The ability of neuropeptide Y to mediate responses in the murine cutaneous microvasculature: an analysis of the contribution of Y1 and Y2 receptors

Abstract

1. The ability of neuropeptide Y (NPY) to modulate skin blood flow, oedema formation and neutrophil accumulation was investigated. Experiments were designed to examine the possible contribution of the Y2 receptor, in addition to the Y1 receptor, through use of Y2 receptor knockout mice (Y2-/-) and selective receptor antagonists. 2. The development of a 99mTc clearance technique for the measurement of microvascular blood flow changes in mouse dorsal skin revealed a dose-dependent ability of picomole amounts of NPY, and also of the Y1-preferred agonist Pro34NPY and the Y2-preferred agonist PYY(3-36) to decrease blood flow. 3. The Y1 receptor antagonist BIBO3304 blocked responses to the Y1 agonist at the lower doses, but only partially inhibited at the higher doses tested in Y2+/+. In Y2-/- receptor mice, the responses to the Y2 agonist were abolished at the lower doses and partially reduced at the highest dose tested, while those to the Y1 agonist were similar in both Y2+/+ and Y2-/-receptor mice. 4. In Y2+/+ receptor mice, the simultaneous injection of the Y2 antagonist BIIE0246 with BIBO3304 abolished Y2 agonist-induced decreases in blood flow over the dose range used (10-100 pmol). When the Y2 receptor antagonist BIIE0246 was given alone, it was not able to significantly affect the PYY(3-36)-induced response, whereas the Y1 receptor antagonist BIBO3304 partially (P<0.001) inhibited the decrease in blood flow evoked by PYY(3-36) at the highest dose. 5. NPY did not mediate either oedema formation, even when investigated in the presence of the vasodilator calcitonin gene-related peptide (CGRP), or neutrophil accumulation in murine skin. 6. We conclude that the major vasoactive activity of NPY in the cutaneous microvasculature is to act in a potent manner to decrease blood flow via Y1 receptors, with evidence for the additional involvement of postjunctional Y2 receptors. Our results do not provide evidence for a potent proinflammatory activity of NPY in the cutaneous microvasculature.

Figure 1

Effect of NPY agonists on blood flow in mouse cutaneous dorsal microvasculature. The responses to increasing doses of (a) NPY (30–1000 pmol), (b) Pro³⁴-NPY (1–1000 pmol) and (c) PYY(3–36) (10–1000 pmol) are shown as change (decrease) in % clearance compared with vehicle (Tyrode-injected) skin. Results are shown as mean±s.e.m., and those that are significantly different from clearance at Tyrode-injected sites are shown. *P<0.05; **P<0.01; ***P<0.001, ANOVA+Bonferroni's modified t-test.

Figure 2

Effect of the Y₁ antagonist BIBO3304 on the ability of Pro³⁴-NPY to reduce blood flow in mouse cutaneous dorsal microvasculature. The responses to increasing doses of Pro³⁴-NPY (1–300 pmol) are shown for mice pretreated (−5 min, i.v.) with vehicle (0.5% DMSO in saline) and with BIBO3304 (0.5 μmol kg⁻¹). Responses are shown as the % change (decrease) in clearance compared to Tyrode-injected sites. Results are shown as mean±s.e.m., n=7–11, and responses that are significantly different from corresponding sites in vehicle-treated mice are shown. *P<0.05 (two-tailed Student's unpaired t-test). ^#P<0.05, ^###P<0.001 show a significant difference in control or treated group, as compared to clearance at Tyrode-injected sites (ANOVA+Bonferroni's modified t-test). Tyrode response (=0) is illustrated by the continuous line.

Figure 3

Responses to a Y₂-preferred agonist in cutaneous dorsal microvasculature of Y₂^+/+ and Y₂^−/− receptor mice. The responses to increasing doses of (a) PYY(3–36) (10–100 pmol), (b) PYY(3–36) (10–100 pmol) in the presence of the Y₁ receptor antagonist BIBO3304 (0.5 μmol kg⁻¹, −5 min i.v.) and (c) Pro³⁴-NPY (1–100 pmol) are shown as change in % clearance (decrease) compared with Tyrode-injected sites. Results are shown as mean±s.e.m. ^#P<0.05, ^###P<0.001 illustrate a significant difference between clearance at Tyrode-injected sites and responses to peptides in both control and treated groups (ANOVA+Bonferroni's modified t-test).

Figure 4

Effect of Y₁ and Y₂ antagonists on the ability of PYY(3–36) to reduce blood flow in mouse cutaneous dorsal microvasculature. The responses to increasing doses of PYY(3–36) (10–100 pmol), in the presence and absence of (a) both the Y₁ receptor antagonist BIBO3304 and the Y₂ antagonist BIIE0246 (0.5 and 3 μmol kg⁻¹, respectively), given i.v. as a 5 min co-pretreatment, (b) the Y₂ receptor antagonist peptide BIIE0246 and (c) Y₁ receptor antagonist BIBO3304 (0.5 μmol kg⁻¹, −5 min i.v.). Results are shown as mean±s.e.m. Responses that are significantly different from corresponding sites in vehicle-treated mice are shown. *P<0.05 and **P<0.01 (ANOVA+Bonferroni's modified t-test) ^#P<0.05, ^###P<0.001 show a significant difference between peptide responses and to the clearance at Tyrode-injected sites (ANOVA+Bonferroni's modified t-test).

Figure 5

The effect of NPY agonists on oedema formation in mouse skin. Oedema formation was assessed in response to NPY (300 pmol), Pro³⁴-NPY (300 pmol), PYY(3–36) (300 pmol) and substance P (100 pmol). The responses were measured in the absence (black columns) or presence (open columns) of a vasodilator dose of CGRP, 30 pmol, coinjected i.d. Results are shown as mean±s.e.m. Responses that are significantly different from Tyrode-injected sites are shown. *P<0.05; **P<0.01; ***P<0.001, and also responses that are significantly different from the corresponding CGRP-treated sites. ^#P<0.05; ANOVA+Bonferroni's modified t-test.

Figure 6

The effect of NPY on neutrophil accumulation in the mouse skin. Neutrophil accumulation was assessed in response to NPY, 300 pmol, and compared with that of the known effect of carrageenan (0.3 mg). Results are shown as mean±s.e.m., n=5. Responses that are significantly different from Tyrode-injected sites are shown. ***P<0.001 compared to Tyrode; ANOVA+ Bonferroni's modified t-test.