Roles of endothelial prostaglandin I2 in maintaining synchronous spontaneous Ca2+ transients in rectal capillary pericytes

Abstract

In hollow visceral organs, capillary pericytes appear to drive spontaneous Ca²⁺ transients in the upstream arterioles. Here, mechanisms underlying the intercellular synchrony of pericyte Ca²⁺ transients were explored. Ca²⁺ dynamics in NG2 chondroitin sulphate proteoglycan (NG2)-expressing capillary pericytes were examined using rectal mucosa-submucosa preparations of NG2-GCaMP6 mice. Spontaneous Ca²⁺ transients arising from endoplasmic reticulum Ca²⁺ release were synchronously developed amongst capillary pericytes in a gap junction blocker (3 μM carbenoxolone)-sensitive manner and could spread into upstream vascular segments. Spontaneous Ca²⁺ transients were suppressed by the Ca²⁺ -activated Cl^- channel (CaCC) blocker niflumic acid and their synchrony was diminished by a TMEM16A inhibitor (3 μM Ani9) in accordance with TMEM16A immunoreactivity in pericytes. In capillaries where cyclooxygenase (COX)-2 immunoreactivity was expressed in endothelium but not pericytes, non-selective COX inhibitors (1 μM indomethacin or 10 μM diclofenac) or COX-2 inhibitor (10 μM NS 398) disrupted the synchrony of spontaneous Ca²⁺ transients and raised the basal Ca²⁺ level. Subsequent prostaglandin I₂ (PGI₂ ; 100 nM) or the K_ATP channel opener levcromakalim restored the synchrony with a reduction in the Ca²⁺ level. PGI₂ receptor antagonist (1 μM RO1138452) also disrupted the synchrony of spontaneous Ca²⁺ transients and increased the basal Ca²⁺ level. Subsequent levcromakalim restored the synchrony and reversed the Ca²⁺ rise. Thus, the synchrony of spontaneous Ca²⁺ transients in pericytes appears to be developed by the spread of spontaneous transient depolarisations arising from the opening of TMEM16A CaCCs. Endothelial PGI₂ may play a role in maintaining the synchrony, presumably by stabilising the resting membrane potential in pericytes. KEY POINTS: Capillary pericytes in the rectal mucosa generate synchronous spontaneous Ca²⁺ transients that could spread into the upstream vascular segment. Spontaneous Ca²⁺ release from the endoplasmic reticulum (ER) triggers the opening of Ca²⁺ -activated Cl^- channel TMEM16A and resultant depolarisations that spread amongst pericytes via gap junctions, establishing the synchrony of spontaneous Ca²⁺ transients in pericytes. Prostaglandin I₂ (PGI₂ ), which is constitutively produced by the endothelium depending on cyclooxygenase-2, appears to prevent premature ER Ca²⁺ releases in the pericytes allowing periodic, regenerative Ca²⁺ releases. Endothelial PGI₂ may maintain the synchrony of pericyte activity by stabilising pericyte resting membrane potential by opening of K_ATP channels.

Keywords: capillary; gastrointestinal tract; intracellular Ca2+ imaging; pericyte; prostacyclin.