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.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

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 PTHrP in attenuating transient pressure rises and associated afferent nerve activity of the rat bladder.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) as the bladder fills acts as an endogenous DSM relaxant to facilitate bladder storage function. Here, the effects of exogenous PTHrP on transient pressure rises (TPRs) in the bladder and associated afferent nerve activity during bladder filling were investigated. In anaesthetized rats, changes in the intravesical pressure were measured while the bladder was gradually filled with saline. Afferent nerve activity was simultaneously recorded from their centrally disconnected left pelvic nerves. In DSM strips, spontaneous and nerve-evoked contractions were isometrically recorded. The distribution of PTHrP receptors (PTHrPRs) in the bladder wall was also examined by fluorescence immunostaining. The bladders in which the contralateral pelvic nerve was also centrally disconnected developed nifedipine, an L-type voltage-dependent Ca2+ channel blocker-sensitive TPRs (< 3 mmHg). Intravenous administration of PTHrP suppressed these TPRs and associated bursts of afferent nerve activity. In the bladders with centrally connected contralateral pelvic nerves, atropine, a muscarinic receptor antagonist-sensitive large TPRs (> 3 mmHg) developed in the late filling phase. PTHrP diminished the large TPRs and corresponding surges of afferent nerve activity. In DSM strips, bath-applied PTHrP (10 nM) suppressed spontaneous phasic contractions, while less affecting nerve-evoked contractions. PTHrPRs were expressed in DSM cells but not in intramural nerve fibers. Thus, PTHrP appears to suppress bladder TPRs and associated afferent nerve activity even under the influence of low degree of parasympathetic neural input during storage phases. Endogenous PTHrP may indirectly attenuate afferent nerve activity by suppressing TPRs to facilitate urinary accommodation.

Mechanosensitive modulation of peristaltic contractions in the mouse renal pelvis.

The renal pelvis develops spontaneous phasic contractions (SPCs) that underlie pyeloureteric peristalsis. Increased urine flow into the renal pelvis mechanically stimulates the contractile machinery within the renal pelvis to facilitate the propagation of peristalsis. Here, the effects of mechanostimulation of the pelvicalyceal junction (PCJ), where SPCs originate from, on the properties of SPCs were investigated. Using the wire myograph, isometric tension changes in tubular preparations of mouse renal pelvis with calyces were circumferentially measured, while mechanostimuli were applied to the PCJ. Immunohistochemistry and intracellular Ca2+ imaging were performed, respectively, to investigate the distribution and functional roles of mechanosensitive TRPV4 channels in the renal pelvis. SPCs periodically originated from PCJ and propagated distally. Mechanostimulation of the PCJ reduced the frequency of SPCs by about 60%, while almost quadrupling their amplitude. Capsaicin (100 nM), an agonist of TRPV1 channels, or calcitonin gene-related peptide (CGRP) (30 nM) also slowed and enlarged SPCs. A prolonged pre-exposure to capsaicin or BIBN4096 (1 µM), a CGRP receptor antagonist, inhibited the mechanostimulation-induced reduction in the SPC frequency, but did not block the increase in SPC amplitude. TRPV4 immunoreactivity was expressed in both atypical (ASMCs) and typical smooth muscle cells (TSMCs). GSK1016790A (100 nM), a TRPV4 agonist, enlarged SPCs independently of TRPV1 or CGRP without increasing the amplitude of spontaneous Ca2+ transients in TSMCs. Thus, mechanostimulation of PCJ appears to activate TRPV1-expressing sensory nerves, releasing CGRP that predominantly reduce the SPC frequency. Activation of TRPV4 may be involved in the mechanosensitive enlargement of SPCs. (247 words).

Functional nitrergic innervation of smooth muscle structures in the mucosa of pig lower urinary tract.

Neurally released nitric oxide (NO) functions as an inhibitory neurotransmitter of urethral but not detrusor smooth muscles while relaxing bladder vasculature and muscularis mucosae (MM). Here, the distribution of nitrergic nerves was examined in the mucosa of pig lower urinary tract using immunohistochemistry, and their vasodilatory functions were studied by measuring arteriolar diameter changes. Properties of smooth muscle cells in the lamina propria (SMC-LP) of urethra and trigone were also investigated using florescence Ca2+ imaging. In the bladder mucosa, neuronal nitric oxide synthase (nNOS)-immunoreactive nitrergic fibres projected to suburothelial arterioles and venules. Perivascular nitrergic nerves were intermingled with but distinct from tyrosine hydroxylase (TH)-immunoreactive sympathetic or calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerves. MM receive a nitrergic but not sympathetic or afferent innervation. In the mucosa of urethra and trigone, nitrergic nerves were in close apposition with sympathetic or afferent nerves around suburothelial vasculature but did not project to SMC-LP. In suburothelial arterioles of bladder and urethra, N ω-nitro-L-arginine (L-NA, 100 µM), an NOS inhibitor, enhanced electrical field stimulation (EFS)-induced sympathetic vasoconstrictions, while tadalafil (10 nM), a phosphodiesterase type 5 (PDE5) inhibitor, suppressed the vasoconstrictions. SMC-LP developed asynchronous spontaneous Ca2+ transients without responding to EFS. The spontaneous Ca2+ transients were enhanced by acetylcholine (1 µM) and diminished by noradrenaline (1 µM) but not SIN-1 (10 µM), an NO donor. In the lower urinary tract mucosa, perivascular nitrergic nerves appear to counteract the sympathetic vasoconstriction to maintain the mucosal circulation. Bladder MM but not SMC-LP receive an inhibitory nitrergic innervation.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Role of enteric dopaminergic neurons in regulating peristalsis of rat proximal colon.

BACKGROUND: Constipation is commonly seen in patients with Parkinson's disease associated with a loss of dopaminergic neurons in both central and enteric nervous systems. However, the roles of enteric dopaminergic neurons in developing constipation remain to be elucidated. Here, we investigated the roles of enteric dopaminergic neurons in the generation of colonic peristalsis. METHODS: Cannulated segments of rat proximal colon were situated in the organ bath, abluminally perfused with physiological salt solution and luminally perfused with 0.9% saline. Drugs were applied in the abluminal solution. Changes in diameter along the length of the colonic segment were captured by a video camera and transformed into spatio-temporal maps. Fluorescence immunohistochemistry was also carried out. KEY RESULTS: Blockade of nitrergic neurotransmission prevented oro-aboral propagation of peristaltic waves and caused a colonic constriction without affecting ripples, non-propagating myogenic contractions. Blockade of cholinergic neurotransmission also prevented peristaltic waves but suppressed ripples with a colonic dilatation. Tetrodotoxin (0.6 µM) abolished peristaltic waves and increased ripples with a constriction. SCH 23390 (20 µM), a D1 -like dopamine receptor antagonist, slowed the peristaltic waves and caused a constriction, while GBR 12909 (1 µM), a dopamine reuptake inhibitor, diminished the peristaltic waves with a dilatation. Bath-applied dopamine (3 µM) abolished the peristaltic waves associated with a colonic dilation in an SCH 23390 (5 µM)-sensitive manner. D1 receptor immunoreactivity was co-localized to nitrergic and cholinergic neurons. CONCLUSIONS AND INFERENCES: Dopaminergic neurons appear to facilitate nitrergic neurons via D1 -like receptors to stabilize asynchronous contractile activity resulting in the generation of colonic peristalsis.

Comparative effects of angiotensin II on the contractility of muscularis mucosae and detrusor in the pig urinary bladder.

To explore contractile actions of angiotensin II (ATII) on the muscularis mucosae (MM) of the bladder, ATII-induced contractions were compared between MM and the detrusor smooth muscle (DSM) of the pig bladder by isometric tension recordings. Effects of ATII on spontaneous Ca2+ transients in MM were visualized using Cal-520 fluorescence. ATII receptor type 1 (ATR1) expression in MM and DSM was also examined by immunohistochemistry. ATII (1 nM-1 µM) caused phasic contractions of MM in a concentration-dependent manner, while ATII (10 nM-10 µM) had no or marginal effects on DSM contractility. ATII (100 nM)-induced MM contractions had an amplitude of approximately 70% of carbachol (1 µM)-induced or 90% of U46619 (100 nM)-induced contractions. Candesartan (10 nM), an ATR1 blocker, prevented the contractile effects of ATII (1 nM) in MM, while ATR1 immunofluorescence was greater in MM than DSM. ATII (10-100 pM) increased the frequency but not the amplitude of spontaneous Ca2+ transients in MM. Both urothelium-intact and -denuded MM strips developed comparable spontaneous phasic contractions, but ATII, carbachol and U46619-induced contractions were significantly larger in urothelium-denuded than urothelium-intact MM strips. In conclusion, the MM appears to have a much greater sensitivity to ATII compared with DSM that could well sense circulating ATII, suggesting that MM may be the predominant target of contractile actions induced by ATII in the bladder while the urothelium appears to inhibit MM contractility.

NO-mediated signal transmission in bladder vasculature as a therapeutic target of PDE5 inhibitors. Rodent model studies.

BACKGROUND AND PURPOSE: While the bladder vasculature is considered as a target of PDE5 inhibitors to improve bladder storage dysfunctions, its characteristics are largely unknown. Thus, the functional and morphological properties of arteries/arterioles of the bladder focusing on the NO-mediated signal transmission were explored. EXPERIMENTAL APPROACH: Diameter changes in rat bladder arteries/arterioles were measured using a video-tracking system. Intercellular Ca2+ dynamics in pericytes or smooth muscle cells (SMCs) of suburothelial arterioles were visualised using transgenic mice expressing GCaMP6 under control of the NG2- or parvalbumin-promoter. The perivascular innervation was investigated using fluorescence immunohistochemistry. KEY RESULTS: In rat suburothelial arterioles and vesical arteries, tadalafil (100 nM) attenuated nerve-evoked sympathetic vasoconstrictions. In both vascular segments, tadalafil-induced inhibition of sympathetic vasoconstriction was prevented by N ω-propyl-l-arginine hydrochloride (l-NPA, 1 µM), an nNOS inhibitor or N ω-nitro-l-arginine (l-NA, 100 µM). Both vascular segments were densely innervated with nNOS-positive nitrergic nerves in close apposition to tyrosine hydroxylase-immunoreactive sympathetic nerves. In pericyte-covered pre-capillary arterioles of the mouse bladder where sympathetic nerves were absent, nerve stimulation evoked transient reductions in pericyte Ca2+ levels that were shortened by l-NPA and abolished by l-NA. In SMC-containing arterioles, tadalafil (10 nM) caused a l-NPA-sensitive suppression of sympathetic Ca2+ transients. In mice, nitrergic perivascular nerves were distributed in the arterioles and the pre-capillary arterioles. CONCLUSION AND IMPLICATIONS: Both nitrergic nerve and nerve-evoked endothelial NO release appear to be involved in vasodilatory signal transmission in bladder vasculature. The NO-mediated signal transmission is a potential target for PDE5 inhibitor therapy in bladder dysfunctions.

Functional heterogeneity of PDGFRα (+) cells in spontaneously active urogenital tissues.

AIMS: As PDGFRα (+) cells appear not to suppress the excitability of detrusor smooth muscle by generating SK3-dependent hyperpolarising as proposed in the gastrointestinal tract, we further explored the functional roles of PDGFRα (+) cells in regulating the spontaneous activity of urogenital tissues. METHODS: Using PDGFRα-eGFP mice, intracellular Ca2+ signaling in PDGFRα (+) cells of the bladder lamina propria, renal pelvis, and seminal vesicle were visualized using Cal-590 fluorescence. The distribution and SK3 expression of PDGFRα (+) cells were also examined by immunohistochemistry. RESULTS: In the bladder lamina propria, SK3 (-) PDGFRα (+) cells exhibited spontaneous Ca2+ transients and responded to stimulation of P2Y1 purinoceptors with MRS2365 (100 nM) or adenosine diphosphate (ADP) (100 µM) by developing Ca2+ transients. In the proximal renal pelvis, PDGFRα (+) cells were distributed in the mucosal, muscular and serosal layers but did not express SK3 immunoreactivity. PDGFRα (+) cells in the musculature resembling atypical smooth muscle cells generated spontaneous Ca2+ transients that were partially suppressed upon P2Y1-stimulation, while vigorously responding to human angiotensin II (100 nM). In the seminal vesicle, PDGFRα (+) cells in the musculature but not mucosa expressed SK3 immunoreactivity. In the mucosa, the P2Y1 stimulation evoked Ca2+ transients in both PDGFRα (+) cells and PDGFRα (-) cells. CONCLUSION: PDGFRα (+) cells in spontaneously active urogenital tissues display heterogeneity in terms of their SK3 expression and P2Y1-induced Ca2+ responses. Muscular PDGFRα (+) cells in the renal pelvis and mucosal PDGFRα (+) cells in the seminal vesicle may generate depolarizing signals to drive smooth muscle cells.

Synchrony of spontaneous Ca2+ activity in microvascular mural cells.

Spontaneous rhythmic constrictions known as vasomotion are developed in several microvascular beds in vivo. Vasomotion in arterioles is considered to facilitate blood flow, while venular vasomotion would facilitate tissue metabolite drainage. Mechanisms underlying vasomotion periodically generate synchronous Ca2+ transients in vascular smooth muscle cells (VSMCs). In visceral organs, mural cells (pericytes and VSMCs) in arterioles, capillaries and venules exhibit synchronous spontaneous Ca2+ transients. Since sympathetic regulation is rather limited in the intra-organ microvessels, spontaneous activity of mural cells may play an essential role in maintaining tissue perfusion. Synchronous spontaneous Ca2+ transients in precapillary arterioles (PCAs)/capillaries appear to propagate to upstream arterioles to drive their vasomotion, while venules develop their own synchronous Ca2+ transients and associated vasomotion. Spontaneous Ca2+ transients of mural cells primarily arise from IP3 and/or ryanodine receptor-mediated Ca2+ release from sarcoendoplasmic reticulum (SR/ER) Ca2+ stores. The resultant opening of Ca2+-activated Cl- channels (CaCCs) causes a membrane depolarisation that triggers Ca2+ influx via T-type and/or L-type voltage-dependent Ca2+ channels (VDCCs). Mural cells are electrically coupled with each other via gap junctions, and thus allow the sequential spread of CaCC or VDCC-dependent depolarisations to develop the synchrony of Ca2+ transients within their network. Importantly, the synchrony of spontaneous Ca2+ transients also requires a certain range of the resting membrane potential that is maintained by the opening of Kv7 voltage-dependent K+ (Kv7) and inward rectifier K+ (Kir) channels. Thus, a depolarised membrane would evoke asynchronous, 'premature' spontaneous Ca2+ transients, while a hyperpolarised membrane prevents any spontaneous activity.

Neural regulation of the contractility of nutrient artery in the guinea pig tibia.

Nutrient arteries provide the endosteal blood supply to maintain bone remodelling and energy metabolism. Here, we investigated the distribution and function of perivascular nerves in regulating the contractility of the tibial nutrient artery. Changes in artery diameter were measured using a video tracking system, while the perivascular innervation was investigated using fluorescence immunohistochemistry. Nerve-evoked phasic constrictions of nutrient arteries were suppressed by phentolamine (1 µM), an α-adrenoceptor antagonist, guanethidine (10 µM), a blocker of sympathetic transmission, or fluoxetine (10 µM), a serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor. In arteries pretreated with guanethidine, residual nerve-evoked constrictions were abolished by a high concentration of propranolol (10 µM) that is known to inhibit 5-HT receptors, or ketanserin (100 nM), a 5-HT2 receptor antagonist, but not SB207216 (1 µM), an antagonist of 5-HT3 and 5-HT4 receptors. Bath-applied 5-HT (100 nM) induced arterial constriction that was suppressed by propranolol (10 µM) or ketanserin (100 nM). Nerve-evoked arterial constrictions were enhanced by spantide (1 µM), a substance P (SP) receptor antagonist, or L-nitro arginine (L-NA; 100 µM), an inhibitor of nitric oxide synthase (NOS). Immunohistochemistry revealed 5-HT-positive nerves running along the arteries that are distinct from perivascular sympathetic or substance P-positive primary afferent nerves. For the first time, functional serotonergic nerves are identified in the tibial nutrient artery of the guinea pig. Thus, it appears that tibial nutrient arterial calibre is regulated by the balance between sympathetic and serotonergic vasoconstrictor nerves and vasodilator afferent nerves that release substance P-stimulating endothelial nitric oxide (NO) release.

Contractile elements and their sympathetic regulations in the pig urinary bladder: a species and regional comparative study.

Contractile behaviour of the urinary bladder and its sympathetic inhibition during storage phases are not well understood. Here, we explore muscularis mucosae (MM) as a predominant mucosal contractile element and the capability of sympathetic nerves to relax detrusor smooth muscle (DSM) or MM. Distribution of α-smooth muscle actin (α-SMA)-immunoreactive cells was compared in pig, human, guinea pig, rat and mouse bladders by immunohistochemistry, while contractility of the bladder mucosa was compared in these species by isometric tension recordings. In pig, human and guinea pig bladders, DSM and MM located in the lamina propria expressed α-SMA immunoreactivity, while both rat and mouse bladders lacked a MM. Consistent with this presence or absence of MM, bladder mucosa of pig, human and guinea pig but not rat and mouse developed spontaneous phasic contractions (SPCs). Distribution of tyrosine hydroxylase (TH)-immunoreactive sympathetic nerve fibres was compared in pig DSM, MM, trigone and urethra, as were their sympathetic nerve-evoked contractile/relaxing responses examined. In pig DSM or MM, where TH-immunoreactive sympathetic fibres exclusively projected to the vasculature, sympathetic relaxations were difficult to demonstrate. In contrast, sympathetic contractions were invariably evoked in pig trigone and urethra where the smooth muscle cells receive TH-immunoreactive sympathetic innervations. Thus, SPCs of bladder mucosa appear to predominantly arise from the MM displaying species differences. Despite the currently accepted concept of sympathetic nerve-mediated DSM relaxation during the storage phase, it is unlikely that neurally released noradrenaline acts on ß-adrenoceptors to relax either DSM or MM due to the anatomical lack of sympathetic innervation.

Role of Pericytes in the Initiation and Propagation of Spontaneous Activity in the Microvasculature.

The microvasculature is composed of arterioles, capillaries and venules. Spontaneous arteriolar constrictions reduce effective vascular resistance to enhance tissue perfusion, while spontaneous venular constrictions facilitate the drainage of tissue metabolites by pumping blood. In the venules of visceral organs, mural cells, i.e. smooth muscle cells (SMCs) or pericytes, periodically generate spontaneous phasic constrictions, Ca2+ transients and transient depolarisations. These events arise from spontaneous Ca2+ release from the sarco-endoplasmic reticulum (SR/ER) and the subsequent opening of Ca2+-activated chloride channels (CaCCs). CaCC-dependent depolarisation further activates L-type voltage-dependent Ca2+ channels (LVDCCs) that play a critical role in maintaining the synchrony amongst mural cells. Mural cells in arterioles or capillaries are also capable of developing spontaneous activity. Non-contractile capillary pericytes generate spontaneous Ca2+ transients primarily relying on SR/ER Ca2+ release. Synchrony amongst capillary pericytes depends on gap junction-mediated spread of depolarisations resulting from the opening of either CaCCs or T-type VDCCs (TVDCCs) in a microvascular bed-dependent manner. The propagation of capillary Ca2+ transients into arterioles requires the opening of either L- or TVDCCs again depending on the microvascular bed. Since the blockade of gap junctions or CaCCs prevents spontaneous Ca2+ transients in arterioles and venules but not capillaries, capillary pericytes appear to play a primary role in generating spontaneous activity of the microvasculature unit. Pericytes in capillaries where the interchange of substances between tissues and the circulation takes place may provide the fundamental drive for upstream arterioles and downstream venules so that the microvasculature network functions as an integrated unit.

Role of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients in the mural cells of rat rectal submucosal arterioles.

Mural cells in precapillary arterioles (PCAs) generate spontaneous Ca2+ transients primarily arising from the periodic release of Ca2+ from sarcoendoplasmic reticulum (SR/ER). The Ca2+ release induces Ca2+-activated chloride channel (CaCC)-dependent depolarisations that spread to neighbouring mural cells to develop the synchrony of their Ca2+ transients. Here, we explored the roles of K+ channels in maintaining the synchrony of spontaneous Ca2+ transients. Intracellular Ca2+ dynamics in mural cells were visualised by Cal-520 fluorescence Ca2+ imaging in the submucosal PCAs of rat rectum. Increasing extracellular K+ concentration ([K+]o) from 5.9 to 29.7 mM converted synchronous spontaneous Ca2+ transients into asynchronous, high-frequency Ca2+ transients. Similarly, the blockade of inward rectifier K+ (Kir) channels with Ba2+ (50 µM) or Kv7 voltage-dependent K+ (Kv7) channels with XE 991 (10 µM) disrupted the synchrony of spontaneous Ca2+ transients, while the blockers for large-, intermediate- or small-conductance Ca2+-activated K+ channels had no effect. Kir2.1 immunoreactivity was detected in the arteriolar endothelium but not mural cells. In the PCAs that had been pretreated with XE 991 or Ba2+, nifedipine (1 µM) attenuated the asynchronous Ca2+ transients but failed to restore their synchrony. In contrast, levcromakalim, an ATP-sensitive K+ channel opener, restored the synchronous Ca2+ transients. Thus, constitutively active Kv7 and Kir channels appear to be involved in maintaining the relatively hyperpolarised membrane of mural cells. The hyperpolarised membrane prevents depolarisation-induced 'premature' Ca2+ transients to ensure sufficient SR/ER Ca2+ refilling that is required for regenerative Ca2+ release resulting in synchronous Ca2+ transients amongst the mural cells.

Exercise-induced sympathetic dilatation in arterioles of the guinea pig tibial periosteum.

Strength training induces not only muscle growth but also increased bone strength, a change that is expected to be associated with increased bone blood flow. However, the effects of exercise on contractile properties of bone microvascultaure have not been investigated. Once-a-week strength training with electrical muscle stimulation was applied unilaterally to tibialis anterior muscle of guinea pigs, while muscle force was measured from both legs to compare their muscle strength and endurance. After 10 weeks of training, changes in the arteriolar diameters of isolated periosteum taken from both trained and non-trained legs were measured using a video tracking system. Electrical field stimulation evoked a phasic constriction followed by a sustained dilatation in periosteal arterioles of trained legs, while triggering only vasoconstriction in the arterioles of non-trained legs. In trained leg arterioles, phentolamine, an α-adrenoceptor antagonist, inhibited both the constriction and dilatation. Prazosin, an α1-adrenoceptor antagonist, inhibited only the constriction, while yohimbine, α2-adrenoceptor antagonist, or l-nitro arginine (L-NA), a nitric oxide (NO) synthase inhibitor, inhibited the dilatation. In non-trained leg arterioles, phentolamine or prazosin largely suppressed the constriction, but failed to unmask any dilatation. Consistently, noradrenaline (NAd)-induced arteriolar constriction was enhanced and prolonged by L-NA in trained but not non-trained side arterioles. Thus, NAd released from sympathetic nerves appears to activate endothelial α2-adrenoceptors to release NO resulting in the sustained dilatation of periosteum arterioles from trained leg. The altered sympathetic vasomotor function would facilitate the blood supply to the bone and may contribute to the exercise-induced bone strength gain.

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.

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.

Contractile properties of periosteal arterioles in the guinea-pig tibia.

The periosteal arterioles of the compact bone may play a critical role in bone growth. To explore the contractile properties of tibial arterioles, spontaneous and nerve-evoked constrictions were compared in preparations from 3-week-old and 1-year-old guinea-pigs. Changes in arteriole diameters were measured using video microscopy. Their innervation was investigated using fluorescence immunohistochemistry. Fifty per cent and 40% of tibial arterioles from 3-week-old and 1-year-old guinea-pigs, respectively, exhibited spontaneous phasic constrictions that were inhibited by 1 µM nifedipine, 10 µM cyclopiazonic acid or 100 µM 2-APB. Nerve-evoked phasic constrictions in both age groups were largely suppressed by phentolamine (1 µM), an α-adrenoceptor antagonist, or sympathetic neurotransmitter depletion using guanethidine (10 µM) but were enhanced by spanttide (1 µM), a substance P receptor antagonist, or L-nitro arginine (L-NA; 100 µM), an inhibitor of nitric oxide synthase (NOS). Nerve-evoked constrictions in 1-year-old animals were smaller than those in younger animals but greatly enhanced by L-NA. Immunohistochemistry revealed sympathetic and substance P-positive primary afferent nerves running along the arterioles as well as endothelial NOS expression in both age groups. Spontaneous arteriolar constrictions appear to rely on both Ca2+ release from the sarcoplasmic reticulum and Ca2+ influx through L-type Ca2+ channels. Noradrenaline released from sympathetic nerves triggers arteriolar constriction, while substance P released from primary afferent nerves dilates the arterioles by releasing nitric oxide (NO), presumably from the endothelium. Thus, the enhanced endothelial NO release in adult guinea-pigs may be important to increase the blood supply to meet the increased metabolic demands during bone growth.