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 ADPbS (3 mM) 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: ADPbS 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 endotheliumdependent dilatation to acetylcholine (1 mM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 mM) or BIS-VIII (1 mM) tended to augment concentration-dependent dilatation to ADPbS (0.1-3 mM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 mM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca 2+ ]i in pressurized arteries confirmed the P2Y1 receptor but not M3 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.

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5-bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

“…In addition, XE-991 also significantly reduced the facilitation by bradykinin (Figures 3b and e, n ¼ 11). In contrast, neither cyclopiazonic acid (3 Â 10 À6 mol l À1 , n ¼ 6), a sarcoendoplasmic reticulum calcium ATPase inhibitor, 21 nor bisindolylmaleimide-I (10 À6 mol l À1 , n ¼ 9), a protein kinase C inhibitor, 25 altered the action of bradykinin (Figure 3c-e).…”

“…The following drugs were used: bradykinin (10 À7 mol l À1 ), 3 U-73122 (10 À6 mol l À1 ), 20 U-73433 (10 À6 mol l À1 ), 20 cyclopiazonic acid (3 Â 10 À6 mol l À1 ), 21 HC-030031 (5 Â 10 À6 mol l À1 ), 22 H-89 (10 À6 mol l À1 ), 8,23 XE-991 (10 À5 mol l À1 ), 24 bisindolylmaleimide-I (10 À6 mol l À1 ), 25 capsaicin (10 À5 mol l À1 ), 26 capsazepine (5 Â 10 À6 mol l À1 ) 27 and phentolamine (10 À4 mol l À1 ) 28 from Sigma-Aldrich (St Louis, MO, USA) arachidonyl trifluoromethyl ketone (3 Â 10 À5 mol l À1 , AACOCF 3 ) 29 from Calbiochem (San Diego, CA, USA) o-conotoxin GVIA (2 Â 10 À9 or 10 À6 mol l À1 ) 30,31 and Calcitonin Gene-Related Peptide (Human, 8-37) (CGRP8-37, 10 À7 mol l À1 ) 32 from Peptide Institute (Osaka, Japan) and wortmannin (10 À5 mol l À1 ) 33 and AP-18 (10 À6 mol l À1 ) 34 from Biomol (Plymouth Meeting, PA, USA). Stock solutions of bradykinin, phentolamine, oconotoxin GVIA, H-89 and CGRP8-37 were dissolved in distilled water.…”

Coordination of Vasomotor Responses by the Endothelium

“…11 which is consistent with expression of M3 muscarinic ACh receptors within this microdomain. 43 Therefore this does not appear to be the explanation for the differential activation of KCa2.3-and KCa3.1-channels. However, changes in extracellular Ca 2 do affect changes in the activity of KCa3.1-channels, indicating quite clearly that the ECP microdomain and the input to EDH and thus vasodilatation is subject to dynamic modulation by the immediate intra-and extracellular environment in a complex fashion.…”

“…The endothelial cell projections (ECPs) form a unique signaling circuit, with certain proteins involved in endothelium-dependent dilatation concentrated in this space. 11,18 Receptors for some agonists (cyan) 43 and inositol trisphosphate (InsP3) receptors (pink) 17,42 appear concentrated within this signaling domain. Agonist-evoked rises in Ca 2+ can activate KCa3.1-channels (green), which are inhibited by rises in extracellular Ca 2+ , likely via the CaSR (orange) and PKA, 11 and KCa2.3-channels (blue).…”

Mechanism Underlying Endothelium-Dependent Relaxation by 2-Methylthio-ADP in Monkey Cerebral Artery

“…1B. In preliminary experiments, relaxation induced by 2MeSADP (1 nM -10 μM) was found to be decreased with repeated applications, probably due to desensitization of endothelial P2Y 1 receptor (14). Therefore, we decided to compare the initial response to 2MeSADP in order to analyze the underlying mechanism as reported previously (15).…”