Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through KATP channel activation

Abstract

The sensory neurotransmitter calcitonin gene-related peptide (CGRP) is associated with vasodilation of systemic arteries through activation of ATP-sensitive K⁺ (K_ATP) channels in smooth muscle cells (SMCs); however, its effects on endothelial cell (EC) membrane potential ( V_m) are unresolved. In pulmonary arteries (PAs) of C57BL/6J mice, we questioned whether CGRP would hyperpolarize ECs as well as SMCs. Intact PAs were isolated and immunostained for CGRP to confirm sensory innervation; vessel segments (1-2 mm long, ∼150 µm diameter) with intact or denuded endothelium were cannulated and pressurized to 16 cmH₂O at 37°C. Increasing concentrations (10^-10-10^-6 M) of CGRP progressively dilated PAs preconstricted with UTP (10^-5 M); SMCs hyperpolarized similarly (Δ V_m ∼20 mV) before and after endothelial denudation. To study native intact PA ECs, SMCs were dissociated to isolate endothelial tubes, and their integrity was confirmed by vital dye uptake, nuclear staining, and reproducible electrical and intracellular Ca²⁺ responses to acetylcholine (10^-5 M) over 2 h. Increasing [CGRP] hyperpolarized ECs in a manner similar to SMCs, with each cell layer demonstrating robust immunostaining for CGRP receptor proteins. Increasing concentrations (10^-10-10^-6 M) of pinacidil, a K_ATP channel agonist, resulted in progressive hyperpolarization of SMCs of intact PAs (Δ V_m ∼30 mV), which was blocked by glibenclamide (10^-6 M), as was hyperpolarization of ECs and SMCs to CGRP. Inhibition of protein kinase A with protein kinase inhibitor (10^-5 M) also inhibited hyperpolarization to CGRP. We demonstrate [CGRP]-dependent hyperpolarization of ECs for the first time while validating freshly isolated PA endothelial tubes as an experimental model. Redundant electrical signaling to CGRP in ECs and SMCs implies an integral role for K_ATP channels in PA dilation.

Keywords: hyperpolarization; lung blood flow; protein kinase A; sensory nerves; vasodilation.

Fig. 1.

Mouse pulmonary arteries are innervated by sensory nerves. Representative calcitonin-gene-related peptide (CGRP) staining (maximum z projections) confirms sensory innervation in larger (~300 μm diameter, A) and smaller (~150 μm diameter, B) pulmonary arteries. Arrows indicate nerve fibers; dotted lines in B indicate vessel edge. Scale bars = 20 µm.

Fig. 2.

Calcitonin-gene-related peptide (CGRP) dilates pulmonary arteries (PAs) preconstricted with UTP. A: concentration response for vasoconstriction to UTP (10⁻⁹–10⁻³ M) in isolated, pressurized (16 cmH₂O), intact PAs. B: UTP (5 × 10⁻⁶ M) constricted intact PAs, and this effect was not different for endothelium-denuded PAs. C: vasodilation of intact PAs preconstricted with UTP (5 × 10⁻⁶ M) to maximal [ACh] (10⁻⁵ M) was abolished following endothelial denudation. D: concentration response for vasodilation to CGRP (10⁻¹⁰–10⁻⁶ M) of intact PAs preconstricted with UTP (5 × 10⁻⁶ M). Values are means ± SE; n = 4–5 vessels per group. *P < 0.05 vs. intact. Details: UTP constricted arteries from 126 ± 12 µm inner diameter (ID) at rest to 19 ± 3 µm ID (maximal response). To preconstrict arteries for dilator studies, 5 × 10⁻⁶ M (EC₅₀) UTP constricted intact PAs from 128 ± 8 to 68 ± 6 µm ID. In the presence of UTP, CGRP dilated intact PAs to maximal ID of 119 ± 7 µm.

Fig. 3.

Live/dead assay and cell morphology of pulmonary artery (PA) endothelial tubes. A: calcein staining of cytoplasm of live cells. B: ethidium bromide staining of dead cells beneath pinning pipettes (located at each end of the endothelial tube in respective panels). C: overlay image of A and B. D: bright-field image of boxed region in C shows endothelial cell (EC) orientation and intact morphology of endothelial tube secured between pinning pipettes. E: staining with Hoechst 33342 illustrates smooth muscle cell nuclei (thin, vertical) and EC nuclei (oval, horizontal) of an intact pressurized PA. F: nuclei staining showing purity for EC composition of endothelial tube. Apparent increase in EC nuclei compared with E reflects “flattened” tube with top and bottom sides within the same focal plane. Scale bars = 50 µm.

Fig. 4.

Stability of electrical and Ca²⁺ signaling in pulmonary artery (PA) endothelial tubes. A: representative continuous recording for membrane potential (V_m) and intracellular Ca²⁺ concentration ([Ca²⁺]_i) responses (see methods) to intermittent exposure to ACh (10⁻⁵ M) during a 2-h protocol. Horizontal bars indicate periods of ACh superfusion. B: summary data. Pre, before ACh; ACh, peak hyperpolarization; Post, at 5-min washout. Values are means ± SE; n = 5 per group.

Fig. 5.

Pulmonary artery (PA) smooth muscle cells (SMCs) and endothelial cells (ECs) are hyperpolarized by calcitonin gene-related peptide (CGRP). A: representative continuous recording of membrane potential (V_m) illustrates concentration-dependent hyperpolarization of SMCs by CGRP in a denuded PA. From baseline (zero), note abrupt change in V_m upon entry and exit from cell. W, washout. B: summary data for V_m of PA endothelial tubes to CGRP. B, baseline. C: summary data for V_m of SMCs of denuded PAs to CGRP. D: summary data for V_m of SMCs of intact PAs to CGRP. E: summary data for V_m during resting baseline, with 5 × 10⁻⁶ M UTP alone and during addition of 10⁻⁸ or 10⁻⁶ M CGRP. Values are means ± SE; n = 5 per group. #P < 0.05 vs. baseline V_m. *P < 0.05 vs. UTP. +P < 0.05 vs. 10⁻⁸ M CGRP. UTP depolarized PAs 10 ± 1 mV, from which 10⁻⁶ M CGRP hyperpolarized PAs 17 ± 2 mV.

Fig. 6.

Immunostaining for calcitonin receptor proteins in pulmonary arteries (PAs). Representative dual staining of PAs for receptor activity-modifying protein 1 (RAMP1, left, green) and calcitonin receptor-like receptor (CRLR; middle, red) confirms CRLR expression in endothelial cells (ECs) and smooth muscle cells (SMCs). Overlay images (right) illustrate corresponding localization (yellow) of respective proteins. Scale bar = 20 µm.

Fig. 7.

ATP-sensitive K⁺ channel activation hyperpolarizes pulmonary artery (PA) endothelial cells (ECs) and smooth muscle cells (SMCs). A: representative continuous recording of membrane potential (V_m) illustrates concentration-dependent hyperpolarization of ECs to pinacidil (10⁻⁹−10⁻⁵ M) in a PA endothelial tube. W, washout. B: summary data for hyperpolarization to pinacidil in endothelial tubes. C: summary data for hyperpolarization to pinacidil in SMCs of intact PAs. Values are means ± SE; n = 4 per group. B, baseline V_m. #P < 0.05 vs. baseline V_m. EC₅₀ values for pinacidil were 7 × 10⁻⁸ M in ECs and 4 × 10⁻⁸ M in SMCs.

Fig. 8.

Glibenclamide reverses ATP-sensitive K⁺ channel-dependent hyperpolarization of pulmonary artery (PA) endothelial cells (ECs) and smooth muscle cells (SMCs). A: representative continuous recording of membrane potential (V_m) from an endothelial tube illustrates concentration-dependent reversal of hyperpolarization to pinacidil (P; 10⁻⁵ M, maintained throughout) by glibenclamide. B: summary data for reversal of hyperpolarization to pinacidil in endothelial tubes with glibenclamide (10⁻¹⁰−10⁻⁶ M). C: summary data for reversal of hyperpolarization to pinacidil in SMCs of intact PAs with glibenclamide (10⁻¹⁰−10⁻⁶ M). Values are means ± SE; n = 4 per group. B, baseline V_m; P, V_m during 10⁻⁵ M pinacidil. #P < 0.05 vs. baseline V_m. EC₅₀ values for glibenclamide were 6 × 10⁻⁹ M in ECs and 2 × 10⁻⁸ M in SMCs.

Fig. 9.

Hyperpolarization to calcitonin gene-related peptide (CGRP) in pulmonary artery (PA) endothelial cells (ECs) and smooth muscle cells (SMCs) requires ATP-sensitive K⁺ (K_ATP) channels. A: representative continuous recording of membrane potential (V_m) from an endothelial tube in the presence of glibenclamide (10⁻⁶ M) illustrates lack of responses to CGRP (10⁻¹⁰−10⁻⁶ M) with maintenance of hyperpolarization to ACh (10⁻⁵ M; 5 min). B: summary data for loss of hyperpolarization to CGRP (but not to ACh) for endothelial tubes in the presence of glibenclamide (10⁻⁶ M). C: summary data for loss of hyperpolarization to CGRP for SMCs of denuded PAs; note final depolarization with KCl (10⁻¹ M). D: summary data for loss of hyperpolarization to CGRP (but not depolarization to KCl) for SMCs of intact PAs. Values are means ± SE; n = 4–5 per group. B, baseline V_m. Details: inhibition of K_ATP channels also attenuated hyperpolarization in female mice: ΔV_m = 3 ± 1 mV for ECs and 4 ± 2 mV for SMCs from intact PAs (n = 3 per group). To confirm electrode placement, from a resting V_m of −43 ± 2 mV, ACh hyperpolarized ECs to −79 ± 4 mV (n = 4), while KCl depolarized SMCs from a resting V_m of −41 ± 3 to −9 ± 3 mV in intact PAs (n = 4) and to −7 ± 2 mV in denuded PAs (n = 2).

Fig. 10.

Genetic deletion of ATP-sensitive K⁺ (K_ATP) channels inhibits hyperpolarization to calcitonin gene-related peptide (CGRP). A: representative continuous recording of membrane potential (V_m) from endothelial tube of a mouse lacking K_ATP channels illustrates absence of responses to CGRP (10⁻¹⁰−10⁻⁶ M) or pinacidil (P; 10⁻⁵ M) followed by hyperpolarization to ACh (10⁻⁵ M; 5 min). B: summary data for responses to CGRP in smooth muscle cells of intact pulmonary arteries from K_ATP^−/− mice. Values are means ± SE; n = 3 per group. B, baseline V_m. #P < 0.05 vs. baseline V_m.

Fig. 11.

Calcitonin gene-related peptide (CGRP)-dependent hyperpolarization in endothelial cells (ECs) and smooth muscle cells (SMCs) requires PKA. A: representative continuous recording of membrane potential (V_m) from a SMC of an intact pulmonary artery (PA) in the presence of PKI (10⁻⁵ M) illustrates lack of response to CGRP (10⁻¹⁰−10⁻⁶ M) followed by depolarization to KCl (10⁻¹ M; 5 min). B: summary data for V_m during exposure of endothelial tubes to CGRP in the presence of PKI (10⁻⁵ M) followed by hyperpolarization to ACh (10⁻⁵ M). C: summary data for V_m during exposure of SMCs of denuded PAs to CGRP followed by depolarization to KCl (10⁻¹ M). D: summary data for V_m during exposure of intact PAs to CGRP followed by depolarization to KCl (10⁻¹ M). ACh hyperpolarized ECs by ∼36 mV, and KCl depolarized SMCs by ∼30 mV. Values are means ± SE; n = 4 per group. B,  baseline V_m. #P < 0.05 vs. baseline V_m.

Fig. 12.

Roles for ATP-sensitive K⁺ channels and PKA in pulmonary artery (PA) dilation to calcitonin gene-related peptide (CGRP). A: vasodilation during EC₅₀ (10⁻⁸ M) and maximal (10⁻⁶ M) [CGRP] for intact PAs preconstricted with UTP (5 × 10⁻⁶ M) in the absence (vehicle) and presence of glibenclamide (10⁻⁶ M). B: changes in vessel wall [Ca²⁺]_i (ΔF₃₄₀/F₃₈₀; see methods) of intact PAs corresponding with A. C: vasodilation to CGRP in the absence and presence of PKI (10⁻⁵ M). D: changes in vessel wall [Ca²⁺]_i (ΔF₃₄₀/F₃₈₀) corresponding with C. Values are means ± SE; n = 4–5 per group. #P < 0.05 vs. vehicle. +P < 0.05 vs. 10⁻⁸ M CGRP. Details: in A and B, baseline diameters [137 ± 9 and 134 ± 8 µm in the absence (vehicle) and presence of glibenclamide, respectively] and resting [Ca²⁺]_i (F₃₄₀/F₃₈₀ = 0.51 ± 0.05 and 0.57 ± 0.06, respectively) were similar between groups. Responses to UTP were not significantly different between PAs during superfusion with respective treatments [inner diameter (ID) = 72 ± 5 µm, constriction = 46 ± 4%, and ΔF₃₄₀/F₃₈₀ = 0.24 ± 0.05 for vehicle; ID = 71 ± 10 µm, constriction = 47 ± 6%, and ΔF₃₄₀/F₃₈₀ = 0.21 ± 0.04 for glibenclamide]. C and D: baseline diameters (134 ± 4 and 124 ± 11 µm for vehicle and PKI, respectively), Ca²⁺ (F₃₄₀/F₃₈₀ = 0.63 ± 0.06 and 0.60 ± 0.08 for vehicle and PKI, respectively), and responses to UTP (ID = 75 ± 5 µm, constriction = 43 ± 3%, and ΔF₃₄₀/F₃₈₀ = 0.17 ± 0.02 for vehicle; ID = 64 ± 8 µm, constriction = 47 ± 2%, and ΔF₃₄₀/F₃₈₀ = 0.20 ± 0.06 for PKI) were similar between conditions.

Fig. 13.

Pulmonary artery (PA) dilation to calcitonin gene-related peptide (CGRP) has both endothelial and smooth muscle components. A: vasodilation to CGRP of UTP-preconstricted (5 × 10⁻⁶ M) intact (±l-NAME, 10⁻⁴ M) and endothelium-denuded PAs during EC₅₀ (10⁻⁸ M) and maximal (10⁻⁶ M) [CGRP]. B: changes in vessel wall [Ca²⁺]_i (ΔF₃₄₀/F₃₈₀; see methods) in response to CGRP were similar for intact (±l-NAME) and denuded arteries. Values are means ± SE; n = 4 per group. #P < 0.05 vs. intact. +P < 0.05 vs. 10⁻⁸ M CGRP. Details: baseline diameter = 132 ± 11, 133 ± 6, and 128 ± 4 µm for intact, denuded, and l-NAME, respectively; resting intracellular [Ca²⁺] (F₃₄₀/F₃₈₀) = 0.62 ± 0.04, 0.58 ± 0.04, and 0.57 ± 0.02 for intact, denuded, and l-NAME, respectively. Responses to 5 × 10⁻⁶ M UTP (ID = 76 ± 11 µm, constriction = 44 ± 5%, and ΔF₃₄₀/F₃₈₀ = 0.19 ± 0.03 (intact); ID = 66 ± 10 µm, constriction = 47 ± 7%, and ΔF₃₄₀/F₃₈₀ = 0.20 ± 0.02 (denuded); and ID = 68 ± 3 µm, constriction = 47 ± 3%, and ΔF₃₄₀/F₃₈₀ = 0.15 ± 0.04 (l-NAME)] were not significantly different between groups.