Nitrergic inhibition of sympathetic arteriolar constrictions in the female rodent urethra.

During the urine storage phase, tonically contracting urethral musculature would have a higher energy consumption than bladder muscle that develops phasic contractions. However, ischaemic dysfunction is less prevalent in the urethra than in the bladder, suggesting that urethral vasculature has intrinsic properties ensuring an adequate blood supply. Diameter changes in rat or mouse urethral arterioles were measured using a video-tracking system. Intercellular Ca2+ dynamics in arteriolar smooth muscle (SMCs) and endothelial cells were visualised using NG2- and parvalbumin-GCaMP6 mice, respectively. Fluorescence immunohistochemistry was used to visualise the perivascular innervation. In rat urethral arterioles, sympathetic vasoconstrictions were predominantly suppressed by α,ß-methylene ATP (10 µM) but not prazosin (1 µM). Tadalafil (100 nM), a PDE5 inhibitor, diminished the vasoconstrictions in a manner reversed by N-ω-propyl-l-arginine hydrochloride (l-NPA, 1 µM), a neuronal NO synthesis (nNOS) inhibitor. Vesicular acetylcholine transporter immunoreactive perivascular nerve fibres co-expressing nNOS were intertwined with tyrosine hydroxylase immunoreactive sympathetic nerve fibres. In phenylephrine (1 µM) pre-constricted rat or mouse urethral arterioles, nerve-evoked vasodilatations or transient SMC Ca2+ reductions were largely diminished by l-nitroarginine (l-NA, 10 µM), a broad-spectrum NOS inhibitor, but not by l-NPA. The CGRP receptor antagonist BIBN-4096 (1 µM) shortened the vasodilatory responses, while atropine (1 µM) abolished the l-NA-resistant transient vasodilatory responses. Nerve-evoked endothelial Ca2+ transients were abolished by atropine plus guanethidine (10 µM), indicating its neurotransmitter origin and absence of non-adrenergic non-cholinergic endothelial NO release. In urethral arterioles, NO released from parasympathetic nerves counteracts sympathetic vasoconstrictions pre- and post-synaptically to restrict arteriolar contractility. KEY POINTS: Despite a higher energy consumption of the urethral musculature than the bladder detrusor muscle, ischaemic dysfunction of the urethra is less prevalent than that of the bladder. In the urethral arterioles, sympathetic vasoconstrictions are predominately mediated by ATP, not noradrenaline. NO released from parasympathetic nerves counteracts sympathetic vasoconstrictions by its pre-synaptic inhibition of sympathetic transmission as well as post-synaptic arteriolar smooth muscle relaxation. Acetylcholine released from parasympathetic nerves contributes to endothelium-dependent, transient vasodilatations, while CGRP released from sensory nerves prolongs NO-mediated vasodilatations. PDE5 inhibitors could be beneficial to maintain and/or improve urethral blood supply and in turn the volume and contractility of urethral musculature.

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

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 KATP 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 KATP channels.

Role of capillary pericytes in the integration of spontaneous Ca2+ transients in the suburothelial microvasculature in situ of the mouse bladder.

KEY POINTS: In the bladder suburothelial microvasculature, pericytes in different microvascular segments develop spontaneous Ca2+ transients with or without associated constrictions. Spontaneous Ca2+ transients in pericytes of all microvascular segments primarily rely on the cycles of Ca2+ uptake and release by the sarco- and endoplasmic reticulum. The synchrony of spontaneous Ca2+ transients in capillary pericytes exclusively relies on the spread of depolarizations resulting from the opening of Ca2+ -activated chloride channels (CaCCs) via gap junctions. CaCC-dependent depolarizations further activate L-type voltage-dependent Ca2+ channels as required for the synchrony of Ca2+ transients in pericytes of pre-capillary arterioles, post-capillary venules and venules. Capillary pericytes may drive spontaneous Ca2+ transients in pericytes within the suburothelial microvascular network by sending CaCC-dependent depolarizations via gap junctions. ABSTRACT: Mural cells in the microvasculature of visceral organs develop spontaneous Ca2+ transients. However, the mechanisms underlying the integration of these Ca2+ transients within a microvascular unit remain to be clarified. In the present study, the origin of spontaneous Ca2+ transients and their propagation in the bladder suburothelial microvasculature were explored. Cal-520 fluorescence Ca2+ imaging and immunohistochemistry were carried out on mural cells using mice expressing red fluorescent protein (DsRed) under control of the NG2 promotor. NG2(+) pericytes in both pre-capillary arterioles (PCAs) and capillaries developed synchronous spontaneous Ca2+ transients. By contrast, although NG2-DsRed also labelled arteriolar smooth muscle cells, these cells remained quiescent. Both NG2(+) pericytes in post-capillary venules (PCVs) and NG2(-) venular pericytes exhibited propagated Ca2+ transients. L-type voltage-dependent Ca2+ channel (LVDCC) blockade with nifedipine prevented Ca2+ transients or disrupted their synchrony in PCA, PCV and venular pericytes without dis-synchronizing Ca2+ transients in capillary pericytes. Blockade of gap junctions with carbenoxolone or Ca2+ -activated chloride channels (CaCCs) with 4,4'-diisothiocyanato-2,2'-stilbenedisulphonic acid disodium salt prevented Ca2+ transients in PCA and venular pericytes and disrupted the synchrony of Ca2+ transients in capillary and PCV pericytes. Spontaneous Ca2+ transients in pericytes of all microvascular segments were abolished or suppressed by cyclopiazonic acid, caffeine or tetracaine. The synchrony of Ca2+ transients in capillary pericytes arising from spontaneous Ca2+ release from the sarco- and endoplasmic reticulum appears to rely exclusively on CaCC activation, whereas subsequent LVDCC activation is required for the synchrony of Ca2+ transients in pericytes of other microvascular segments. Capillary pericytes may drive spontaneous activity in the suburothelial microvascular unit to facilitate capillary perfusion.