Tetrodotoxin-resistant contractions induced by electrical stimulation of bladder muscle from man, rabbit and rat

Isolated detrusor preparations from man, rabbit and rat were suspended in an organ bath and isometric tension was recorded. The preparations were stimulated electrically in the presence of Bay K8644 and nifedipine before and after neuronal blockade with tetrodotoxin. Transmural electrical stimulation produced frequency-dependent contractions in all preparations. Bay K8644 significantly increased and nifedipine decreased these contractions. TTX effectively suppressed the response to electrical field stimulation in all species. When Bay K8644 was added to TTX blocked preparations, the responses to electrical stimulation were partly restored in bladder strips from man and rat. No increase in response was seen in the rabbit preparations. However, if the extracellular K+-concentration was increased to 10 mM (which per se did not affect the response) Bay K8644 significantly increased the contractions. All responses elicited by electrical stimulation in the presence of TTX were abolished by nifedipine. It is concluded that if the bladder smooth muscle is exposed to factors that can increase its sensitivity to contractile agents, this may result in uncontrolled (unstable) bladder contractions. Such contractions may use the 'normal' transmitter substances, but may be triggered at a lower stimulus intensity than normal. As a non-specific increase in membrane excitability seems to be associated with an influx of calcium through voltage-sensitive calcium channels, calcium antagonists, together with agents specifically blocking relevant transmitter substances, would offer an effective therapy against the unstable bladder.     

Oestrogen-induced changes in muscarinic receptor density and contractile responses in the female rabbit urinary bladder

Mature ovariectomized rabbits were treated for 1, 4 or 8 weeks with oestrogen, and the effects on contractile responses and on muscarinic receptor density in the isolated urinary bladder were studied. Oestrogen treatment caused a significant increase in the weight of the bladders. The responses to K+ (124 mM) were depressed, but not the maximum responses to carbachol. The frequency-response curve to electrical stimulation was shifted to the right after 4 and 8 weeks of treatment, but the maximum response was not reduced. There was also a (non-significant) shift to the right of the carbachol concentration-response curve. Pre-treatment with scopolamine revealed a significant reduction of the non-cholinergic response to electrical stimulation after oestrogen treatment compared to controls. Binding of [3H]quinuclidinyl benzilate (QNB) was saturable and of high affinity. There were no changes in apparent dissociation constant after oestrogen treatment. However, the muscarinic receptor density decreased already after 1 week of treatment and was only 10% of the control after 4 weeks. It is concluded that oestrogen treatment causes a down-regulation of muscarinic receptors in the rabbit urinary bladder, but the consequences for contractile activation through muscarinic receptors seem to be small.     

The role of muscarinic receptor subtypes in acetylcholine release from urinary bladder obtained from muscarinic receptor knockout mouse

We investigated the subtype of prejunctional muscarinic receptors associated with inhibition of acetylcholine (ACh) released from the mouse bladder. We measured endogenous ACh release in the bladder obtained from the wild-type mice and muscarinic 1-5 (M1-M5) receptor knockout (KO) mice. Electrical field stimulation increased ACh release in all bladder preparations obtained from wild-type and M1-M5 receptor KO mice. The amount of ACh released from M1-M3 and M5 receptor KO mice was equal to that in the wild-type mice. In contrast, the amount of electrical field stimulation-induced ACh release in M4 receptor KO mice was significantly larger than that in the wild-type mice, but the extent of increase was small. Atropine increased electrical field stimulation-induced ACh release to levels found in wild-type mice in all M1-M5 receptor KO mice. In M2/M4 receptor double KO mice, the amount of electrical field stimulation-induced ACh release was equivalent to that in the M4 receptor KO mice. The cholinergic component of electrical field stimulation-induced contraction (in the presence of alpha,beta-methylene ATP) in the detrusor of M4 receptor KO mice was no different from that in the detrusor of wild-type mice. M4 receptor immunoreactivity was located between smooth muscle cells, colocalized with choline acetyltransferase immunoreactivity. These results indicate that the prejunctional inhibitory muscarinic receptors are of the M4 and non-M2 receptor subtypes. The nature of the non-M2 receptors remains unknown.     

Mechanism of the reflex inhibition of micturition contractions of the urinary bladder elicited by acupuncture-like stimulation in anesthetized rats.

The effects of acupuncture-like stimulation of various segmental areas on the rhythmic micturition contractions (RMCs) of the urinary bladder were examined in anesthetized rats. The urinary bladder was cannulated via the urethra and expanded by infusing saline until the urinary bladder produced micturition contractions rhythmically as a consequence of the rhythmic burst discharges of the vesical pelvic efferent nerves. An acupuncture needle, having a diameter of either 160 or 340 microns, was inserted to a depth of about 4-5 mm into the skin and underlying muscles at various segmental areas, rostrally from the face then caudally to the hindlimb. Once being inserted, the needle was twisted left and right with the fingers about once every second for 1 min. (1) Acupuncture-like stimulation applied to the perineal area inhibited both the RMCs and the rhythmic burst discharges of vesical pelvic efferent nerves without any significant changes in the hypogastric efferent nerve activity. By contrast, stimulation applied to the face, neck, forelimb, chest, abdomen, back, and hindlimb areas was ineffective. (2) After surgically separating the perineal skin from the underlying muscles with the main cutaneous nerve branches intact, stimulation of either the perineal skin or the perineal muscles inhibited the RMCs. Stimulation of the perineal muscles produced a stronger inhibition of the RMCs than that of the perineal skin. (3) Stimulation of the perineal area increased afferent nerve activity, either recorded from the pudendal nerve branches innervating the perineal skin or underlying muscles, or recorded from the pelvic nerve branches innervating the perineal muscles. (4) The stimulation-induced inhibition of the RMCs was abolished after surgically severing both pudendal and pelvic nerve branches that innervated the perineal skin and underlying muscles. (5) The present findings indicate that the inhibition of the RMCs following acupuncture-like stimulation of the perineal area is a reflex response characterized by segmental organization. The afferent arcs of the reflex are both pelvic and pudendal nerve branches innervating the perineal skin and underlying muscles, while the efferent arcs are pelvic nerve branches innervating the urinary bladder.     

Contraction wave in the chick blastoderm induced by muscarinic stimulation

Summary The mechanism of excitation contraction coupling during morphogenetic movements is unknown. We describe a contraction wave in the chick blastoderm after muscarinic stimulation, which indicates that an autocrine cholinergic mechanism might be involved in the induction of morphogenetic movements during embryogenesis. Chick blastoderms were explanted in a modified new culture and the cellular movements were recorded by time lapse video filming. Perfusion with acetylcholine or carbachol induced a contraction wave in the blastoderm which started in the periphery at the point of entrance of the drug, and proceded within 8–10 min through the area pellucida to the opposite side of the blastoderm. Perfusion with the muscarinic antagonist pilocarpine in turn induced relaxation. Atropine inhibited the effect of the agonists acetylcholine and carbachol. From earlier studies we know that in the chick embryo a muscarinic system is present, the expression of which correlates with morphogenetic movements. The induction of a contraction wave in the chick blastoderm by muscarinic agonists supports our hypothesis that embryonic cell movements might be regulated via muscarinic receptors.     

Microvasculature of the rabbit urinary bladder

Background: The urinary bladder requires a rich blood supply to maintain its functions, the storage and release of urine. Specialized properties of the bladder vasculature might be anticipated to ensure the integrity of this blood supply, because it is known that blood flow is reduced by distension during bladder filling. However, the bladder vasculature has been described in detail only at the gross level. A comprehensive, threedimensional view of the blood supply to the bladder wall is presented here. Methods: The microvasculature of the bladder of male New Zealand white rabbits was described using the combination of vascular corrosion casting, alkali digestion, light microscopy, and scanning and transmission electron microscopy. Following administration of an anticoagulant and an overdose of anesthetic, the abdominal aorta was cannulated just above the inferior mesenteric artery to permit flushing of the distal vasculature. The bladder vasculature was cleared of blood with buffered saline and then either perfuse-fixed with buffered 2% glutaraldehyde and sectioned, or filled with “Mercox” resin to prepare vascular corrosion casts. Casts were cleaned with NaOH, formic acid, and water. In some cases fixed bladders were partially digested with NaOH to expose the mucosal capillary plexus. Results: The bladder is supplied with blood by single, left and right vesicular branches of the internal or external iliac arteries. The serpentine vesicular arteries extend along the lateral borders of the bladder from base to apex just deep to the serosal surface and send dorsal and ventral branches to supply the dorsal and ventral bladder walls. Veins accompany the arteries and exhibit numerous valves. A very dense complex of vessels at the apex of the bladder apparently serves to accommodate bladder distension. The muscularis and submucosa contains few vessels, but the mucosa is well vascularized. An especially dense capillary plexus is present in the lamina propria at its junction with the transitional epithelium. In the relaxed bladder these capillaries lie in grooves formed by the basal layers of the epithelium. The endothelial cells of these capillaries display few cytoplasmic vesicles and are continuous or fenestrated. These capillaries are often invested with pericytes. The mucosal capillary plexus may be associated with an epithelial transport function or may be necessary for urothelial metabolism or maintenance of the barrier function of the urothelium. Unusual capillary tufts, possibly associated with vascular lymphatic tissue, are found associated with the main vessels on the lateral walls in the basal half of the bladder. Conclusions: These methods present a clear, comprehensive, three-dimensional view of the microvasculature of the bladder wall. They also identify several unique features of this vasculature and provide a basis for studies of the response of this vasculature to pathologic states and experimental manipulation. © 1995 Wiley-Liss, Inc.     

Na transport compartment in rabbit urinary bladder

Electron microprobe analysis was used to determine cellular electrolyte concentrations in rabbit urinary bladder. Under control conditions the mean cellular electrolyte concentrations were for Na 11.6±2.0, for K 124.1±15.3, and for Cl 26.0±5.1 mmol/kg wet weight. The dry weight content was 19.0±2.0 g/100 g. Inhibition of the Na/K-pump with ouabain resulted in drastic changes of the cellular element concentrations. Similar changes also occurred when in addition to ouabain the apical side was kept Na-free. In all epithelial layers the Na and Cl concentrations increased by 90 and 30 mmol/kg wet weight, whereas the K concentration and the dry weight content decreased by 90 mmol/kg wet weight and 6 g/100 g wet weight, respectively. With Na-free choline-Ringer's solution on the basal side ouabain led to a decrease in the K concentration by about 60 mmol/kg wet weight while the Na and Cl concentrations remained unchanged. These data indicate that the basolateral membrane is permeable to Na, choline, Cl, and K. Nystatin produced drastic changes in the cellular electrolyte concentrations when Na- or Rb-sulfate Ringer's solutions were present on the apical side. With Na-sulfate Ringer's solution the Na concentration increased by about 25, the Cl concentration by 30 mmol/kg wet weight and the dry weight content decreased by 4.5 g/100 g, respectively. With Rb-Ringer's solution about 20 mmol/kg wet weight of the cellular K was exchanged against Rb. The concentration changes were identical in all epithelial layers supporting the idea that the rabbit urinary bladder represents a functional syncytium with regard to the transepithelial Na transport.     

Rat urinary bladder epithelial lesions induced by acrolein

Acrolein, a constituent of cigarette smoke and a metabolite of cyclophosphamide, has been shown to induce acute cytotoxicity of the rat urinary bladder mucosa when instilled directly into the bladder lumen. To evaluate the effects of systemic administration, we examined the rat urinary bladder following intragastric or intraperitoneal administration of acrolein to male F344 rats. In an initial experiment, acrolein was administered at a dose of 25 mg/kg, which proved to be extremely toxic. Five of 12 rats injected intragastrically and 5 of 12 injected intraperitoneally died within 24 hrs. After 2 days, 3 of the 3 surviving rats injected intraperitoneally had focal simple hyperplasia of the urinary bladder. None of the rats injected intragastrically had bladder hyperplasia. In a second experiment, acrolein was administered by intraperitoneal injection at doses of 0.5, 1, 2, 4, and 6 mg/kg. Five days later, the labeling index of the bladder mucosa was evaluated by autoradiography. In rats injected with 6 mg/kg of acrolein, the labeling index was significantly increased compared to the other doses and compared to a vehicle injection control group. These data indicate that sufficient acrolein reaches the urinary bladder to induce a proliferative response following intraperitoneal administration.     

Quantitative analysis of muscle hardness in tetanic contractions induced by electrical stimulation in rats

The purpose of this study was to investigate the in vivo relation between muscle hardness during an electrically induced contracting state and neuromuscular functions (M-wave and developed tension). Sixteen Sprague-Dawley rats were deeply anesthetized with urethane. Muscle hardness was measured quantitatively at the mid-portion of the gastrocnemius (GS) muscle during tetanic contractions induced by electrical stimulation (50 Hz, 100 μs duration) of the sciatic nerve or of the muscle directly. The M-wave was recorded with a pair of wire electrodes inserted into the muscle, and the developed tension was monitored with a push–pull gauge. Muscle hardness, M-wave amplitude and developed tension increased rapidly with the onset of nerve stimulation. Similar but intensity-dependent increases in muscle hardness and tension were observed following direct tetanic stimulation of the muscle. The hardness measured during nerve stimulation was correlated with the amplitude of the M-wave (r = 0.62, P < 0.0001) and the developed tension (r = 0.85, P < 0.0001). These phenomena were suppressed by pancuronium treatment (2 mg/ml, i.v.). These results suggest that muscle tension might be the most important factor for transcutaneously measured muscle hardness induced by tetanic muscle contraction.     

Ultrastructure of tumors induced in the rat urinary bladder by nitrosomethyldodecylamine

Summary N-Nitroso-N-methyl-n-dodecylamine (NMDA) is a powerful carcinogen in the rat and the Syrian golden hamster. In both species the urinary bladder is the main target organ. We studied the ultrastructure of these bladder tumors in the Fischer rat in some detail, since this compound provides an interesting model for carcinogenesis in the urinary bladder. We found that the proliferating basal layers of the transitional cell carcinomas were undergoing squamous metaplasia, which indicates that squamous carcinomas in the organ may arise from pre-existing transitional cell tumors.The author gratefully acknowledges the excellent technical assistance of Mark ShermanThis work was supported by Contract No. N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20205, USABy acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article     

Supraspinal and spinal mechanisms in morphine-induced inhibition of reflex urinary bladder contractions in the rat

The supraspinal and spinal mechanisms of morphine-induced inhibition of isometrically recorded reflex urinary bladder contractions were studied in rats anesthetized with urethan. Chronic intracerebroventricular administration of 5,7-dihydroxytryptamine (200 micrograms) or 6-hydroxydopamine (150 micrograms), to selectively deplete central serotoninergic and noradrenergic systems, attenuated the intracerebroventricular effect but not the intrathecal effect of morphine. The intracerebroventricular effect of morphine was reversibly attenuated or abolished by an intrathecal injection of the novel delta-receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Arb-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) (1-3 micrograms) and by intrathecal methysergide (4-10 micrograms), phentolamine (5-10 micrograms), and yohimbine (5-10 micrograms) but not by intrathecal propranolol (10 micrograms), atropine (8 micrograms) or saline (2 micrograms) administered at similar molar concentrations and volumes respectively. These observations support the hypothesis that supraspinal and spinal mechanisms involved in morphine-induced inhibition of reflex urinary bladder contractions can be dissociated. The supraspinal actions of morphine were mediated indirectly via descending 5-hydroxytryptamine and noradrenergic pathways which activated specific 5-hydroxytryptamine and alpha-adrenergic but not beta-adrenergic receptor in the spinal cord. In addition, supraspinal morphine indirectly activated a spinal opioid system which could be directly activated by intrathecal morphine. The similarities between these observations and studies of central pathways mediating nociception and opioid analgesia suggest that similar physiological mechanisms control certain somatic and visceral activity.     

Operant discrimination of interoceptive urinary bladder stimulation in the monkey

The interoceptive stimulus of fluid in the urinary bladder of a female rhesus monkey was manipulated so as to function as a discriminative stimulus, such that lever-presses emitted in the absence of the stimulus (S^D) were reinforced on FR 24, and those emitted in the presence of the stimulus (S^Δ) were extinguished. Successful discrimination was achieved. Results were in agreement with those of previous research, in which stimulus onset was quickly discriminated but stimulus offset was not. Implications of visceral stimulus functions for basic psychophysiology and practical applications for the study and modification of behavior were considered.     

Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells

The mechanism underlying the regulation of the K-channel by the muscarinic receptor was examined with patch-clamp experiments in atrial cells isolated enzymatically from the rabbit heart. The patch-electrode and the recording chamber were perfused with various solutions while the activity of the K-channels in the membrane-patch was recorded continuously. In the absence of muscarinic agonists, opening of K-channels occurred at a low frequency (basal activity). Application of ACh to the bath did not affect the basal activity, but perfusion of the patch electrode with ACh markedly increased the channel activity in the "cell-attached" patch. Application of oxotremorine, i.e. a specific muscarinic agonist, via the pipette also opened K-channels. When the membrane patch was isolated from the cell body ("inside-out" patch), ACh-induced single K-channel currents were still observed, but the frequency was reduced. Perfusion of atropine or scopolamine, two muscarinic antagonists, through the patch-electrode depressed the basal activity. In the case of scopolamine, channel-activity recovered after washing out the drug. The current voltage relationship determined from the basal activity was similar to that of ACh-induced single K-channel currents. The mean open time was 0.49 ms at basal activity and 1.35 ms during the application of 0.1 microM ACh via the patch electrode. Application of oxotremorine via the pipette hardly affected the open-time, it remained at 99 +/- 4% (n = 7) of the control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Shortening induced deactivation in the guinea-pig urinary bladder

Shortening induced deactivation, the depressant effect of active muscle shortening on the subsequently measured isometric force, has been shown in smooth muscle strips and rings. The guinea-pig bladder permits the investigation of this phenomenon in a whole organ preparation. Previous work in our laboratory showed that shortening of the in vitro guinea-pig detrusor muscle had a depressant effect on the isovolumetric pressure that could be generated immediately afterwards. To test the hypothesis that this was caused by deactivation, the effects of active and passive detrusor shortening on the subsequently measured isovolumetric pressure were compared. The isovolumetric pressures measured after 5 min periods of recovery were taken as control values. It was found that the isovolumetric pressure after passive shortening was 7% smaller than the isovolumetric pressure without preceding shortening. This difference was ascribed to viscoelastic relaxation during shortening. Active shortening had an additional 8% depressant effect on isovolumetric pressure compared with passive shortening. The effects of active and passive shortening differed significantly. It was concluded that shortening induced deactivation in the guinea-pig urinary bladder smooth muscle in toto can be considered proven. The fact that deactivation is shown both by striated and smooth muscle preparations is in line with the assumption that it is caused by reduced actin-myosin interaction. The hypothesis that (in striated muscle) the latter is effected by a decrease in troponin–calcium binding, however, needs reconsideration.     

Induction of cellular contractions in the human melanoma cell line SK-mel 28 after muscarinic cholinergic stimulation

In a previous immunohistochemical study we observed muscarinic acetylcholine receptors in primary and metastatic human melanomas, which were not present in normal skin melanocytes. In the present study we demonstrated the endogenous expression of muscarinic receptors, of choline acetyltransferase and of cholinesterase activity in the human melanoma cell line SK-mel 28. We tested the effect of muscarinic agonists on cellular movements of the melanoma cells in a perfusion chamber by digital video time-lapse microscopy. Within 3 to 10 min after onset of muscarinic perfusion cell body contractions and retraction of cell processes of more than 5 μm occurred in about 30% of the melanoma cells. The effect disappeared after addition of atropine. The proportion of reacting cells corresponded to the endogenous expression of muscarinic receptors revealed by immunocytochemistry with the monoclonal antibody M35. The experiments indicate the presence of an autocrine muscarinic cholinergic system in the melanoma cells and demonstrate a direct link between muscarinic receptors and the contractile apparatus. Melanocytes are derived from neural crest cells that express cholinesterase activity and muscarinic receptors during their migratory phase in the embryo. Therefore, re-expression of the muscarinic cholinergic system in tumour cells may be involved in invasive growth. Accepted: 10 June 1999     

Alteration of muscarinic and purinergic receptors in urinary bladder of rats with cyclophosphamide-induced interstitial cystitis

We characterized muscarnic and purinergic receptors and urodynamic parameters in the bladder of cyclophosphamide (CYP)-treated rats to clarify the mechanisms involved in the pathophysiology of interstitial cystitis (IC). In the cystometry of CYP-treated rats compared with control rats, the micturition interval and micturition volume were significantly (55% and 77%, respectively) decreased and the frequency of micturition and basal pressure were significantly (3 and 2.3 times, respectively) increased. These changes in urodynamic parameters may characterize the detrusor overactivity occurring in CYP-treated rats. The maximal number of binding sites (B(max)) for specific binding of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) and alphabeta-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]alphabeta-MeATP) was significantly (43% and 31%, respectively) decreased in the bladder of CYP-treated rats compared with control rats. On the other hand, the apparent dissociation constant (K(d)) for neither radioligand was significantly altered by the CYP treatment. K(i) value for the inhibition of bladder [(3)H]NMS binding by antimuscarinic agents (oxybutynin, tolterodine, darifenacin, and AF-DX 116) did not differ significantly between control and CYP-treated rats. The inhibition constant (K(i)) for the inhibition of bladder [(3)H]alphabeta-MeATP binding by purinergic antagonists (A-317491, PPADS) was significantly higher in CYP-treated rats than control rats. In conclusion, CYP treatment has been shown to cause down-regulation of pharmacologically relevant (muscarinic and purinergic) receptors in the bladder of rats. Thus, the present study offers further pharmacological evidence that both muscarinic and purinergic mechanisms contribute significantly to the urinary dysfunction due to IC.