Direct inhibition of rat detrusor muscle contraction by erythromycin

PURPOSE: Detrusor instability is a common problem in the elderly, which is usually treated with anti-cholinergic medication. This study investigates the effect of erythromycin on rat detrusor muscle contractile response to characterise its potential as an alternative inhibitor of bladder muscle contraction. MATERIALS AND METHODS: Strips of rat detrusor muscle were suspended in a perfusion organ bath. The contractile response to direct muscle stimulation, electrical field stimulation (EFS, 0.5-60 Hz), carbachol (10(-5) M), and potassium (10-80 x 10(-3) M) were determined before and after the addition of erythromycin (10(-4)-10(-3) M). The contractile response to carbachol (10(-5) M) in the presence of nifedipine (10(-8) or 10(-6) M) or in calcium-free Kreb's solution was also determined in the absence and presence of erythromycin. RESULTS: Erythromycin 5 x 10(-4) M inhibited the maximum contractile response to EFS, carbachol, and potassium by 38% (P < 0.01), 62% (P < 0.001), and 17% (P < 0.05), respectively, but did not significantly reduce the response to direct muscle stimulation. The atropine-resistant component of EFS-evoked contraction was inhibited by 19.5% (P < 0.01) in the presence of erythromycin. In calcium-free Krebs solution, the maximum contractile response to carbachol was reduced by 42% of control (P < 0.0001) and nifedipine 10(-8) M had no additional effect. When erythromycin 5 x 10(-4) M was added together with nifedipine 10(-8) M, the response to carbachol was inhibited by a further 25% (P < 0.005). CONCLUSIONS: Erythromycin inhibits rat detrusor muscle contraction through the inhibition of calcium influx and the modulation of intracellular calcium movement.     

Carbachol-induced sustained tonic contraction of rat detrusor muscle.

OBJECTIVE: To investigate the underlying contractile mechanism of the sustained tonic contraction (SuTC) induced by repetitive carbachol application in rat detrusor muscles. MATERIALS AND METHODS: Longitudinal muscle strips with no mucosa were obtained from the anterior wall of the urinary bladder in 12-week-old Sprague-Dawley rats. Carbachol (5 micromol/L) was applied repetitively to induce SuTC. The carbachol-induced SuTC was assessed in the presence of various Ca2+-channel blockers and drugs affecting intracellular Ca2+ concentration. RESULTS: The first application of carbachol elicited a large phasic contraction followed by a tonic contraction (TC); the carbachol-induced contraction was completely reversed by washing out the solution. However, the initial phasic contraction was not reproduced after a second or further application of carbachol. There was consistently only a SuTC with no phasic contraction. The amplitude of the SuTC was 85% of the TC induced by the first carbachol application. The application of atropine (1 micromol/L) to the bath completely blocked SuTC. The carbachol-induced SuTC was insensitive to nicardipine (5 micromol/L) and extracellular polyvalent cations (1 mmol/L, La3+, Co2+, Cd2+, Ni2+ ). Moreover, a similar SuTC was induced even after the complete elimination of extracellular Ca2+ by adding 2 mmol/L EGTA to the Ca2+-free Tyrode solution. To exclude intracellular Ca2+ sources related to the sarcoplasmic reticulum (SR), the effects of SR Ca2+ pump inhibitors, cyclopiazonic acid (CPA, 10 micromol/L) and thapsigargin (0.5 micromol/L) were tested. The carbachol-induced SuTC was insensitive to pretreatment with CPA and/or thapsigargin. To deplete the ryanodine-sensitive Ca2+ pool, muscle strips were repetitively stimulated with caffeine (10 mmol/L) in the presence of 10 micromol/L ryanodine, which did not affect the carbachol-induced SuTC. CONCLUSIONS: Although the characteristics of the carbachol-induced SuTC have not been defined, these results show that a significant proportion of the carbachol-induced contraction in rats is contributed by the SuTC, which is present even in the complete absence of external Ca2+. The SuTC was not affected by limiting the contributions of internal Ca2+ sources. This suggests that the SuTC in rat bladders is unrelated to known Ca2+ mobilization mechanisms.     

The routine assessment of detrusor contraction strength

A method is described for the semiquantitative assessment of the strength of the detrusor contraction during any voiding for which the detrusor pressure, flow rate, and residual urine are measured. A computer is used to produce plots of detrusor pressure vs detrusor shortening velocity throughout voiding. The adequacy of the contraction can be followed through micturition until the bladder is nearly empty. In some cases the isovolumetric detrusor pressure and the maximum potential velocity of shortening of the detrusor can be estimated. The ranges of values found are comparable with those deduced from the stop test, although there are slight discrepancies. The median values of these two parameters form the basis of the routine method that can be used for any detrusor contraction.     

Cooling-induced bladder contraction: studies on isolated detrusor muscle preparations in the rat

OBJECTIVES: Detrusor muscle contraction and uninhibited micturition after intravesical instillation of ice water is interpreted as a sign of upper motor neuron lesions. The basic mechanism of cooling-induced contraction (CIC) at the level of smooth muscle, however, has not been satisfactorily explained. We therefore designed model experiments with cooling of rat detrusor muscle. METHODS: We recorded isometric tension from strips of rat urinary detrusor muscle in organ baths during stepwise cooling. CIC was tested before and after addition of various standard agents interfering with known neurogenic (autonomic blockers, tetrodotoxin, capsaicin) and myogenic mechanisms of contraction (calcium channel blockers). RESULTS: Stepwise cooling (37 degrees to 5 degrees C) of detrusor muscle induced reproducible graded contractions, inversely proportional to temperature. CIC was not dependent on a neural mechanism (not blocked by tetrodotoxin or capsaicin) or the release of neurotransmitters but was linked to translocation of calcium. It was reduced by calcium channel blockers and Ca(2+)-free solution. Blockage of the Ca(2+)-adenosine triphosphatase pump, which inhibits the extrusion of calcium, also plays a significant role in the process and enhances CIC. CONCLUSIONS: Cooling of detrusor muscle preparations induces a graded myogenic contraction inversely proportional to the temperature. The mechanism is not dependent on local nervous control but is related to calcium translocation.     

Effects of magnesium ions on the contraction of the rat detrusor muscle]

Effects of magnesium (Mg) ions on the contraction of the rat detrusor muscle induced by intramural nerve stimulation were investigated in vitro. 1. Frequency-response curve demonstrated that contractile responses increased in magnesium-free Krebs' solution and decreased in high magnesium solution (MgCl2 5 mM) in comparison with those in normal Krebs' solution. 2. When MgCl2 was added into an organ bath with cumulative way, the magnitude of detrusor contraction decreased dose-dependently. The contractile response was completely abolished by 30 mM MgCl2. 3. Mg ions suppressed the detrusor contraction induced by a high KCl concentration. 4. The effect of Mg ions on the detrusor contraction was enhanced by verapamil but was inhibited by Bay-K8644. 5. Procaine agonized the effect of Mg ions on the detrusor contraction. On the other hand, caffeine did not modify the effect of Mg ions. These results suggest that Mg ions seem to play an inhibitory role on 2 types of Ca-channels existing on the cell membrane and to inhibit release of Ca ions from the intracellular deposit.     

A proposed mechanism of bupivacaine-induced contraction of human umbilical artery smooth muscle cells

This in vitro study using ring preparations of human umbilical vessels and cultured human umbilical artery smooth muscle cells was designed to determine: (a) the mechanism of bupivacaine-induced contraction of ring preparations, and (b) whether similar concentrations of bupivacaine release Cal(2+) in cultured smooth muscle cells. Isometric tension was recorded from ring preparations of human umbilical veins and arteries in an isolated tissue chamber. Separate fluorescence and electrophysiology studies were done with cultured human umbilical artery smooth muscle cells. Bupivacaine-evoked contractions of ring preparations were either tonic or twitch in nature. The contraction of ring preparations was dependent on extracellular Cal(2+) and sensitive to nifedipine inhibition. Bupivacaine also increased intracellular Cal(2+) in patterns consistent with tonic or phasic tension responses seen in isometric recordings. In addition, the membrane-resting potential was depolarized by bupivacaine. Since similar concentrations of bupivacaine caused both contraction and a rise in intracellular Ca(2+), the bupivacaine-evoked contraction was the result of increased cell Cal(2+) and the source of this Ca(2+) was the extracellular space.     

Monocyte chemoattractant protein-1 production by human detrusor smooth muscle cells

PURPOSE: Most recently attention has turned to the secretory properties of smooth muscle cells. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that causes mast cells recruitment and provokes mast cells activation in vitro. We investigated whether MCP-1 is produced by human detrusor smooth muscle cells (HDSMCs) cultured under inflammatory conditions. MATERIALS AND METHODS: Using an explantation technique HDSMCs were isolated and short-term cultured. HDSMCs were incubated at 37C for 24 hours with the proinflammatory mediators interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), lipopolysaccharide, histamine, leukotriene D4 and prostaglandin E2. The level of MCP-1 in cell supernatants were measured by enzyme linked immunoassay. RESULTS: There was basal secretion of MCP-1 in unstimulated cultures. Following 24-hour incubation with IL-1 beta or TNF-alpha (1 pg/ml to 100 ng/ml) the level of MCP-1 increased in a dose dependent manner. IL-1 beta was more potent at inducing MCP-1 release in 8 of 10 experiments. Lipopolysaccharide (10 microg/ml), histamine (100 microM), leukotriene D4 (50 nM), prostaglandin E2 (1 microM) and KCl (30 to 100 mM) failed to induce MCP-1 production. When IL-1 beta (10 ng/ml) and TNF-alpha (10 ng/ml) were given in combination, a highly synergistic effect on MCP-1 production was observed. CONCLUSIONS: To our knowledge this study shows for the first time that human detrusor smooth muscle cells cultivated under inflammatory conditions produce significant amounts of MCP-1. In addition to contractile function, HDSMCs have synthesis and secretory potential with the release of MCP-1. Thus, MCP-1 may contribute to the local inflammatory process by producing proinflammatory mediators. The release of cytokines and chemokines by human detrusor muscle even in small amounts may have important functional consequences.     

Effects of verapamil and sodium nitroprusside on acetylcholine-induced contraction of the rabbit detrusor muscle

Summary Effects of extracellular and intracellular Ca²⁺ on acetylcholine-induced contraction of the bladder detrusor muscle were studied in vitro, utilizing two types of Ca²⁺ antagonists of different mechanisms of action; verapamil and sodium nitroprusside (NP). Acetylcholine (10^-8 to 10^-2 M) caused dose-dependent contractions of the detrusor muscle strips. Pretreatment of the strips with verapamil (10^-7, 10^-6 M) significantly inhibited the acetylcholine-induced contraction in a dose-dependent manner, whereas NP (10^-7 to 10^-5 M) failed so suppress the contraction. The contraction of the strips once elicited by acetylcholine (10^-6 M) could be completely relaxed by verapamil (10^-5 M) addition, but only incompletely by NP (10^-5, 10^-4 M). In Ca²⁺-free solution containing 0.01 mM EGTA, replenishment of Ca²⁺ (2.5 mM) to the medium caused contractions of the strips. Addition of acetylcholine (10^-6 M) to the medium enhanced the Ca²⁺-induced contration, which was significantly inhibited by pretreatment with verapamil (10^-6 M), but not affected by NP (10^-6 M). In Ca²⁺-free medium containing 0.1 mM EGTA, acetylcholine caused a slight degree of tension increase of the strips in a dosedependent fashion, at higher concentrations exceeding 10^-6 M. These results suggest that the detrusor muscle contraction induced by acetylcholine is mostly dependent of extracellular Ca²⁺ influx both in its initiation and maintenance. It is also supposed, however, that in tracellular Ca²⁺ fractions will partly participate in the acetylcholine-induced contration and possibly in its maintenance.     

Inhibition of human detrusor contraction by a urothelium derived factor

PURPOSE: Stimulating muscarinic receptors in pig bladder urothelium causes the release of a diffusable factor that inhibits contractions of the underlying detrusor muscle. We investigated whether the contractions of human detrusor strips elicited by the muscarinic agonist carbachol, electrical field stimulation, KCl or the neurokinin receptor agonist neurokinin A are affected by the urothelium. MATERIALS AND METHODS: Paired intact and urothelium denuded muscle strips were placed in modified gassed Tyrode's solution at 37C. Cumulative concentration-response curves to carbachol or KCl were constructed. In other tissues the strips were stimulated electrically (1 to 40 Hz) with trains of square wave pulses 20 seconds in duration at 5-minute intervals. RESULTS: Cholinergic contractions evoked by electrical field stimulation at 10 and 30 Hz or by carbachol were significantly inhibited in the presence of an intact urothelium. Contractions elicited by KCl and by 10 microM neurokinin A were not modified by the urothelium. The urothelium mediated inhibition of contractions induced by carbachol was not affected by 300 microM L-NG-nitroarginine, 1 microM ODQ (1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one), 1 microM propranolol or 5 microM indomethacin. CONCLUSIONS: Muscarinic agonists stimulate the release of an inhibitory factor from the human urothelium. The factor is distinct from nitric oxide and it persists in the presence beta-adrenoceptor blockade or cyclooxygenase inhibition.     

The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells

PURPOSE: We generated and characterized a convenient isolated cell model of human detrusor smooth muscle to understand mechanisms that may underlie detrusor instability and provide a suitable model to test potentially useful drugs. MATERIALS AND METHODS: The electrophysiological properties of freshly isolated detrusor smooth muscle cells from human and guinea pig biopsies were compared with those undergoing cell culture to document in detail the changes that occur during primary culture and subsequent passages as well as the differences in the 2 species. RESULTS: Resting electrical characteristics were changed in the cultured cells. Membrane potential was less negative (guinea pig -59 versus -42 mV.) and membrane resistance was less (138 versus 124.5 Omegacm.(2)). Regenerative action potentials were recorded in cultured and freshly isolated cells. In guinea pig cells the overall duration and initial rate of depolarization (upstroke) was slower in cultured than in freshly isolated cells, indicative of a decreased magnitude of ionic current in cultured cells. Human cells had a similar prolongation in culture but no decrease in the upstroke rate. Experiments with selective blockers indicated that depolarization is due to influx through L-type Ca2+ channels and repolarization occurred via Ca2+ dependent K+ channels in freshly isolated and cultured cells. No further changes to properties were observed in cells passaged up to 3 times from primary cultured cells. CONCLUSIONS: Cell culture qualitatively preserves the electrophysiological properties of detrusor smooth muscle cells, although there is some decrease in channel density.     

Innervation of the detrusor muscle bundle in neurogenic detrusor overactivity

OBJECTIVE: To evaluate the peripheral anatomical distribution of innervation within muscle bundles of the detrusor and the changes arising in neurogenic detrusor overactivity (DO). PATIENTS AND METHODS: Full-thickness samples from the bladder dome of three cadaveric transplant organ donors and four people with neurogenic DO caused by spinal cord injury were compared. Systematic serial cryostat sections were stained using Masson trichrome and elastin techniques, and vimentin immunohistochemistry. A coherent image stack was generated for three-dimensional image reconstructions, which were displayed using mixed rendering (i.e. differing graphics for separate tissue components) to show peri- and intra-bundle innervation against the muscle fascicle framework. RESULTS: Control specimens had a dense nerve supply. Muscle bundle innervation was derived by dichotomous branching from peri-bundle nerve trunks in the inter-bundle connective tissue. Transverse interfascicular branches entered bundles perpendicular to the long axis at the midpoint of the bundle. They gave rise to axial interfascicular branches, which distributed to the pre-terminal and terminal nerve fibres. All samples from patients with neurogenic DO had patchy denervation. The primary deficit was predominantly at the level of the terminal axial innervation and was cross-sectionally consistent along the longitudinal axis of the muscle bundle. CONCLUSION: Patchy denervation may reflect a deficit at the level of the peripheral ganglia. Any contraction in the areas of denervation either occurs out of co-ordination with the rest of the bladder, or is co-ordinated by means of non-neural structures. The observation of fine muscle strands running between fascicles, and connective tissue anchoring structures, represent two hypothetical mechanisms by which such co-ordination might be effected.     

The control of muscle contraction

Skeletal muscle is also known as voluntary muscle and, as its name implies, it is under volitional control, unlike smooth or cardiac muscle. Adult skeletal muscle fibres are each innervated by a single branch of the axon arising from an α-motoneuron in the spinal cord. The α-motoneuron and all the fibres it innervates constitute a motor unit, and this is the functional unit of the muscle. α-Motoneurons differ in size and excitability and it is the recruitment of these cell bodies in the spinal cord that determines which fibres within the muscle are active during a movement. The axonal branches generally make contact with their muscle fibre about halfway along its length, this being the ‘motor point’ at which the muscle can most easily be stimulated by an external electrical stimulus during muscle testing. Correct functioning of the neuromuscular junction is clearly critical for muscle action and it is a site at which many drugs affecting muscle have their action.