Intramural Nerves Research Articles

β−Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder

β(3)-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β(3)-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β(1), β(2), and β(3) were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β(3)-receptor in the human urothelium appears to be functional, as two different selective β(3)-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in URO tsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β(2)-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β(3)-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.

Properties of spontaneous contractions and their modulation by transmural nerve stimulation in circular smooth muscle isolated from the pacemaker area in the flexure region of the guinea-pig colon

The properties of spontaneous contractions and their modulation by transmural nerve stimulation (TNS) were investigated in circular smooth muscle preparations isolated from the "pacemaker area" in the flexure region of the guinea-pig colon. Four types of preparation were made; mucosal layer removed preparations, mucosal and submucosal layer removed preparations (submucosal layer removed preparations), longitudinal muscle removed preparations, and preparations with all layers attached (intact preparations). Intact and mucosal layer removed preparations periodically generated two types of phasic contractions; large contractions at a low frequency (about 2 times per min) and small contractions at a higher frequency (about 13 times per min). Submucosal layer removed preparations only produced large contractions, while longitudinal muscle removed preparations only produced small contractions. Both the amplitude and frequency of the large contractions were significantly inhibited by atropine, but were not markedly modulated by N(ω)-nitro-L-arginine (L-NA) in either intact, mucosal layer removed or submucosal layer removed preparations. Small contractions were not significantly modulated by atropine or L-NA in any type of preparation. In both intact and mucosal layer removed preparations, TNS (1 Hz frequency, for 2-3 min) increased the amplitude and/or frequency of large contractions, but caused no significant change in small contractions. TNS also increased the amplitude and/or frequency of large contractions in submucosal layer removed preparations, but did not significantly modulate small contractions in longitudinal muscle removed preparations. Exogenously applied acetylcholine (>10(-7) M) enhanced, while sodium nitroprusside (>10(-8) M) inhibited, the amplitude and frequency of both types of contraction, in a concentration-dependent manner. The results suggest that the large contractions were elicited by interstitial cells distributed in the myenteric layer (ICC-MP), while the small contractions were elicited by ICC distributed in the submuscular plexus layer (ICC-SMP). Intramural nerves mainly modulated the activity of ICC-MP, while neural regulation of ICC-SMP was weak.

Interstitial cells in the urinary bladder—localization and function

This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC). On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review. The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca2+-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder. There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation.

Endo- and exocytosis of vesicles in the intramural nerve fibers of the rat right atrium

Endo- and exocytosis of vesicles in the intramural nerve fibers of the rat right atrium

Embryology and anatomy of the vesicoureteric junction with special reference to the etiology of vesicoureteral reflux

Concerning the ureterovesical junction - the region most important for the anti-reflux mechanism - there is still a lot of misunderstanding and misinterpretation with regard to normal fetal development. Data are scarce on possible causes of primary vesicoureteral reflux and on involved mechanisms of the so-called maturation process of refluxing ureteral endings. The ratio of the intravesical ureteral length to the ureteral diameter is obviously lower than assumed so far, as clearly revealed by some studies. Therefore it can be doubted that the length and course of the intravesical ureter is of sole importance in the prevention of reflux. Additionally refluxing intravesical ureteral endings present with dysplasia, atrophy, and architectural derangement of smooth muscle fibers. Besides, a pathologically increased matrix remodeling combined with deprivation of the intramural nerve supply has been confirmed. Consequently, symmetrical narrowing of the very distal ureteral smooth muscle coat creating the active valve mechanism to defend reflux is not achievable. It is apparent that primary congenital vesicoureteral reflux seems to be the result of an abnormality within the ureterovesical junction with an insufficient muscular wrap. Nature is believed to establish much more sophisticated mechanisms than the so-called passive anti-reflux mechanism. Remodeling processes within the ureterovesical junction of refluxing ureteral endings support that maturation itself is nothing else than wound or defect healing and not a restitution of a morphological normal ureterovesical junction. Lacking the nerve supply a restoration of any muscular structure can not be achieved.

Effect of Aerobic Training on Innervation Density and Neurogenic Responses of Skin Afferent Blood Vessels

We studied the effect of 8-week aerobic training (treadmill running) on neurogenic responses and density of sympathetic innervation of subcutaneous artery in rats. In trained rats, the artery response to stimulation of intramural sympathetic nerve decreased, but the sensitivity of vascular smooth muscles to norepinephrine was not changed. The density of adrenergic nerve fibers in the arterial wall after training was also lower than in the control group. This effect of training can be explained by the need in increased heat emission during physical activity.

Postganglionic nerve cell bodies and neurotransmitter localization in the teleost heart

A study was undertaken to determine the distribution of specific types of autonomic nerves and the presence of various transmitter substances in the heart of two teleost species: the mullet (Mugil cephalus) and the Nile catfish (Synodontis nigriventris). Large nerve trunks in the sinus venosus were shown to contain tyrosine hydroxylase immunoreactivity and indicate the location of adrenergic nerve fibers, which are also associated with a coronary circulation to the ventricular myocardium in the mullet heart. Fluorescence immunolabelling methods revealed that the atrium and the outer and inner compact muscle of the ventricle have nerves in which substance P and galanin (GA) are localized. It seems likely that the cell bodies (perikarya) of the substance P and GA-immunopositive axons are located at sites outside the heart. The GA-immunopositive nerve fibers may represent a population of axons of intramural postganglionic nerve cell bodies. Most intracardiac nerve cell bodies are located in the sinus venosus and in the sinoatrial junction and reveal immunoreactivity to substance P, GA, neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). Furthermore, substance P immunoreactivity is present in the cardiac cells intermingled with the substance P-immunopositive nerve fibers. A nerve plexus consisting of a well-developed network of nerve fibers and nerve cell bodies may possibly correspond to a cardiac pacemaker, but its function in fish cardiac regulation is unknown and remains to be elucidated.

Hypogenesis of intramural vascularity and perivascular plexuses of gallbladder in patients with congenital biliary dilatation

We hypothesized neuronal disorders of the biliary tract as the cause of congenital biliary dilation (CBD). Gallbladders were removed from a total of 15 patients who were categorized into two study groups: a CBD group (eight patients) and in a control group (neuroblastoma, acute myelogenous leukemia, wandering gallbladder, Wilms' tumor, glycogen storage disease, familial amyloid polyneuropathy; seven patients). Whole-mount preparations of the gallbladders were made to immunostain the intramural nerves. The intramural vascularity in the gallbladders of the CBD group (5.5 +/- 1.9/cm(2)) was significantly lower than that in the control group (27.6 +/- 14.4/cm(2)). The rate of perivascular plexuses on the surface of intramural vessels was also significantly lower in the CBD group than in the controls (37.7 +/- 18.1 vs. 80.2 +/- 17.4%, respectively). The numbers of ganglion cells per visual field were 38.5 +/- 24.0 and 42.3 +/- 20.6, respectively, in the CBD and control groups; this difference was not statistically significant. There may be a mechanism in CBD causing contractile failure and dilatation of the biliary tract as a result of decreased intramural blood flow that accompanies the diminished vascularity.

227 CHOLINERGIC MODULATION OF SPONTANEOUS ACTIVITY IN BLADDER STRIPS FROM THE DIABETIC RAT: EFFECT OF THE MUCOSA

Spontaneous contractions (SCs) have been shown to be increased in the overactive bladder and animal models of detrusor overactivity and may underlie the abnormal increases in intravesical pressure which lead to urgency, frequency and urge incontinence. We have previously shown increased SCs in bladder strips from a diabetic rat model. It has been suggested that SCs may be enhanced by leakage of small amounts of acetylcholine from intramural nerves (1). The aim was to study cholinergic modulation of SCs in bladder strips from streptozotocin-induced diabetic rats and the role of the mucosa.

Modulation of the activity of two pacemakers by transmural nerve stimulation in circular smooth muscle preparations isolated from the rat proximal colon

Circular smooth muscle preparations isolated from the rat proximal colon periodically generated two different amplitudes and frequencies of phasic contractions: large phasic contractions (LPC) with a frequency of about 1.5 times/min and small phasic contractions (SPC) with a frequency of about 9 times/min. Preparations with no attached longitudinal smooth muscle layer (and also myenteric layer) generated SPC alone, while those with no attached submucosal layer generated only LPC, indicating that the pacemakers of the LPC and SPC are distributed in the myenteric and submucosal layers, respectively. In intact preparations, transmural nerve stimulation (TNS) applied for 1-2 min with different frequencies (0.2-2 Hz) inhibited the phasic contractions. The amplitude of LPC was reduced at >0.25 Hz and abolished at >0.3 Hz, while the amplitude but not the frequency of SPC was reduced at >0.5 Hz (in a frequency-dependent way). The TNS-induced inhibitory responses were augmented by atropine and attenuated by N(omega)-nitro-L-arginine (L-NA). In the presence of L-NA and atropine, TNS elicited biphasic (inhibitory and following excitatory) responses. The former were not antagonized by apamin, guanethidine or suramin, while the latter were antagonized by capsaicin, suggesting an innervation by non-adrenergic non-cholinergic non-nitrergic (NANCNN) inhibitory and peptidergic excitatory nerves, respectively. In preparations with the longitudinal muscle layer removed, TNS inhibited only the amplitude of SPC, which was augmented by atropine and antagonized by L-NA. In intact preparations, muscarinic stimulation with acetylcholine increased the frequency of LPC, while nitrergic stimulation with sodium nitroprusside reduced the amplitude and frequency of LPC, and also the amplitude but not the frequency of SPC. These results indicate that the rat proximal colon has two types of pacemaker cells. Myenteric pacemaker cells which receive predominantly nitrergic, but also cholinergic, peptidergic and NANCNN innervation, and submucosal pacemaker cells that are not markedly influenced by intramural nerves.

Effect of Dai-kenchu-to on gastrointestinal motility and gastric emptying

Background/aims The gastrointestinal symptoms accompanying dysfunction of the remnant stomach were seen after pylorus-preserving operation. Against such complications, Dai-kenchu-to (DKT) is used, but scientific evidences for efficacy are poor. The effect of DKT on gastrointestinal motility and gastric emptying after pylorus-preserving operation was investigated. Methods Using beagle dogs, the experimental models mimicking the state after pylorus-preserving pancreaticoduodenectomy were prepared. We sutured strain gauge transducers to the stomach, duodenum and jejunum and inserted indwelling tubes into the stomach. About 4 weeks after operation, DKT 0.1 g/kg was administered during the fasting or fed state. At the same time, the gastric emptying was evaluated by the acetoaminophene method. Results In the fasting state, administration of DKT enhanced the gastrointestinal motility and accelerated gastric emptying. In the postprandial state, no apparent effect on motility was seen. Conclusion DKT enhances the gastrointestinal motility after pylorus-preserving pancreaticoduodenectomy in the fasting state. The effect of DKT may not be related to the continuity of the intramural nerve.

Effect of ischemia/reperfusion on bladder nerve and detrusor cell damage

Ischemia, reperfusion, and subsequent free radical damage have been implicated in many voiding disorders. Our goal was to investigate further the mechanisms of these disorders, with particular emphasis on nerve and mitochondrial function and on detrusor smooth-muscle cells. The effects on contractile responses to various stimulations, citrate synthase, choline acetyltransferase activities, and vesicular acetylcholine transporter were evaluated after ischemia alone and ischemia/reperfusion 2 h, 7 days, and 14 days. Nerve density and detrusor cell apoptosis were also measured. The contractile responses were significantly decreased at both 7 and 14 days reperfusion, although at 14 days some recovery was observed. Similar patterns were seen for the intramural nerves, both nerve cell cytoskeletal structures and cholinergic neurotransmitters. Citrate synthase activity was also depressed by ischemia and 2 h reperfusion, but the activity recovered by 7 days. Detrusor cell apoptosis was not significantly affected by ischemia and 2 h reperfusion; but showed an approximately 14-fold increase at both 7 and 14 days reperfusion. Reperfusion following ischemia resulted in worsening intramural bladder nerve dysfunction, nerve fiber injury, mitochondrial injury, and damaged detrusor muscle cells. However, at 14 days reperfusion, nerve and mitochondrial regeneration occurred and resulted in partial recovery of contractile function.

Effects of Exogenous Glutamate and Kainate on Electric Field-stimulated Contractions of Isolated Human Ureter

A neurotransmitter role for glutamate in the autonomous nervous system was recently demonstrated in the gastrointestinal tract, and its stimulatory effect on spontaneous motility of human ureter was shown. The aim of our study was to investigate the effects of glutamate on the release of neurotransmitters from intramural nerves of the human ureter. The effects of exogenous glutamate were tested on electric field-stimulated contractions of isolated human ureter, taken from 16 adult patients after nephrectomy. The longitudinal tension and intraluminal pressure of the isolated ureter were recorded simultaneously. The electric field stimulation was done with square wave pulses (20 V through electrodes, 400 mA, duration 1 ms, frequency 16 Hz). The pulse trains lasted for 30 s, a with 30-s pause. Glutamate (7.9 x 10(-6) M/L to 10.6 x 10(-3) M/L) and kainic acid (6.3 x 10(-8) M/L to 2.2 x 10(-5) M/L) produced a concentration-dependent decrease in the electric field-stimulated activity of the isolated preparations. However, N-methyl-D-aspartic acid (9.1 x 10(-8) M/L to 3.1 x 10(-5) M/L), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (7.2 x 10(-8) M/L to 3.2 x 10(-6) M/L) and (+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (7.7 x 10(-8) M/L to 6.5 x 10(-5) M/L) were ineffective. The electric field-stimulated contractions of isolated ureter were also inhibited by lidocaine (3.70 x 10(-4)M/L) and atropine (1.00 x 10(-6)M/L). The results of our study suggest that glutamate inhibits electric field-stimulated release of acetylcholine in the human ureter through activation of kainate ionotropic receptors, located on the intramural nerve fibers.

Innervation and Neurotransmitter Localization in the Lung of the Nile bichir Polypterus bichir bichir

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P(2)X(2), and 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P(2)X(2) receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase-immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods.

Effect of exogenous glutamate and N‐Methyl‐D‐aspartic acid on spontaneous activity of isolated human ureter

While the neurotransmitter role of glutamate in the gastrointestinal tract has been shown, its effects on smooth muscle of the human ureter have not previously been investigated. In our study we have investigated the effects of exogenous glutamate on the spontaneous activity of isolated human ureter, taken from 14 adult patients after nephrectomy. The segment of ureter, excised 3 cm distal from the pyeloureteral junction, was isolated in an organ bath. Both longitudinal tension and intraluminal pressure of the segment were recorded simultaneously. Glutamate administered in the lumen of the isolated ureteral segments (7.8 x 10(-7) M/L-3.5 x 10(-2) M/L) was ineffective. When added to the isolated organ bath from the serous side of the ureteral segment, glutamate (7.9 x 10(-6) M/L-10.6 x 10(-3) M/L) and N-Methyl-D-aspartic acid (NMDA) (9.1 x 10(-8) M/L-3.1 x 10(-5) M/L) produced a concentration-dependent increase in spontaneous activity of the isolated preparations, while kainic acid (6.3 x 10(-8) M/L-10.5 x 10(-5) M/L) and (+/-)-trans-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) (7.7 x 10(-8) M/L -6.5 x 10(-5) M/L) were ineffective. The results of our study suggest that an excitatory neurotransmitter glutamate stimulates spontaneous activity of the human ureter through activation of NMDA ionotropic receptors, located on smooth muscle cells or intramural nerve fibers.

Pre‐junctional α2‐adrenoceptors modulation of the nitrergic transmission in the pig urinary bladder neck

To investigate the nitric oxide (NO)-mediated nerve relaxation and its possible modulation by pre-junctional alpha2-adrenoceptors in the pig urinary bladder neck. Urothelium-denuded bladder neck strips were dissected, and mounted in isolated organ baths containing a physiological saline solution (PSS) at 37 degrees C and continuously gassed with 5% CO2 and 95% O2, for isometric force recording. The relaxations to transmural nerve stimulation (electrical field stimulation [EFS]) or exogenously applied NO were carried out on strips pre-contracted with 1 microM phenylephrine (PhE) and treated with guanethidine (10 microM) and atropine (0.1 microM), to block noradrenergic neurotransmission and muscarinic receptors, respectively. EFS (0.2-1 Hz, 1 msec duration, 20 sec trains, current output adjusted to 75 mA) evoked frequency-dependent relaxations which were abolished by the neuronal voltage-activated Na+ channel blocker tetrodotoxin (TTX, 1 microM). These responses were potently reduced by the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOARG, 30 microM) and further reversed by the NO synthesis substrate L-arginine (L-ARG, 3 mM). The alpha2-adrenoceptor agonist BHT-920 (2 microM) reduced the electrically evoked relaxations, its effectiveness being higher on the responses induced by low frequency stimulation. BHT-920-elicited reductions were fully reversed by the alpha2-adrenoceptor antagonist rauwolscine (RAW, 1 microM). Exogenous NO (1 microM-1 mM) induced concentration-dependent relaxations which were not modified by BHT-920, thus eliminating a possible post-junctional modulation. These results indicate that NO is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission in the pig urinary bladder neck, the release of NO from intramural nerves being modulated by pre-junctional alpha2-adrenoceptor stimulation.

Human Heart Glutamate Receptors—Implications for Toxicology, Food Safety, and Drug Discovery

Excitatory amino acids (EAAs) mediate their effects through the glutamate receptors (GluRs) in the brain. GluRs play an important role in the treatment of a variety of neuropsychiatric conditions and are central to the neurotoxicity of EAAs such as domoic and kainic acid. Unstained histological preparations of human heart tissues were used for the histopathological assessment, the anatomical identification of specific cardiac structures and the presence of the GluRs. Immunohistochemical stains with the biomarkers protein gene product (PGP 9.5) and the neurofilaments (NF 160 and NF 200) were used to identify neural structures and the components of the conducting system. Several subtypes of GluRs were differentially expressed and each had a specific distribution. In contrast to nonhuman primates, GluRs are more widely expressed in humans, where the working myocardium and the wall of blood vessels stained for GluRs. The immunolabelling was observed within the specialized structures of the conducting system, intramural nerves, and ganglia cells. These receptors may be involved in important cardiac functions such as contraction, rhythm, coronary circulation, and thus may be implicated in the pathobiology of some cardiac disease. The GluRs in the heart could be targets for the effects of excitatory compounds and is therefore an important consideration for the safety evaluation of foods and therapeutic products.

The Distribution and Chemical Coding of Intramural Neurons Supplying the Porcine Stomach – the Study on Normal Pigs and on Animals Suffering from Swine Dysentery

The present study was designed to investigate the expression of biologically active substances by intramural neurons supplying the stomach in normal (control) pigs and in pigs suffering from dysentery. Eight juvenile female pigs were used. Both dysenteric (n = 4; inoculated with Brachyspira hyodysenteriae) and control (n = 4) animals were deeply anaesthetized, transcardially perfused with buffered paraformalehyde, and tissue samples comprising all layers of the wall of the ventricular fundus were collected. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against vesicular acetylcholine (ACh) transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), Leu(5)-enkephalin (LENK), substance P (SP) and calcitonin gene-related peptide (CGRP). In both inner and outer submucosal plexuses of the control pigs, the majority of neurons were SP (55% and 58%, respectively)- or VAChT (54%)-positive. Many neurons stained also for CGRP (43 and 45%) or GAL (20% and 18%) and solitary perikarya were NOS-, SOM- or VIP-positive. The myenteric plexus neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (7%), SP (6%) or CGRP (solitary neurons), but they were SOM-negative. No intramural neurons immunoreactive to LENK were found. The most remarkable difference in the chemical coding of enteric neurons between the control and dysenteric pigs was a very increased number of GAL- and VAChT-positive nerve cells (up to 61% and 85%, respectively) in submucosal plexuses of the infected animals. The present results suggest that GAL and ACh have a specific role in local neural circuits of the inflamed porcine stomach in the course of swine dysentery.

Kit‐positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle

To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra.

Smooth muscle and neural mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development

Spontaneous bladder contractions (SBCs) in the neonatal rat urinary bladder change from a high-amplitude, low-frequency pattern to a low-amplitude, high-frequency pattern during the first 6 wk of life. Understanding the mechanism of this developmental change may provide insights into the causes of bladder overactivity in adults. In vitro whole bladder preparations from Sprague-Dawley rats were used to study the modulation of SBCs by calcium-activated potassium channels (K(Ca)) and electrical field stimulation from 3 days to 6 wk of life. SBCs in 3-day-old bladders were unmasked by treatment with iberiotoxin (100 nM), an inhibitor of large conductance K(Ca) (BK) channels, or apamin (100 nM), an inhibitor of small conductance K(Ca) (SK) channels. Iberiotoxin significantly increased the magnitude of SBCs at 2-3 wk, whereas apamin was only effective at 6 wk. In 1-2 wk bladders, exposure to room temperature Krebs solution decreased SBCs. This decrease was reversed by activating intramural nerves with electrical field stimulation. The effect of electrical field stimulation was inhibited by atropine (1 microM), suramin (10 microM), or pretreatment with tetrodotoxin (1 microM) but was not reversed by tetrodotoxin applied after electrical field stimulation. BK-alpha mRNA increased threefold, and BK-alpha protein increased fivefold from 3 days to 6 wk. These data suggest that BK channels play an important role in the regulation of SBCs in the neonatal bladder and that both increased BK channel activity, as well as changes in smooth muscle sensitivity to locally released neurotransmitters contribute to the downregulation of SBCs during early postnatal development.