thap (30nM) or GSK101 (3nM) in the presence (blue traces) or absence (red traces) of extracellular calcium. Responses were normalized to reduction (%) in the PE-contraction in each condition. (G) Area under a curve (AUC) of the ACh, Thap and GSK101 responses obtained in calcium-containing and calcium-free medium. (H) ACh and Thap transient relaxations parameters: (1) peak, (2) area under curve, (3) time to reach peak and (4) to return to basal (N=3-6 experiments)
Figure 2
Figure 2. Effect of selective inhibitors for ORAI, TRPCs and TRPV4 channels upon endothelium-dependent relaxations elicited by ACh, Thap and GSK101 in rat thoracic aorta. (A) Original recordings showing the effect of Pyr2 and Pyr3 (1 to 3μM) on the phenylephrine (PE)-induced contraction in endothelium-denuded rings (-E). (B) Concentration-response curve for the inhibitory effect of pyrazole compounds (1 to 10μM) on PE-contraction. (C to G) Original recordings showing the effect of addition of Pyr3 (1μM), Pyr2 (3μM), Pyr10 (3μM), Pyr6 (1μM) or HC-067047 (3 or 10μM) on the plateau of relaxations produced by ACh or Thap. Respective bar graphs summarize the effect of pyrazole compounds and HC-067047 added on the plateau of ACh (1μM), Thap (30nM) or GSK (3nM) relaxations. Results are presented as changes in the tension (Δ) expressed as percentage of PE-evoked contraction in rat aortic rings (N=3-6 experiments)
Figure 3
Figure 3. Effects of selective TRPC4 channel inhibitor in rat thoracic aorta. (A) Representative tracings of the effect of ML204 (3μM) added on the plateau of the relaxations produced by ACh (left) or Thap (right) in rat thoracic aortic rings pre-constricted with phenylephrine (PE). (B) ML204 effect presented as tension (%). **p<0.01 and ***p<0.001, Student t-test (paired, two tailed). (C) Original recordings showing the relaxing responses to ACh (1 to 3μM) or Thap (30nM) observed in rat thoracic aortic rings pre-incubated with ML204 (+ML204, 3μM, for 5 minutes). (D) Representative time course of ACh and Thap relaxations before (control, blue traces) and after (red traces) pre-incubation of arterial rings with ML204. (E) Changes in tension (Δ) in percentage of PE-contraction produced by ACh or Thap in the absence (-) and presence (+) of ML204. (F) Left: time-course of PE-contraction in endothelium-intact rings (ACh relaxation >95%) before (control) and after pre-incubation with ML204. Right: PE-contraction in grams (g) for these rings. **p<0.01, Student t-test (paired, two tailed) (N=5-11 experiments)
Figure 4
Figure 4. Kinetics of the reversal produced by ORAI and TRPCs channels inhibitors. (A to B) Representative time course for the reversing effect of pyrazole compounds and ML204 on ACh (1μM, A) and Thap (30nM, B) responses. Plateau of the ACh or Thap relaxations was taken as 100%. Concentrations tested: Pyr2 (3μM), Pyr6 (1μM), Pyr3 (1μM) and ML204 (3μM). (C) X-Y plot comparing the time required for ORAI and TRPCs inhibitors to produce a reversal of 20%, 50%, 80% and 100% of ACh or Thap relaxations. (D) Latencys to start the reversal of ACh (1μM) and Thap (30nM) responses. Inhibition was considered initiated when drugs caused a 10% reversal. (E) Velocity (dT/dt, mg.s−1) of the reversal produced by each compound on endothelium-dependent relaxations indicated (ACh, Thap) (N=3-6 experiments)
Figure 5
Figure 5. Effects of the pre-incubation with ORAI, TRPCs and TRPV4 channels blockers on endothelium-dependent relaxations. (A to C) Original recordings in endothelium-intact rings of rat thoracic aorta (ACh relaxation >95%) showing the effect of a (5 minutes) pre-incubation with Pyr2 (3μM, A) and Pyr6 (1μM, B and C) on endothelium-dependent relaxations elicited by ACh, Thap or GSK101. (D to E) Representative time course of the relaxations exhibited by ACh (1μM, D) or Thap (30nM, E) in the presence of Pyr2 (3μM), Pyr6 (1μM), Pyr3 (1μM), Pyr10 and HC-067047 (both at 3μM). Arrows indicate the moment of ACh or Thap application. Responses were normalized to reduction (%) in the phenylephrine (PE)-induced contraction in each preparation. (F) Parameters of the transient relaxations produced by ACh and Thap in the presence of Pyr2 or Pyr6: (1) peak, (2) area under curve (AUC), (3) time to reach peak and (4) to return to basal (N=3-5 experiments)
Figure 6
Figure 6. The pre-incubation of rat aortic rings with Pyr3 facilitated the reversal of ACh relaxations by Pyr2. (A to B) Original recordings showing the reversal of ACh-induced relaxations by Pyr2 (0.3μM) in rat aortic rings pre-constricted with phenylephrine (PE). In (B), arterial rings were pre-incubated with Pyr3 (1μM, 5 minutes). (C) Changes in the tension (Δ) of PE-contraction produced by ACh before (-) and after (+) pre-incubation of rat thoracic aortic rings with Pyr3. (D) Representative time course comparing the reversal produced by Pyr2 (0.3μM, arrow) in the absence (-) and presence (+) of Pyr3 (1μM). Plateau of the ACh relaxation was taken as 100%. (E) Parameters of the Pyr2 reversal measured in the absence (-) and presence (+) of Pyr3: (E1) latency in seconds to start (stated as the interval between the addition of Pyr2 and the production of 10% of reversal) (E2) time (minute) needed to produce 50% of reversal of ACh response (n=4 experiments). (F) Schematic representation showing the involvement of ORAI and TRPCs channels in the different phases of endothelium-dependent relaxations produced by ACh or Thap. It was proposed considering kinetics properties of the reversal caused by pyrazole compounds and the effect of their pre-incubation on ACh/Thap relaxant effect
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