Structure-activity relationships of vanilloid receptor agonists for arteriolar TRPV1

Abstract

Background and purpose: The transient receptor potential vanilloid 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli and is a therapeutic target. However, functional TRPV1 that affect microvascular diameter are also expressed in peripheral arteries and we attempted to characterize this receptor.

Experimental approach: Sensory TRPV1 activation was measured in rats by use of an eye wiping assay. Arteriolar TRPV1-mediated smooth muscle specific responses (arteriolar diameter, changes in intracellular Ca(2+)) were determined in isolated, pressurized skeletal muscle arterioles obtained from the rat and wild-type or TRPV1(-/-) mice and in canine isolated smooth muscle cells. The vascular pharmacology of the TRPV1 agonists (potency, efficacy, kinetics of action and receptor desensitization) was determined in rat isolated skeletal muscle arteries.

Key results: Capsaicin evoked a constrictor response in isolated arteries similar to that mediated by noradrenaline, this was absent in arteries from TRPV1 knockout mice and competitively inhibited by TRPV1 antagonist AMG9810. Capsaicin increased intracellular Ca(2+) in the arteriolar wall and in isolated smooth muscle cells. The TRPV1 agonists evoked similar vascular constrictions (MSK-195 and JYL-79) or were without effect (resiniferatoxin and JYL-273), although all increased the number of responses (sensory activation) in the eye wiping assay. Maximal doses of all agonists induced complete desensitization (tachyphylaxis) of arteriolar TRPV1 (with the exception of capsaicin). Responses to the partial agonist JYL-1511 suggested 10% TRPV1 activation is sufficient to evoke vascular tachyphylaxis without sensory activation.

Conclusions and implications: Arteriolar TRPV1 have different pharmacological properties from those located on sensory neurons in the rat.

Figure 1

Functional effects of TRPV1 stimulation in skeletal muscle arteries. Internal diameter of cannulated gracilis arteries were measured at 80 mmHg intraluminal pressure before treatments (control). The existence of spontaneous myogenic tone and viability of endothelium was determined by ACh (10 µM)-evoked dilatations. The constrictor response to TRPV1 agonist capsaicin (1 µM) was compared with the effect of NA (10 µM). Experiments were performed on the same (n = 7) arteries. Values are mean ± SEM. Significant differences are represented by asterisks (*P < 0.05 or **P < 0.01).

Figure 2

TRPV1 specificity of capsaicin-evoked vasoconstriction. Internal diameter of cannulated gracilis arteries was measured at 80 mmHg intraluminal pressure upon addition of different doses of capsaicin (cumulative dose-response curve) in the absence (control) and presence of the TRPV1 antagonist AMG9810 (100, 300 and 1000 nM, A). Symbols are means ± SEM of five to nine independent determinations. The equilibration dissociation constant of AMG9810 was determined by the conventional Schild plot (x-intercept, B). Finally, gracilis arterioles isolated from control (wild-type) and TRPV1 knockout (TRPV1^−/−) mice were also tested for capsaicin-mediated vasoconstriction (C). Symbols are mean ± SEM of five to six independent determinations.

Figure 3

Mechanism of capsaicin-mediated vasoconstriction: skelatal muscle arteries. Capsaicin-evoked changes in arteriolar diameter were recorded in parallel with changes in intracellular Ca²⁺ concentrations of the vascular wall. An individual experiment is shown in (A) (the full recorded experiment also available in the supplementary movie). Solid line represents the arteriolar diameter (please note that in this specific case, the outer diameter is plotted), while dotted line shows intracellular Ca²⁺ concentrations expressed as 340/380 ratio. Capsaicin was administered in a cumulative fashion (indicated by the arrows, the applied capsaicin doses were: 3 × 10⁻¹⁰, 10⁻⁹, 3 × 10⁻⁹, 10⁻⁸, 3 × 10⁻⁸, 10⁻⁷, 3 × 10⁻⁷, 10⁻⁶, 3 × 10⁻⁶ M). (B)The mean responses ±SEM of n = 5 single determinations.

Figure 4

Mechanism of capsaicin-mediated vasoconstriction: isolated arteriolar smooth muscle cells. Canine, freshly isolated coronary arteriolar smooth muscle cells were loaded with fura-2 fluorescent Ca²⁺-sensitive dye and treated with capsaicin (1 µM) and KCl (100 mM). Changes in intracellular Ca²⁺ concentrations were detected as changes in the 340/380 fluorescence ratio (a representative experiment is shown in A, where green pixels represent low values and red represent high values). Capsaicin evoked a fast increase in the intracellular Ca²⁺ concentrations in some cells, which was not increased further upon the addition of KCl (B). These observations were confirmed when responses of the capsaicin-sensitive cells (10 out of 28 viable cells) were evaluated (C). Columns represent mean ± SEM.

Figure 5

Pharmacological characterization of vascular responses to capsaicin. Experiments were performed on cannulated arteries as mentioned above. First, a cumulative dose-response curve was obtained (A, n = 7). Next on a separate set of arteries, the kinetics of response were measured by the application of 1 µM capsaicin for 20 min. Arteriolar diameter was measured at 10 s intervals (B, n = 9). After this 20 min treatment, the arteries were washed and were incubated in PSS solution for 40 min (regeneration). At the end of regeneration, vasoconstriction to the same dose of capsaicin (1 µM) was measured to determine tachyphylaxis (C, n = 7). Values are mean ± SEM, significant difference (P < 0.05) is represented by an asterisk.

Figure 6

Arteriolar response to resiniferatoxin. Experiments were performed as mentioned in Figure 4 with resiniferatoxin. Responses to cumulative doses are shown in (A) (n = 3). No functional response was detected after 20 min of resiniferatoxin (B, 10 nM, n = 5). However, this treatment desensitized the receptors to capsaicin (1 µM) measured after regeneration (C, n = 5).

Figure 7

Arteriolar response to JYL-273. Experiments were performed as mentioned in Figure 4 with JYL-273. Responses to cumulative doses are shown in (A) (n = 7). No functional response was detected after 20 min of JYL-273 (B, 1 µM, n = 5). However, this treatment desensitized the receptors to capsaicin (1 µM) measured after regeneration (C, n = 4).

Figure 8

Arteriolar response to MSK-195. Experiments were performed as mentioned in Figure 4 with MSK-195. Responses to cumulative doses are shown in (A) (n = 5). A transient vasoconstriction was observed upon MSK-195 stimulation for 20 min (B, 1 µM, n = 6). In addition, this treatment desensitized the receptors to capsaicin (1 µM) measured after regeneration (C, n = 6).

Figure 9

Arteriolar response to JYL-79. Experiments were performed as mentioned in Figure 4 with JYL-79. Responses to cumulative doses are shown in (A) (n = 8). A transient vasoconstriction was observed upon JYL-79 stimulation for 20 min (B, 1 µM, n = 5). In addition, this treatment desensitized the receptors to capsaicin (1 µM) measured after regeneration (C, n = 5).

Figure 10

Arteriolar response to JYL-1511. Experiments were performed as mentioned in Figure 4 with JYL-1511. Responses to cumulative doses are shown in (A) (n = 6). No functional response was detected after 20 min of JYL-1511 (B, 1 µM, n = 6). However, this treatment desensitized the receptors to capsaicin (1 µM) measured after regeneration (C, n = 6).

Figure 11

Partial agonism/antagonism of JYL-1511 on TRPV1 located in vascular smooth muscle. Changes in internal diameter of the arteries were measured before treatments (control, n = 6) and after addition of 1 µM JYL-1511 (JYL-1511, n = 6). Capsaicin responses were also determined in the presence of 1 µM JYL-1511 (JYL-1511 + capsaicin, n = 6). Finally, capsaicin responses alone (1 µM, without any pretreatment) were also measured on a different set of arteries (capsaicin, n = 7). The efficacy of JYL-1511 as a partial agonist was expressed as the percentage of decrease in arteriolar diameter evoked by the application of capsaicin alone (100%, capsaicin). Its efficacy as a partial antagonist was expressed as the percentage of decrease in capsaicin constriction (100%, capsaicin) in the presence of JYL-1511 (1 µM, JYL-1511 + capsaicin).

Figure 12

Sensory neuronal irritation evoked by application of the TRPV1 ligands. TRPV1 agonists or vehicle were applied in the eye of rats to determine their ability to evoke sensory neuronal irritation. Number of eye wipes was counted for 60 s after application of 10 µL of the drugs onto the conjunctiva of the rats. Concentrations of the drugs were chosen to represent the highest dose used in the vascular experiments (indicated in the figure). Columns represent mean ± SEM (n = 5), significant differences (P < 0.05) from the control (wipes upon administration of the vehicle alone) are represented by asterisks.