Guinea-pig Urethra Research Articles

Evaluation of the potentiating effects of antidepressants on the contractile response to noradrenaline in guinea pig urethra smooth muscles.

We investigated the potential augmenting effects of 19 clinically available antidepressants on noradrenaline (NA)-induced contractions in guinea pig urethra smooth muscle (USM). Concentration-response curves for NA-induced contractions in guinea pig USM strips were obtained in the absence or presence of selected antidepressants. Desipramine, an active metabolite of imipramine, produced a contraction and potentiated NA-induced contraction at the distal urethra without affecting the proximal urethra. Further, nortriptyline and amoxapine, tricyclic antidepressants, produced a contraction and potentiated NA-induced contraction at the distal urethra. NA-induced contraction was unaffected or reduced by imipramine, clomipramine, trimipramine, and amitriptyline at the proximal and distal urethra. Maprotiline, a tetracyclic antidepressant, potentiated NA-induced contraction at the distal urethra. NA-induced contraction was unaffected by mianserin at the proximal and distal urethra. Paroxetine, a selective serotonin reuptake inhibitor (SSRI), potentiated NA-induced contraction at the distal urethra, while NA-induced contraction was unaffected by fluvoxamine, sertraline, and escitalopram at the proximal and distal urethra. Milnacipran, a serotonin-noradrenaline reuptake inhibitor (SNRI), potentiated NA-induced contraction at the proximal and distal urethra, whereas duloxetine potentiated it at the distal urethra. Mirtazapine slightly inhibited NA-induced contraction at the distal urethra. Aripiprazole and sulpiride did not affect NA-induced contractions at the proximal nor distal urethra. Trazodone inhibited NA-induced contraction at both urethras. Desipramine, nortriptyline, amoxapine, maprotiline, paroxetine, milnacipran, and duloxetine likely induce urinary disturbance by increasing urethral resistance and augmenting NA-induced contraction, which should be carefully considered when delivering guidance for drug administration to patients.

Mechanisms of regulation electric and contractile activity smooth muscle cells: the role of cytoskeleton

The influence of modulation of cytoskeleton by colchicine, vinblastine, cytochalasine B and docetaxel on contractile reactions of smooth muscle cells caused by electric stimulus, depolarization, phenylephrine has been investigated by the mechanographical method, by the methods of the double sucrose gup junction. It is established, that induced by a isoosmotic hyperpotassium solution of reduction of smooth muscle of the rat’s aorta, and also caused depolarization stimulus potentials of action and reductions smooth muscle cells from guinea pig urethra, depend more on the condition of microfilaments cytoskeleton than on microtubules. The reduction of smooth muscles cells of an aorta of the rat, caused by isoosmotic striction, is suppressed under the destruction microfilaments whereas the reduction in a hyperosmotic solution depends on a condition of both microfilaments, and microtubules. Cytoskeleton’s microfilaments of aorta’s smooth muscles and microtubules of smooth muscles of cells ureter are involved in mechanisms of action phenylephrine’s action on contractile activity of smooth muscle cells of an aorta and ureter.

Histological Characterization of the Urethral Edges in Patients Who Underwent Bulbar Anastomotic Urethroplasty

Meticulous excision of the stricture and any associated spongiofibrosis is critical to the success of urethral reconstruction. However, normal urethral limits are determined during surgical reconstruction based on macroscopic appearance only. We evaluated structural changes of the presumed healthy urethral ends. Samples were obtained from 29 patients with a mean age of 40 years who underwent end-to-end anastomotic urethroplasty. Suprapubic catheter drainage was performed for at least 30 days in 15 patients. After the urethral stricture was excised biopsy was performed of the proximal and distal healthy ends. Structural characterization was evaluated by staining histological sections with Masson's trichrome, Sirius red and Weigert's resorcin-fuchsin method. The control group consisted of 10 cadaver bulbar urethras. All samples of presumed normal urethral ends showed histological changes. Chronic and acute inflammatory reactions were the most common findings. In the proximal urethral end intense cellularity was found in 18 patients (62.1%). Fibroblast and inflammatory cells were most common and related to the lack of suprapubic cystostomy (p = 0.001). Between proximal urethral edges with and without a suprapubic urinary catheter the elastic fiber distribution and the ratio of collagen types III and I showed a significant change (p = 0.045 and <0.001, respectively). The supposedly healthy urethral ends of end-to-end anastomotic urethroplasty show structural changes. Urethral obstruction and its relief by suprapubic urinary drainage can affect extracellular matrix turnover regulation. Therefore, these changes can lead to urethral remodeling in the proximal bulbar urethra.

Pudendal nerve stimulation of a preparation of isolated guinea‐pig urethra

To assess the functional response of the urethral striated muscle to activation of its nerves, using a novel isolated organ-bath preparation. The urethra of the female guinea-pig was chosen as a suitable model for investigation, as it is functionally and structurally similar to the human urethra. Female Dunkin-Hartley guinea-pigs (400-500 g) were used; for the histochemical and immunohistochemical experiments, unfixed urethras were cryo-sectioned (14 microm thick) and were stained using established methods. For in vitro experiments, whole urethras were suspended vertically, with pudendal nerves intact, for isometric tension and intraluminal pressure recording in a 40-mL organ bath. Drugs were applied directly to the bathing solution. In the striated muscle layer of the urethra there was positive beta-NADPH-diaphorase activity. In organ-bath studies the pudendal nerve-evoked contractions (0.2 ms pulses, 5 s trains, 70 V and 1-100 Hz) were abolished in the presence of tubocurarine (10(-6)m), and unaffected by guanethidine and atropine (both 10(-6)m). Pre-incubation with sodium nitroprusside and SIN-1 chloride significantly reduced the initial peak pressure responses (P < 0.05, anova for paired data) evoked by electrical field stimulation of the pudendal nerves at stimulus parameters of 0.2 ms pulses, 5 s trains, 70 V and 25 Hz. Electrically induced contractions were abolished by tubocurarine, confirming that the pudendal nerve innervates the striated muscle of the guinea-pig external urethral sphincter via nicotinic receptors. beta-NADPH-diaphorase histochemistry gave positive staining around guinea-pig striated muscle cells and possibly identified neuromuscular junction sites staining positively for the nitric oxide synthase marker. Together with the results of the organ-bath experiments, the results suggest that the striated muscle cells of the guinea-pig urethra have the machinery to respond to nitric oxide.

In vitro alpha-adrenoceptor autoradiography of the urethra and urinary bladder of the female pig, cat, guinea-pig and rat.

The aim of this study was to investigate the distribution of alpha1- and alpha2-adrenoceptors in the urethra and urinary bladder of the female pig, cat, guinea-pig and rat. The binding distributions of an alpha1-adrenoceptor ligand (3H-prazosin) and an alpha2-adrenoceptor ligand (3H-rauwolscine) were determined using in vitro autoradiography. Autoradiograms were analysed by combining computer-based image analysis and light microscopy. In the pig, guinea-pig and rat urethra 3H-prazosin binding was highest in the muscle layer. In the cat urethra 3H-prazosin binding could not be analysed due to a negative chemography artefact. In the pig, cat and guinea-pig urethra 3H-rauwolscine binding was highest in the urothelium, followed by the sub-mucosa, with low levels in muscle. Little 3H-rauwolscine binding was observed in the rat urethra. In the urinary bladder of all species 3H-prazosin binding was low. In the rat bladder, binding was higher in the trigone than in the dome. In the pig, cat and guinea-pig bladder 3H-rauwolscine binding was highest in the mucosa. with little binding in muscle or lamina propria. In the rat bladder, there was little binding and no regional differences. Alpha1-adrenoceptors were predominantly located in urethral smooth muscle, indicating their contractile importance in maintaining continence. Alpha2-Adrenoceptors were present in the urethral submucosa and bladder mucosa, but not in muscle, suggesting a role in regulation of blood flow, urethral lubrication and tumescence, but not in contraction.

Elastic fibres in the vesicourethral junction and urethra of the guinea pig: quantification with computerised image analysis.

Elastic fibres, which are intimately associated with collagen, a major component of the urethra, have been assumed to contribute to the resting urethral closure pressure. The Miller stain for elastin was used to demonstrate elastic fibres in cryostat sections of guinea pig bladder base, vesicourethral junction (VUJ) and urethra. Computerised image analysis was employed to objectively quantify these fibres. Both male and female guinea pigs showed significantly greater amounts of circularly disposed elastic fibres in the VUJ than in the other 2 regions examined. This particular disposition of fibres may be responsible for imparting resiliency and plasticity to the VUJ, allowing it to distend and recoil repeatedly in response to urine outflow. Furthermore, the elastic fibres may be partly responsible for the passive occlusive force in this region. Elastic fibres in the distal urethra were not quantified because of their relative paucity. Sagittal sections of the urethra revealed a mass of longitudinally arranged elastic fibres localised almost exclusively within the mucosa, submucosa and longitudinal smooth muscle layer. Functionally, this arrangement may exist to facilitate urethral length changes that occur in micturition.

Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.

1. Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea-pig urethra using conventional microelectrode techniques. 2. Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3. STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4. When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5. Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either alpha,beta-methylene-ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co-application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6. In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+-activated chloride channels. STDs sum to activate L-type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.

Inhibitory innervation of the guinea-pig urethra; roles of CO, NO and VIP

The inhibitory innervation of guinea-pig urethral smooth muscle was investigated histochemically and functionally. The distribution of immunoreactivities to haem oxygenases (HO), neuronal NO synthase (nNOS), and vasoactive intestinal polypeptide (VIP) was studied, and the functional effects of the corresponding putative transmitters, CO, NO, and VIP, were assessed. HO-2 immunoreactivity was found in all nerve cell bodies of intramural ganglia, localized between smooth muscle bundles in the detrusor, bladder base and proximal urethra. About 70% of the ganglionic cell bodies were also NOS-immunoreactive (IR), whereas a minor part was VIP-IR. Some ganglion cells exhibiting tyrosine hydroxylase (TH) activity were demonstrated. Rich numbers of NOS-IR varicose nerve terminals could be found innervating the smooth muscle of the urethra, whereas VIP-IR terminals were less numerous. A rich number of TH-IR terminals were observed. The bladder showed a similar distribution of nerves, although only a few number of TH-IR nerves could be found. In bladder preparations exposed to sodium nitroprusside, cGMP-IR cells could be seen, forming an interconnecting network with long spindle-shaped processes. The cGMP-IR cells were especially abundant in the outer smooth muscle layers of the bladder, but less numerous in the urethra. In urethral strip preparations, electrical field stimulation evoked long-lasting frequency-dependent relaxations. The relaxations were not inhibited by the NO-synthesis inhibitor, l-NOARG, or enhanced by the NO-precursor, l-arginine. The haem precursor, 5-aminolevulinic acid (5-ALA), or the inhibitor of guanylate cyclase, ODQ, did not affect the urethral relaxations. Exogenously applied NO, SIN-1, and VIP relaxed the preparations by approximately 50%, whereas the relaxation evoked by exogenous CO was minor. These results suggest that CO probably is not involved in non-adrenergic, non-cholinergic inhibitory control of the guinea-pig urethra, where a non-NO/cGMP mediated relaxation seems to be predominant.

A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra.

It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.

Characterizing biological tissue using scanning laser acoustic microscopy

The scanning laser acoustic microscopy (SLAM) technique offers high resolution for determining the properties of individual layers. To introduce the SLAM technique as a method for measuring the speed of sound in tubular layered organs, and possibly distinguish between the layers by this measure, the authors chose the guinea-pig urethra, due to its relatively simple layered structure. To obtain more data of the acoustic properties of tissue, the attenuation coefficient was determined for the wall in toto.

Presynaptic α2A‐Adrenoceptors Regulate the 3H‐Noradrenaline Secretion in the Guinea‐Pig Urethra

The subtype of the functional presynaptic autoreceptor in adrenergic nerves of the male guinea-pig urethra was pharmacologically characterised. The urethra was incubated with 3H-noradrenaline and superfused with Tyrode solution in vitro and the fractional secretion of 3H-noradrenaline evoked by 300 electrical shocks at 5 Hz was measured. alpha-Adrenoceptor antagonists enhanced the 3H-noradrenaline secretion. The effects of BRL 44408 ((+/-)-2-[(4,5-dihydro-1H-imidazol-2yl)methyl]-2,3- dihydro-1-methyl-1H-isoindole), BRL 41992 ((-)-1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazo[1,5- a] azepine), and WB-4101 (2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4- benzodioxane) were tested. The rank order of apparent EC50 values was BRL 44408 < BRL 41992 < WB-4101, and correlated best with constants for the alpha 2A-C10/alpha 2A adrenoceptor subtype. The results suggest that 3H-noradrenaline secretion in guinea-pig urethra is regulated by a presynaptic adrenoceptor of the alpha 2A subtype.

Species differences in characteristics and distribution of [ 3H]α,β-methylene ATP binding sites in urinary bladder and urethra of rat, guinea-pig and rabbit

The characteristics and distribution of [ 3H]α, β-methylene ATP ([ 3H]α, β-MeATP, a radioligand for P 2x-purinoceptors) binding sites in the urinary bladder of rat, guinea-pig and rabbit were examined. Autoradiographic localization of [ 3H]α, β-MeATP binding sites was also carried out on the urethra of the three species. Receptor binding assay showed that the rat bladder possesses the highest density of specific binding sites, followed by rabbitand guinea-pig bladder. Semi-quantitative analysis of autoradiograms showed that the grain densities in the bladders of rat, guinea pig, and rabbit were parallel to those obtained from receptor binding assay. The grain densities were greatly reduced in the presence of β,γ-methylene ATP (β,γ-MeATP). No significant specific binding was detected in the smooth muscle of rat and guinea-pig urethra, while a very low level of specific binding was observed in the rabbit urethra. Differences of grain densities different regions (dome, body, and trigone) of the same bladder were also observed, but they were not as remarkable as those between species. The results of this study demonstrate species differences of P 2x-purinoceptor densities in the urinary bladder, which may reflect differing degrees of purinergic neurotransmitter control of the bladder detrusor muscle.

Subtype classification of the presynaptic alpha-adrenoceptors which regulate [3H]-noradrenaline secretion in guinea-pig isolated urethra.

1. The following experiments were carried out to investigate the presence and type of functional presynaptic receptors in adrenergic nerves of the guinea-pig urethra. 2. The urethra from male guinea-pigs was incubated with [3H]-noradrenaline and superfused with Tyrode solution in vitro. The fractional secretion of [3H]-noradrenaline evoked by 300 electrical pulses was measured. 3. The [3H]-noradrenaline secretion was positively frequency-dependent, yielding a half-maximal secretion at 8 +/- 5 Hz. Stimulation was usually applied at 5 Hz. 4. The [3H]-noradrenaline secretion was not altered by noradrenaline (1 or 100 microM), norephedrine (1 microM), isoprenaline (0.1 microM), 5-hydroxytryptamine (10 microM), oxotremorine (10 microM), adenosine (0.2 mM), propranolol (1 microM), atropine (1 microM) or 8-phenyltheophylline (10 microM). 5. The [3H]-noradrenaline secretion was enhanced by clonidine (3 microM), chlorpromazine (10 microM), metitepine (1 microM), 4-aminopyridine (0.5 mM), tetraethylammonium (2 mM), 3-isobutyl-1-methylxanthine (4 mM), 8-bromo cyclic AMP (1 mM) and forskolin (25 microM). 6. The alpha-adrenoceptor antagonists rauwolscine, yohimbine, phentolamine, prazosin and AR-C 239 maximally enhanced the [3H]-noradrenaline secretion to about 300% of control. The partial alpha-adrenoceptor agonist oxymetazoline maximally enhanced the secretion to about 200% of control. The order of apparent EC50 values was rauwolscine less than yohimbine less than phentolamine less than oxymetazoline less than prazosin less than AR-C 239.7. The enhancing effects of yohimbine (1 microM) with tetraethylammonium (2mM), 8-bromo cyclic AMP (1 mM), or forskolin (25,microM) were additive, but not those of yohimbine (1 microM) with prazosin (10 microM), 4-aminopyridine (0.5 mM), or 3-isobutyl-1-methylxanthine (4 mM).8. These results suggest that the [3H]-noradrenaline secretion in the guinea-pig urethra is regulated by presynaptic alpha2A-adrenoceptors which may, in a cyclic AMP-independent manner, be coupled to a 4-aminopyridine-sensitive potassium channel. The secretion is not influenced by compounds acting at beta-adrenoceptors, muscarinic cholinoceptors or adenosine receptors.

Early events in stricture formation in the guinea pig urethra.

The early stages of urethal stricture formation have been examined by light and electron microscopy after injury of the anterior urethra in the guinea pig. It was found that initially an ulcerated surface formed which was subsequently covered by simple then stratified squamous epithelium. The tissue of the stricture contained elongated myofibroblasts and clumps of multinucleated giant cells. It is suggested that the myofibroblasts are responsible for the formation of the stricture and that the giant cells may be involved in continued collagen synthesis.

Light and electron microscopic observations on noradrenergic nerves and striated muscle cells of the guinea pig urethra.

Neurohistochemical and electron microscopic techniques have been used to determine the arrangement and distribution of noradrenergic nerves and striated muscle in the wall of the guinea pig urethra. Fluorescent, catecholamine - containing nerves were observed among striated muscle cells only in the junctional zone between an inner layer of smooth muscle and an outer layer of striated cells. In this region considerable intermixing of the two types of muscle cell occurred. Electron microscopy has shown that noradrenergic terminal regions containing characteristic small dense-cored vesicles were invariable associated with smooth muscle cells; such nerves were never observed forming neuroeffector relationships with striated cells. These findings indicate that noradrenergic 'sympathetic' fibres are not directly involved in the functional control of urethral striated muscle.

Functional role of an adrenergic sphincter in the female urethra of the guinea-pig.

By means of a microtransducer catheter, the urethral pressure profile was recorded in female guinea-pigs. The maximum urethral pressure averaged 24 mmHg (range 18-27 mmHg) and was found 4 to 7 mm from the vesico-urethral junction. Fluorescence microscopy of the urethra showed an irregular distribution of fluorescent nerve terminals in the most proximal part of the organ. In a distal direction, there was a continuous increase in the number of adrenergic nerves, with a maximum 4 to 6 mm from the internal meatus, corresponding to the maximum intraurethral pressure point. In this region, a dense plexus of nerves supplied thick muscle coats of smooth muscle having an outer circular and an inner longitudinal orientation. The findings suggest that a specific region of the female guinea-pig urethra may serve as an "internal sphincter".

The morphology and innervation of subepithelial "striated" muscle cells in the male guinea-pig urethra.

Light and electron microscopic techniques have been used to determine the distribution, morphology and innervation of subepithelial striated muscle cells in the wall of the proximal urethra of the male guinea-pig. These cells form a continuous layer, immediately beneath the urethral epithelium extending from the bladder neck to the termination of the ejaculatory ducts into the proximal urethra. They differ from "typical" striated muscle fibres (as seen in the external urethral sphincter) by their small size, rich acetylcholinesterase content and the irregular arrangement of intracellular myofilaments and sarcoplasmic reticulum. In addition, motor end plate regions have not been observed on these striated cells when examined using a light microscopic histochemical technique. The cells are related to acetylcholinesterase positive nerves which run between them in a manner compatible with the occurrence of "en passant" synaptic interactions. Using electron microscopy, axonal varicosities containing small (50 nm diameter) agranular vesicles are encountered 50 nm from the striated cells; membrane specialisations characteristic om motor end plates have not been observed on the cells. The findings are discussed, particularly in relation to the distribution, unusual morphology and innervation of these subepithelial muscle cells.