Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries

Abstract

Background and purpose: Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.

Experimental approach: The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC).

Key results: ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization.

Conclusions and implications: These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.

Figure 2

Dilatation responses to luminal perfusion of adenosine 5′-[β-thio]diphosphate (ADPβS) (0.1–3 µM, A–D) in rat pressurized small mesenteric artery in the absence or the presence of the selective protein kinase C inhibitors bisindolylmaleimide I (BIS-I) (1 µM), bisindolylmaleimide VIII (BIS-VIII) (1 µM) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö 6976) (1 µM), under submaximal levels of phenylephrine-evoked contraction (n= 3–6). Paired responses in the presence of BIS-I (1 µM) (E) or BIS-VIII (1 µM) (F) from the same arteries before (control) and after N^ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Bars represent periods of ADPβS luminal perfusion. **P < 0.01 and ***P < 0.001 versus L-NAME curve (A–D) or BIS-I control curve (E). L-NAME was present throughout in A–D.

Figure 1

Dilatation responses to luminal perfusion of adenosine 5′-[β-thio]diphosphate (ADPβS) (0.1–3 µM, A–D) in rat pressurized small mesenteric artery in the absence or the presence of the selective protein kinase C inhibitor bisindolylmaleimide I (BIS-I) (1 µM) under submaximal levels of phenylephrine-evoked contraction (n= 4–7). Bars represent periods of ADPβS luminal perfusion. ***P < 0.001 versus control curve. N^ω-nitro-L-arginine methyl ester hydrochloride was not present. BIS-VIII, bisindolylmaleimide VIII.

Figure 3

Effects of protein kinase C (PKC) inhibitors on adenosine 5′-[β-thio]diphosphate (ADPβS) or acetylcholine (ACh)-evoked dilatation. Effects of selective inhibition of PKC by bisindolylmaleimide I (BIS-I) (1 µM), bisindolylmaleimide VIII (BIS-VIII) (1 µM) or 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö 6976) (1 µM) in the dilatation evoked by luminal perfusion of 1 µM ADPβS in the absence (A) or presence (B) of N^ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Values are given for the maximal dilatation peak and 8 min after luminal perfusion in phenylephrine-contracted arteries (n= 4–7). (C) Responses to cumulative additions of ACh to the bath in the absence (n= 18) and presence of luminal BIS-I (n= 15) and BIS-VIII (n= 3). *P < 0.05 and **P < 0.01 versus L-NAME. ^†P < 0.05, ††P < 0.01 versus maximal dilatation peak.

Figure 4

Endothelial cell [Ca²⁺]_i in rat pressurized small mesenteric artery following P2Y1 receptor stimulation by adenosine 5′-[β-thio]diphosphate (ADPβS). (A) Confocal micrograph of endothelial cells loaded with Oregon Green 488 BAPTA-1. Bar = 50 µm. Average data showing the time course of increases in F/F₀ in response to luminal perfusion of 1 µM (B) and 3 µM ADPβS (C) in the absence or the presence of the protein kinase C inhibitor bisindolylmaleimide I (BIS-I) (1 µM; n= 4–7). Bars represent periods of ADPβS luminal perfusion. ***P < 0.001 versus control curve. N^ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (100 µM) was present throughout the experiments.

Figure 6

Dilatation responses and endothelial cell [Ca²⁺]_i to luminal perfusion of adenosine 5′-[β-thio]diphosphate (ADPβS) (3 µM) followed by ADP (1 µM, A–D) and UTP (3 µM, E–F) in pressurized rat small mesenteric arteries (n= 5–7). Arterial preparations were pre-contracted with phenylephrine (PE) for dilatation experiments (only). Bars represent the duration of the luminal perfusion of the agonist. Black triangles indicate addition of acetylcholine (ACh) (1 µM). N^ω-nitro-L-arginine methyl ester hydrochloride was present throughout.

Figure 5

Dilatation responses to luminal perfusion of uridine triphosphate (UTP) (3 µM) and adenosine diphosphate (ADP) (1 µM) in pressurized rat small mesenteric artery. (A) Representative trace showing 3-[N-morpholino]propane-sulphonic acid alone (grey bar) had no effect on diameter, whereas each agonist stimulated rapid dilatation, which reversed rapidly upon washout or cessation of flow. Arterial preparations were precontracted by phenylephrine (PE). Bars represent the duration of the luminal perfusion of the agonist. (B) Summary of dilatation responses to ADP and UTP (n= 4–5). N^ω-nitro-L-arginine methyl ester hydrochloride was present throughout. ACh, acetylcholine.

Figure 7

Immunohistochemical localization of endothelial P2Y1 and M₃ muscarinic acetylcholine receptors (M₃-mAChRs) in pressurized rat mesenteric arteries. Confocal images from the same region of a vessel stained with two P2Y1 receptor antibodies (raised in rabbit) targeting the C-terminus (A), M₃-mAChR antibody (B) or an non-specific normal rabbit immunoglobulin G (C) followed by incubation of an Alexa Fluor 488 secondary antibody (green). The internal elastic lamina (IEL) was stained with Alexa Fluor 633 hydrazide (grey) showing IEL holes. An overlay of the IEL visualization (grey) with P2Y1 receptor staining (green) demonstrated a positive correlation between strong punctate P2Y1 receptor expression and IEL holes (A). Note a weak co-localization of M₃-mAChR at IEL holes (B). Nuclei of endothelial (horizontally aligned cells, arrow) and smooth muscle cells (vertically aligned cells, asterisk) were labelled with propidium iodide (red) (A–C). White arrowheads correspond to the same region in each panel. Bar = 20 µm.