Urinary Bladder Muscle Research Articles

The inhibitory mechanisms of nicorandil in isolated rat urinary bladder and femoral artery

Nicorandil or cromakalim inhibited contractile responses to acetylcholine and KCl in detrusor muscles of rat urinary bladder, whereas nitroglycerin inhibited only the responses to acetylcholine. In the detrusor muscles contracted by electrical stimulations, relaxations caused by nicorandil and cromakalim were inhibited by glyburide, but not by nitroglycerin or apamin. Methylene blue slightly potentiated the nicorandil-relaxation without affecting the cromakalim-relaxation. N G-Monomethyl- l-arginine also did not affect the relaxation induced by nicorandil. The level of cGMP was increased by both nicorandil and nitroglycerin. In rat femoral arteries contracted by phenylephrine, the relaxation induced by nicorandil was inhibited by methylene blue, glyburide and apamin. The relaxation induced by cromakalim was inhibited by glyburide, but not by apamin or methylene blue. These results suggest that the effect of nicorandil is due to activation of K ATP channels in rat detrusor muscles and is due to the activation of guanylate cyclase, K ATP and K Ca channels in rat femoral arteries. The effect of cromakalim is due to the activation of K ATP channels in both smooth muscles.

Reversibility of muscle and heart lesions in chronic, Trypanosoma cruzi infected mice after late trypanomicidal treatment.

The effect of trypanomicidal treatment upon established histopathological Trypanosoma cruzi induced lesions was studied in Swiss mice. The animals were inoculated with 50 trypomastigotes and infection was allowed to progress without treatment for 99 days. After this period, the animals were divided in three groups, treated for 30 days with either placebo, benznidazole (200 mg/kg/day) or nifurtimox (100 mg/kg/day). These treatments induced 94 and 100% cure rates respectively as detected by xenodiagnosis and reduction of antibody levels. Autopsies and histopathological studies of heart, urinary bladder and skeletal muscle performed on day 312 after infection showed almost complete healing without residual lesions. As long periods were allowed between infection, treatment and autopsy, the results indicate that tissue lesions depend, up to advances stages, on the continuous presence of the parasite.

Adrenergic innervation of the urinary bladder body in the cat with special reference to structure of the detrusor muscle: an immunohistochemical study of noradrenaline and its synthesizing enzymes.

The distribution of adrenergic nerves in the body detrusor muscle of the cat urinary bladder was studied by means of the immunohistochemical identification of noradrenaline (NA) and the NA synthesizing enzymes tyrosine hydroxylase, aromatic L-aminoacid decarboxylase and dopamine beta-hydroxylase. We identified the basic structural organization of the detrusor muscle, which had previously been described as lacking discernible layers. In the lateral wall, both outer longitudinal and inner circular muscle bundles were present, the latter extending in both anterior and posterior directions. The posteriorly running bundles came to lie on the outside of the posterior wall where they enabled recognition of inner longitudinal muscle bundles. Those running anteriorly were dispersed to enter the longitudinal bundles in the anterior wall. NA-immunoreactive nerve fibers in the detrusor muscle of the bladder were found to be similar to those immunoreactive for NA synthesizing enzymes in both distribution and density. In the upper and middle bladder body--including the dome (apex)--immunoreactive nerve fibers were always more abundant in the outer part of the detrusor muscle than in the inner part, regardless of the course of muscle bundles. Even in individual muscle bundles running from the inside to the outer surface, the outer part was more richly innervated by immunoreactive fibers than the inner part. In the bladder dome, a moderate number of immunoreactive nerve fibers preferentially innervated the outer part of the muscle layer. In the lower bladder body, these nerve fibers increased in density in the inner part of the detrusor muscle. There was no sexual difference in density or distribution of nerve fibers. NA- and NA synthesizing enzyme-immunoreactive nerve fibers were markedly decreased in number after 6-hydroxydopamine treatment. No dopamine- or phenylethanolamine-N-methyltransferase-immunoreactive nerve fibers were present in the bladder. The findings of this study indicate that the cat bladder musculature includes longitudinal and circular muscle bundles, both of which are extensively innervated by adrenergic nerves, particularly in the outer part of the bladder.

Alterations in the expression of the beta-cytoplasmic and the gamma-smooth muscle actins in hypertrophied urinary bladder smooth muscle.

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle gamma-actin, shows over 200% increase in the gamma-actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in gamma-actin, since the 2D gel electrophoresis does not distinguish gamma-smooth muscle actin from gamma-cytoplasmic actin. The bladder smooth muscle alpha-actin and the smooth muscle alpha-actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the beta-cytoplasmic actin (37%) and a concomitant diminution in the beta-cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.

Inhibitory effects of propiverine on rat and guinea-pig urinary bladder muscle.

In muscle strips isolated from guinea-pig and rat urinary bladder, propiverine (3-10 microM) inhibited carbachol-induced contractions in the presence of verapamil and Ca(2+)-induced contractions in excess K+ medium containing atropine, suggesting it has both anticholinergic and Ca2+ channel blocking actions. The Ca2+ channel blocking action was also demonstrated by recording inward Ca2+ currents in single cells dispersed from both species. The inhibition of inward currents by propiverine was three times stronger in the rat than the guinea-pig, ID50 being 7 microM for rat and 21 microM for guinea-pig. The recovery of the current after washout was faster than that of mechanical inhibition. It is concluded that propiverine blocks not only muscarinic receptors, but also Ca2+ channels at similar concentrations.

Effect of decentralization or contralateral ganglionectomy on obstruction-induced hypertrophy of rat urinary bladder muscle and pelvic ganglion.

After urethral obstruction the musculature of the rat urinary bladder undergoes extensive hypertrophy, irrespective of whether its innervation is intact or whether one of the two pelvic ganglia has been decentralized or excised. Even the excision of both ganglia does not inhibit muscle hypertrophy. The presence of nerves is not a prerequisite for the muscle growth to occur. The stimulus for growth resides in the bladder itself, and the present and previous observations are in agreement with the notion that the distension of the muscle is a primary stimulus for muscle growth. With bladder hypertrophy, the pelvic ganglion neurons undergo hypertrophy, even when they are devoid of their preganglionic input. Synaptic connection with the preganglionic fibres and stimuli from the spinal cord are not prerequisites for neuronal hypertrophy. However, the hypertrophy is less marked in the decentralized ganglion neurons than in the neurons of the contralateral intact ganglion. With bladder hypertrophy and contralateral ganglionectomy, the neuronal hypertrophy is greater than with either procedure alone, suggesting that the two forms of neuronal growth stimulation can be added to each other.

Species differences in cAMP production and contractile response induced by β‐adrenoceptor subtypes in urinary bladder smooth muscle

The contractile activity of urinary bladder smooth muscle has been shown to be inhibited by beta-adrenoceptor agonists. beta-Adrenoceptor subtypes in the rabbit, canine, and human urinary bladder smooth muscles were studied by measuring changes in contractile forces and in intracellular cAMP concentrations on administration of beta-adrenoceptor agonists. In the rabbit bladder, only beta 2-receptors may have functional roles in the detrusor muscle, while both beta 1- and beta 2-receptors may have functional roles in the detrusor muscles of the canine bladder. Only beta 2-receptors may have functional roles in the human detrusor muscles, similar to the rabbit detrusor muscles. Thus, species differences may exist in beta-adrenoceptor subtypes in smooth muscles of urinary bladder.

Expression of constitutive heat shock protein-70 in normal (non-stressed) rabbit urinary bladder tissue.

The expression of constitutive HSP-70 in the urinary bladder was determined by SDS-PAGE and western blotting using a mouse monoclonal antibody against HSP-70. The western blot analysis showed that the mouse anti-HSP-70 cross-reacted with a 70 kDa protein present in the extracts of the urinary bladder muscle and mucosa. Densitometric scanning of the western blots allowed us to specifically quantitate the relative amounts of the HSP-70. The quantitation of the HSP-70 by combining immunoblotting and densitometry using a laser scanner is reproducible and this technique requires only a small amount of tissue. The amounts of HSP-70 can be estimated from a standard curve of nanogram(ng) of HSP-70 vs absorption from the immunoblots. The amounts of HSP-70 in the muscular and mucosal layers in the body of the urinary bladder are more than those in the base of the bladder. The presence of HSP-70 in the muscle and mucosal epithelium of the bladder was demonstrated by immunohistochemical analysis of freshly removed tissue from the base and the body of bladder from normal animals.

Endothelin in the Urinary Bladder. II. Characterization of Endothelin Receptor Subtypes

Endothelins (ET-1, ET-2, ET-3) are a family of regulatory peptides with diverse biological functions, including modulation of smooth muscle tone. To evaluate the possible role of endothelins in the control of the detrusor smooth muscle of urinary bladder, we have investigated the responses of isolated strips of bladder dome and base to endothelins, in organ chambers, and characterized the receptors for these isopeptides in bladder membranes. ET-1, ET-2 and to a lesser extent ET-3 caused sustained concentration-dependent contraction of bladder dome and base. The contraction to endothelins, unlike the short-lived contraction produced by cholinergic agents, was long lasting and difficult to wash out. Equilibrium binding studies demonstrated that endothelin isoforms bound to bladder membrane receptors specifically, with high affinity (KD of 0.4–0.6nM) and limited capacity (60-420 fmol/mg protein). Competition analysis showed two populations of receptors: one with high affinity for ET-1 and ET-2 and low affinity for ET-3 and another with high affinity for ET-1, ET-2 and ET-3. Chemical affinity labeling of endothelins to bladder membranes demonstrated that ET-1 and ET-2 were cross-linked to three proteins (75, 52 and 34kDa, respectively), whereas ET-3 was cross-linked mainly to a 34kDa protein. The data obtained from equilibrium binding studies, competition analysis and cross-linking experiments suggest that at least two endothelin receptor subtypes exist in bladder tissue. These observations further suggest that bladder smooth muscle tone may be modulated by endothelin or an endothelin-like substance via interaction with specific receptor sites.

Lactate dehydrogenase activity and isoform distribution in normal and hypertrophic smooth muscle tissue from the rat

The lactate dehydrogenase (LDH) activity and isoform distribution of LDH were investigated in tissue samples from the rat portal vein, aorta and urinary bladder. In addition, samples were obtained from hypertrophic urinary bladder. The total LDH activity per unit smooth muscle volume was higher in the urinary bladder compared to that in portal vein and aorta. Five LDH isoforms, reflecting different combinations of the two polypeptide chains denoted H and M, could be separated by agarose gel electrophoresis. The aorta contained more of the H form compared to the portal vein and urinary bladder. This difference suggests that the aorta, which is a slow smooth muscle, is more adapted for aerobic metabolism than the faster muscles of portal vein and urinary bladder. In the hypertrophic urinary bladder a shift in LDH isoform pattern towards less of the H form was found, which correlates with a better maintenance of contraction in anoxia in this type of hypertrophic smooth muscle.

Excitatory pathways in smooth muscle investigated by phase-plot analysis of isometric force development

Excitatory pathways in the smooth muscle of the pig urinary bladder were investigated using phase-plot analysis of isometric contractions. The phase plots, plots of the rate of change of the force as a function of the force itself, were dominated by a straight line described by the horizontal intercept (Fiso) and the vertical intercept (U). The quotient Fiso/U is a time constant that characterizes the rate-limiting step in isometric force development in the muscle. Bladder strips of 1 mm diameter were activated by electrical field stimuli, acetylcholine, potassium, and ATP in combination with selective pathway inhibitors such as verapamil, atropine, or a calcium-free solution containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When pathways that depended significantly on depolarization or intracellular calcium release were selected, the time constant was significantly smaller, indicating a faster process. The results indicated that the rate-limiting step in force development was determined by the influx of extracellular calcium.

Antispasmodic effects of flavoxate, MFCA,and REC 15/2053 on smooth muscle of human prostate and urinary bladder

The antispasmodic effects of the flavone compounds flavoxate hydrochloride, 3-methylflavone carboxylic acid (MFCA), and REC 15/2053 (and in the case of the detrusor, oxybutynin), on the human detrusor, prostatic adenoma, prostatic capsule, and bladder neck, were studied by the in vitro isometric method. All the compounds inhibited, in different orders of potency, potassium-induced contractions of the tissues. Flavoxate showed a slightly greater activity than the other two compounds in the prostatic and bladder neck tissues. However, REC 15/2053 displayed greater activity in the detrusor than in the other tissues. The relaxant effect on the prostatic tissues suggests a potential use for these compounds in benign prostatic obstruction.

Existance of cholinergic and purinergic receptor on the detrusor muscle of rat urinary bladder

This study was aimed at investigation of the stimulatory innervations on the rat urinary bladder. Detrusor muscle strips of 15 mm long were suspended in isolated muscle chambers containing 1 ml of PSS maintained at and aerated with 95% . Isometric myography was perfomed, and the results were as followings : Muscle strips showed on-contraction by electric field stimulation (EFS) frequency-dependently. The EFS-induced contraction was not affected by hexamethonium, a ganglion blocker, but abolished, by tetrodotoxin, a nerve conduction blocker. Physostigmine, a cholinesterase inhibitor enhanced the EFS-induced contraction which was inhibited by hemicholinium, an inhibitor of choline uptake at the cholinergic nerve ending. Such an EFS-induced contraction was antagonized by atropine only partially, and the atropine-resistant portion was completely abolished by the desensitization of purinergic receptors by prolonged incubatin of the strips in the presence of high concentratin of ATP. Bethanechol, a cholinergic agonist, elicited concentration-dependent contraction. Adenosine triphosphate (ATP), a purinergic agonist, induced a weak but concentration-dependent contraction of short duration. Bethanechol-induced contraction was not affected by ATP-desensitization, and ATP-induced contraction was not affected by tetrodotoxin. These results suggest that there are at least two main stimulatory components of innervations in the detrusor muscle, cholinergic muscarinic and purinergic ; and those receptors are independent each other.

Pharmacological characterization of tachykinin‐stimulated inositol phospholipid hydrolysis in peripheral tissues

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H]-inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L-Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea-pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists.

Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats

The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats. The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a Vmax = 390 nmoles/mg protein/min, and a Km = 275 microM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with Vmax = 65 and 324 nmoles/mg protein/min, and Km's = 10 microM and 190 microM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate Vmax of 800 nmoles/mg protein/min and a Km of 280 microM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distinct component forms. 5) The Km for ADP for both skeletal muscle and bladder was approximately 10 microM. These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.

Formula omitted] and [formula omitted] antimuscarinic effects of (-)cis 2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1, 5 benzothiazepin-4-(5H)one HC1 (BTM-1086) in guinea pig peripheral tissues

The potency and selectivity of (-)cis-2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1, 5 benzothia]epin-4-(5H) one HCl (BTM-1086) for muscarinic receptor subtypes was compared in functional assay systems, in guinea pig peripheral tissues, to known reference drugs: atropine (nonselective), pirenzepine (M1), AF-DX 116 (M2) and HHSiD (M3). Like atropine, BTM-1086 was a potent, nonselective, competitive muscarinic antagonist with no detectable antispasmodic activity in urinary bladder or ileal muscle. Invivo, in the guinea pig cystometrogram, BTM-1086 depressed intravesical bladder pressure (PvesP) with the same efficacy and potency as oxybutynin, a drug used clinically for the treatment of urinary incontinence. The pharmacological profile of BTM-1086, however, suggests that it may not be suitable for development for bladder dysfunction disorders.

Synchronization of spontaneous contraction activity in smooth muscle of urinary bladder

Synchronization of spontaneous contraction activity in smooth muscle of urinary bladder

Localization of muscarinic receptors on somatostatin-like immunoreactive neurones of the newborn guinea pig urinary bladder in culture

Muscarinic receptors were localized on cells cultured from the detrusor muscle of the newborn guinea pig urinary bladder by autoradiography using the irreversible muscarinic antagonist [ 3H]propylbenzilylcholine mustard, before being immunostained with an antibody to somatostatin. Many mononucleate and binucleate intramural neurones immunoreactive for somatostatin were observed (60–75% of the total population), a subpopulation of which (40–60%) expressed muscarinic receptors. Autoradiographic grains were distributed over the whole cell body surface and the entire length of the neurites. An even distribution of silver grains was also seen on cultured smooth muscle cell surfaces, but not on other cell types present in the culture preparations. The demonstration of muscarinic receptors on specific neuropeptide-containing cells in culture is consistent with the existence of specialized cholinergic, intraganglionic circuits within the bladder wall, and suggests that somatostatin may also be involved in the integration and/or modulation of bladder function.

Multiple sources of calcium for contraction of the human urinary bladder muscle

1. KCl, carbachol, neurokinin A and endothelin produced concentration-dependent contractions of mucosa-free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2. Nifedipine (1 microM) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 microM) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3. Superfusion of the strips with a nominally calcium (Ca)-free medium containing EDTA (1 mM) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mM) Ca-free medium plus EDTA (1 mM) these three agonists still produced a small contractile response. 4. The nifedipine (1 microM) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mM). In contrast, the nifedipine-(1 microM) resistant response to carbachol was not modified by NiCl2 (0.1 mM) or omega-conotoxin (0.1 microM). 5. Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37 degrees C and a concentration-dependent (2.5-20 mM) contraction at 25 degrees C. The latter was markedly inhibited by procaine (3 mM) but unaffected by nifedipine (1 microM). 6. After a prolonged (60 min) superfusion with a high K, Ca-free medium containing EDTA the response to carbachol (100 microM) was abolished by previous exposure to procaine (3 mM). Conversely, the response to endothelin (1 microM) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mM). 7. These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine- and voltage-sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK-2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3-sensitive but nifedipine-resistant. Neither T- nor N-type channels appear to be involved in the response to carbachol. In addition, these agents mobilize a tightly bound Ca pool independently from membrane depolarization. This latter pool is probably a procaine-sensitive intracellular source of activator Ca mobilized by caffeine and carbachol. The failure of procaine to prevent the response to endothelin in high K, Ca-free medium raises the possibility that this peptide mobilizes an intracellular source of activator Ca, distinct from the caffeine- and carbachol-sensitive pool.

The effect of cromakalim on the smooth muscle of the guinea-pig urinary bladder.

1. The actions of cromakalim were studied on the detrusor muscle from guinea-pig urinary bladder. Cromakalim reduced the frequency and amplitude of spontaneous contractile activity of the smooth muscle of the guinea-pig urinary bladder at 5 x 10(-8)M and abolished the activity at concentrations above 5 x 10(-7)M. 2. Electrophysiological experiments demonstrated that cromakalim increased membrane conductance, caused a dose-dependent hyperpolarization of the cell membrane and loss of spike activity. These events are consistent with the opening of K+ channels. 3. The effects of 10(-6)M and 10(-5)M cromakalim on the contractile responses to carbachol, potassium and transmural nerve stimulation were studied. Cromakalim did not prevent the detrusor from responding to these agents, although it significantly reduced the contractile response to K+ at concentrations below 70 mM. 4. Uptake and efflux experiments using 86Rb+ were unable to demonstrate any significant effect on transmembrane movement produced by cromakalim (10(-5)M). 5. 43K+ efflux showed a dose-dependent increase in the rate constant on addition of cromakalim. The difference in the selectivity for K+ over Rb+ was confirmed in dual label uptake experiments. 6. Substitution experiments in which the K+ ions in the tissue were gradually replaced by Rb+ demonstrated that cromakalim had a progressively decreasing effect on spontaneous activity as internal K+ was lowered. When all the K+ was replaced by Rb+, cromakalim no longer inhibited spontaneous activity, confirming that the channel opened by cromakalim appears relatively impermeant to Rb+.