Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

Abstract

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca²⁺-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine (n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 (n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS (n = 4-7, P = nonsignificant), it did inhibit calcium oscillations (n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.

Keywords: asthma; calcium oscillations; capsazepine; organ bath; precision-cut lung slice.

Fig. 1.

Detection of transient receptor potential channel V1 (TRPV1) mRNA and protein in human ASM (hASM). A: RT-PCR analysis of TRPV1 expression in multiple preparations of hASM cells and tissue. RT-PCR sample devoid of cDNA (water blank) served as negative control, and whole human brain served as positive control. B: immunohistochemical analysis of TRPV1 in human tracheal airway tissue with DAPI nuclear staining. C: actin. D: merging of TRPV1 and actin. E: staining control. Bar: 35 µm.

Fig. 2.

Primary human ASM cell representative immunocytochemistry demonstrating TRPV1 colocalization with SERCA2. TRPV1 (red; A) and SERCA2 (green, sarcoplasmic reticulum marker; B) are detected in human ASM cells. C: the merged image (×20) demonstrates TRPV1 and SERCA2 expression in the perinuclear region, showing TRPV1 expression on sarcoplasmic reticulum. D: staining control. Bar: 100 µm; n = 3.

Fig. 3.

Proximity ligation assay (PLA) demonstrating TRPV1 and SERCA2 colocalization in primary human ASM cells. A: positive (red dots) PLA (PLA; Duolink, Invitrogen) in primary human ASM cells performed using primary antibodies against TRPV1 and SERCA2. This demonstrates that the proteins exist within 40 nm of each other, suggesting that TRPV1 expression is in the SR membrane. B: negative control with primary antibodies omitted. Bar: 100 µm; n = 3.

Fig. 4.

Capsaicin-mediated contraction of guinea pig tracheal rings. A: representative tracings of 10 µM capsaicin-induced contraction of tracheal rings in the presence of 1 µM tetrodotoxin, 100 µM Na⁺-channel inhibitor bupivacaine, 20 µM NK-2 receptor antagonist GR-159897, or vehicle. B: quantification of capsaicin-induced contraction force as a fraction of 100 µM acetylcholine-induced force. Pretreatment with bupivacaine and GR-159897 significantly inhibited contraction force. Values are means ± SE; n = 4 for all groups. *P < 0.05 compared with untreated controls by ANOVA with Bonferroni post hoc correction.

Fig. 5.

Contraction force tracings of guinea pig tracheal rings with repeated exposure to capsaicin. A: contraction force tracing of tracheal ring receiving two successive exposures to 10 µM capsaicin. B: contraction force tracing of tracheal ring receiving vehicle followed by 10 µM capsaicin. A second exposure to capsaicin shows tachyphylaxis compared with the first, suggesting a “depletable” mechanism of contraction consistent with the activation of nonadrenergic, noncholinergic (NANC) nerves.

Fig. 6.

Capsazepine-mediated attenuation of acetylcholine-induced contraction of guinea pig tracheal rings. A: representative tracings of guinea pig tracheal ring contractile force receiving pretreatment with 100 µM capsazepine vs. vehicle. All rings were contracted with an EC₅₀ concentration of acetylcholine calculated for each individual ring. B: quantification of contractile force 15 min after acetylcholine exposure (as a percentage of peak force). Capsazepine pretreatment significantly attenuated the maintenance of contractile force in guinea pig ASM. Values are means ± SE; n = 4 animals. ***P < 0.001 by Student’s t-test.

Fig. 7.

Capsazepine-mediated attenuation of acetylcholine-induced contraction of human ASM in organ bath preparations. A: representative tracings of human ASM strip contraction force receiving pretreatment with 100 µM capsazepine vs. vehicle. B: quantification of contractile force 60 min after acetylcholine exposure (as a percentage of force at 10 min). Capsazepine pretreatment significantly attenuates the maintenance of contractile force in human ASM. Values are means ± SE; n = 4 human donors. ***P < 0.001 by Student’s t-test.

Fig. 8.

Capsazepine-mediated inhibition of methacholine-induced airway contraction in mouse precision-cut peripheral lung slice. A: combined tracings of the area of peripheral airways (normalized to baseline area) in mouse precision-cut lung slice (PCLS) preparations. The airways were contracted with 400 nM methacholine (MCh) and subsequently exposed to a concentration range of capsazepine. B: capsazepine led to a significant relaxation (increase in airway area; measured at 30 min) of the MCh-induced airway contraction in a dose-dependent manner. Values are means ± SE; n = 3. *P < 0.05 compared with untreated controls by ANOVA with Bonferroni post hoc correction.

Fig. 9.

Capsazepine-mediated inhibition of methacholine-induced calcium oscillations in ASM cells in mouse PCLS. A: mouse PCLS preparations were loaded with a calcium indicator dye (Oregon Green-BAPTA) and imaged in real-time using a custom two-photon confocal microscope and software. A: exposure of the slices to methacholine (MCh; 400 nM) led to sustained, rapid calcium oscillations over a 20-min recording period (representative tracing). B: 100 µM capsazepine significantly inhibited these MCh-induced oscillations. C: quantification of oscillation frequency demonstrates capsazepine led to a near abolishment of oscillations by 15 min. Values are means ± SE; n = 3. ***P < 0.01 compared with untreated controls by ANOVA with Bonferroni post hoc correction.

Fig. 10.

Absence of capsazepine effect on store-operated calcium entry (SOCE) in ASM cells in mouse PCLS. Mouse peripheral lung slices loaded with an intracellular calcium indicator dye were exposed to caffeine (20 mM) and ryanodine (25 μM) simultaneously to “lock” the ryanodine receptor of sarcoplasmic reticulum (SR) in the open state, to irreversibly deplete the SR of calcium, and to induce SOCE in the presence of zero-calcium external buffer. The slices were then reexposed to 1.3 mM Ca²⁺ extracellular buffer, and the resultant increase in ASM calcium-mediated fluorescence was considered a measure of SOCE. GSK 7575A, 100 µM, a SOCE inhibitor, significantly inhibited SOCE in this assay; however, 100 µM capsazepine did not. This suggests SOCE inhibition is not the mechanism by which capsazepine inhibits calcium oscillations in ASM. A: combined Ca²⁺-mediated fluorescent tracings. B: quantification of SOCE-mediate calcium influx. Values are means ± SE; n = 3. ***P < 0.001 and *P < 0.05 compared with untreated controls by ANOVA with Bonferroni post hoc correction.