Retsu Mitsui

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder.

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca2+ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder

The morphological characteristics of smooth muscle cells (SMCs) and their innervation of the suburothelial microvasculature of the mouse bladder were investigated by immunohistochemistry. Whole mount bladder mucosal preparations were immune-stained for α-smooth muscle actin (α-SMA) and/or neuronal markers and examined using confocal laser scanning microscopy. Suburothelial arterioles consisted of α-SMA-immunopositive circular smooth muscle cells, while the venular wall composed of α-SMA-positive SMCs that displayed several processes which extended from their cell bodies to form an extensive meshwork. In larger venules, a complex meshwork of stellate-shaped SMCs were observed. NG2 chondroitin sulphate proteoglycan-immunoreactive cell bodies of capillary pericytes were not immunoreactive for α-SMA. In the rat bladder suburothelial venules, circular SMCs were the dominant cell type expressing α-SMA-immunoreactivity. Since α-SMA-positive SMCs in suburothelial arterioles and venules in the mouse bladder had quite distinct morphologies, the innervation of both vessels could be examined by double labelling for α-SMA and various neuronal markers. Varicose nerve bundles immunoreactive for tyrosine hydroxylase (sympathetic nerves), choline acetyltransferase (cholinergic nerves) or substance P (primary afferent nerves) were all detected along side suburothelial arterioles. Single varicose nerve fibres positive for these three neuronal markers were also detected around the venules. Thus, whole mount preparations are useful when examining the morphology of α-SMA-positive SMCs of the microvasculature in the suburothelium of mouse bladder as well as their relationship with their innervations. In conclusion, arterioles and venules of the bladder suburothelium are the target of sympathetic, cholinergic and primary afferent nerve fibres.

Potassium and ANO1/TMEM16A chloride channel profiles distinguish atypical and typical smooth muscle cells from interstitial cells in the mouse renal pelvis

BACKGROUND AND PURPOSE Although atypical smooth muscle cells (SMCs) in the proximal renal pelvis are thought to generate the pacemaker signals that drive pyeloureteric peristalsis, their location and electrical properties remain obscure.

Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder.

Venules of the bladder suburothelium develop spontaneous phasic constrictions that may play a critical role in maintaining venular drainage of tissue metabolites. We aimed to investigate neurohumoral regulation of the spontaneous venular constrictions (SVCs). Changes in venular diameter of the rat bladder suburothelium were monitored using a video tracking system, whilst the effects of electrical field stimulation (EFS) and bath-applied bioactive substances were investigated. The innervation of the suburothelial microvasculature was examined by immunohistochemistry. EFS (10Hz for 30s) induced an increase in the frequency of SVCs that was prevented by phentolamine (1μM). In phentolamine-pretreated venules, EFS suppressed SVCs with a venular dilatation in a manner attenuated by propranolol (1μM) or l-nitro arginine (LNA, 10μM). BRL37344 (1μM), a β3 adrenoceptor agonist, dilated venules and reduced the frequency of SVCs in an LNA-sensitive manner. ACh (1-10μM) increased the frequency of SVCs. ATP (1μM) transiently constricted venules and then caused LNA-sensitive cessation of SVCs associated with a dilatation. Substance P (100nM) caused a venular constriction, whilst calcitonin gene related peptide (CGRP, 100nM) caused a dilatation of venules and suppression of SVCs that were not inhibited by LNA. Immunohistochemical staining demonstrated sympathetic as well as substance P- and CGRP-containing nerves running along the venules. Spontaneous constrictions of suburothelial venules are accelerated by sympathetic α-adrenergic stimulation, but suppressed upon β-adrenergic stimulation. In addition, suburothelial venular constrictions appear to be modulated by several bioactive substances that could be released from urothelium or suburothelial sensory nerves.

Spontaneous transient hyperpolarizations in the rabbit small intestine

Recently, it was shown that fibroblast‐like cells (FLCs) possess the apparatus to mediate purinergic motor neurotransmission in the gastrointestinal tract. However, the electrophysiological properties of FLCs in situ have not been determined. We recorded two patterns of slow waves from longitudinal smooth muscle cells and circular smooth muscle cells, large amplitude slow waves from interstitial cells of Cajal, and spontaneous transient hyperpolarizations (STHs) from FLCs in the rabbit small intestine using intracellular recording combined with dye injection to identify the cellular morphology of impaled cells. Drugs that inhibit the signalling pathway involved in purinergic neurotransmission inhibited STHs in FLCs. Small amplitude STHs were recorded in smooth muscle cells but not in interstitial cells of Cajal, suggesting that STHs from FLCs were conducted passively to smooth muscle cells. We conclude that FLCs display the molecular apparatus necessary to mediate purinergic neurotransmission and may tonically dampen smooth muscle excitability in the rabbit small intestine by an ongoing discharge of STHs.

Properties of submucosal venules in the rat distal colon

Venules within the gut wall may have intrinsic mechanisms for maintaining the circulation even upon the intestinal wall distension. We aimed to explore spontaneous and nerve‐mediated contractile activity of colonic venules.

Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum.

Properties of spontaneous Ca(2+) transients in the myenteric microvasculature of the guinea-pig stomach were investigated. Specifically, we explored the spatio-temporal origin of Ca(2+) transients and the role of voltage-dependent Ca(2+) channels (VDCCs) in their intercellular synchrony using fluorescence Ca(2+) imaging and immunohistochemistry. The microvasculature generated spontaneous Ca(2+) transients that were independent of both Ca(2+) transients in interstitial cells of Cajal (ICC) and neural activity. Spontaneous Ca(2+) transients were highly synchronous along the length of microvasculature, and appeared to be initiated in pericytes and spread to arteriolar smooth muscle cells (SMCs). In most cases, the generation or synchrony of Ca(2+) transients was not affected by blockers of L-type VDCCs. In nifedipine-treated preparations, synchronous spontaneous Ca(2+) transients were readily blocked by Ni(2+), mibefradil or ML216, blockers for T-type VDCCs. These blockers also suppressed the known T-type VDCC dependent component of ICC Ca(2+) transients or slow waves. Spontaneous Ca(2+) transients were also suppressed by caffeine, tetracaine or cyclopiazonic acid (CPA). After the blockade of both L- and T-type VDCCs, asynchronous Ca(2+) transients were generated in pericytes on precapillary arterioles and/or capillaries but not in arteriolar SMCs, and were abolished by CPA or nominally Ca(2+) free solution. Together these data indicate that pericytes in the myenteric microvasculature may act as the origin of synchronous spontaneous Ca(2+) transients. Pericyte Ca(2+) transients arise from Ca(2+) release from the sarco-endoplasmic reticulum and the opening of T-type Ca(2+) VDCCs is required for their synchrony and propagation to arteriolar SMCs.

Role of PTHrP and Sensory Nerve Peptides in Regulating Contractility of Muscularis Mucosae and Detrusor Smooth Muscle in the Guinea Pig Bladder

PURPOSE We investigated the neurohumoral modulation of the contractility of bladder muscularis mucosae (mucosa) compared with that of detrusor smooth muscle. MATERIALS AND METHODS Changes in the contractility of mucosal and detrusor bundles from guinea pig bladders were measured using isometric tension recording. The morphological relationship between the muscularis mucosae and blood vessels, and their sensory innervation was examined by fluorescence immunohistochemistry. RESULTS Meshworks of muscularis mucosae with numerous branches and anastomosis preferentially ran parallel with suburothelial blood vessels. Although PTHrPRs (parathyroid hormone-related peptide receptors) were expressed in detrusor and mucosa, the endogenous detrusor relaxant PTHrP (parathyroid hormone-related peptide) (1 nM) suppressed spontaneous contractions in detrusor but not in mucosa. A higher concentration of PTHrP (10 nM) was required to inhibit mucosal contractility. Capsaicin (1 μM) abolished spontaneous contractions in mucosa but had an excitatory action on detrusor contractility. hCGRP (human calcitonin gene-related peptide) (1 nM) attenuated spontaneous mucosal contractions. Pretreatment with the CGRP (calcitonin gene-related peptide) antagonist hCGRP 8-37 (2 μM) inhibited CGRP or capsaicin induced suppression of spontaneous contractions. Consistently, CGRP immunoreactive primary afferent nerves were abundant in muscularis mucosae. CONCLUSIONS Co-localization of muscularis mucosae with the suburothelial microvasculature suggests that spontaneous contractions of mucosa might function to prevent microvasculature stretching upon bladder wall distension during the storage phase. It is likely that PTHrP selectively suppresses spontaneous contractions in detrusor but not in mucosa. Thus, endogenous PTHrP may well increase bladder compliance without an associated distension induced deformation of mucosal elements. Excessive stimulations of sensory nerves may suppress mucosal contractility by releasing CGRP.

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

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.

Nerve-induced responses of mouse vaginal smooth muscle

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.