Voiding and the sacral reflex arc: lessons from capsaicin instillation.
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Afferent Pathways↗
Biomedical subjects
C H Fry
Publications and source records attributed to C H Fry.
Intracellular sodium and contractile function in hypertrophied human and guinea-pig myocardium.
We tested the hypothesis that in left ventricular myocardial hypertrophy (LVH) the positive staircase effect is impaired and is related to a raised intracellular [Na+] ([Na+]i). Human myocardial specimens were obtained from patients undergoing mitral and aortic valve surgery, the latter group had LVH. LVH was induced in guinea-pigs by ascending aortic constriction. The extent of hypertrophy was quantified by measuring myocyte cross-section area, echocardiographic mass (in humans) and heart-to-body weight ratio (in guinea-pigs). The response to increasing stimulation frequency was expressed as the ratio of tension generated at 1.6 and 0.8 Hz (T1.6/0.8); ratios greater and less than 1.0 equate with positive and negative force/frequency relationships respectively. [Na+]i was measured using ion-selective microelectrodes. In human and guinea-pig myocardium T1.6/0.8 values decreased and [Na+]i increased with hypertrophy. For guinea-pig myocardium T1.6/0.8 decreased from 1.39 +/- 0.05 to 1.02 +/- 0.05 and [Na+]i increased from 7.3 +/- 1.4 to 12.1 +/- 1.3 mM in LVH. There was a close relationship between the reduction of T1.6/0.8 and increase of [Na+]i which was also observed when the [Na+]i was increased with strophanthidin in normal myocardium. The recovery of a raised [Na+]i after an acute acidosis was slowed in hypertrophied myocardium and stabilised at a higher level, suggesting that the membrane mechanisms that regulate [Na+]i are reset.
Acidosis↗
Reduced anisotropy of action potential conduction in left ventricular hypertrophy.
INTRODUCTION: The aim of this study was to determine if anisotropic action potential conduction was altered during development of left ventricular hypertrophy (LVH). METHODS AND RESULTS: Isolated guinea pig left ventricular preparations from hearts that had developed LVH were used to measure conduction velocity in longitudinal and transverse orientations to the fiber direction. A variable degree of LVH was induced by placing a ring around the ascending aorta for 50 to 250 days. Results were compared with an age-matched control group that underwent a similar operation but with no ring placement. LVH was measured as the heart-to-body-weight ratio (HBR), which correlated with an increase of mean myocyte cross-sectional area. Longitudinal conduction velocity (LCV) declined progressively as HBR increased (mean +/- SD: sham vs LVH: HBR 3.74 +/- 0.30 g/kg vs 4.53 +/- 0.52 g/kg; LCV 72.8 +/- 15.5 vs 63.6 +/- 11.1 cm/sec). Mean transverse conduction velocity (TCV) was greater in LVH compared with control (20.5 +/- 4.7 cm/sec vs 25.4 +/- 8.1 cm/sec), but there was no significance in the trend as a function of HBR. The anisotropic ratio (LCV/TCV) significantly declined as HBR increased. The time constant of the foot of the action potential was smaller in the transverse compared with the longitudinal dimension. There was no influence of hypertrophy. CONCLUSION: The decrease of LCV and reduction of the anisotropic conduction ratio suggest remodeling of the tissue in LVH. The consequences for the generation of arrhythmias are discussed.
Action Potentials↗
The action of isoprenaline on the electrophysiological properties of hypertrophied left ventricular myocytes.
The electrophysiological effects of the beta-agonist, isoprenaline, on hypertrophied left ventricular myocardium were measured to understand better the arrhythmic effects of beta-stimulation on the hypertrophied heart. Left ventricular hypertrophy was induced in guinea-pigs by constriction of the thoracic aorta. An age-matched sham-operated group served as controls. Isolated myocytes were held under voltage- and current clamp and the effect of isoprenaline on the L-type Ca2+ current, I(Ca), a Cl- current, I(Cl), and action potential morphology were measured. Cardiac growth was mirrored by cellular hypertrophy. I(Ca) and I(Cl) current density were reduced as myocyte hypertrophy progressed. The augmentation of I(Ca) and I(Cl) by isoprenaline was also reduced in hypertrophy, but no other characteristics of the two currents, or the dose-dependency of the action of isoprenaline were a function of cardiac growth. Isoprenaline prolonged the action potential, but to a smaller extent in hypertrophied myocytes. This difference in action potential prolongation was abolished by glibenclamide. The changes to I(Ca) and I(Cl) in hypertrophy would not tend to increase triggered activity in this situation. Under maximum inotropic stimulation hypertrophied myocytes show action potential changes which are consistent with intracellular ATP depletion, and which could enhance the likelihood of re-entrant circuits. A simple diffusion model for oxygen is constructed to demonstrate the possibility of cellular hypoxia in hypertrophied myocytes.
Action Potentials↗
Inward calcium currents in cultured and freshly isolated detrusor muscle cells: evidence of a T-type calcium current.
PURPOSE: We carefully examined the possible routes of Ca2+ influx, and determined whether cultured cells retain Ca2+ channels and whether the culturing process changes their properties. MATERIALS AND METHODS: Inward currents were measured under voltage clamp in freshly isolated cells and myocytes from confluent cell cultures of detrusor smooth muscle. RESULTS: In guinea pig and human cells mean peak inward current density plus or minus standard deviation decreased significantly in cell culture (2.0 +/- 0.9 versus 4.5 +/- 2.2 pA.pF.(-1)) but there was no species variation. In primary cultured and passaged guinea pig cells an inward current was identified as L-type Ca2+ current. In freshly isolated cells another component to the inward current was identified that was insensitive to 20 micromol. l(-1) verapamil and 20 to 50 micromol. l(-1) cadmium chloride but abolished by 100 micromol. l(-1) nickel chloride and identified as T-type Ca2+ current. In addition, total inward current was greater at a holding potential of -100 than -40 mV., also indicating a component of current activated at negative voltage. Steady state activation and inactivation curves of the net inward current were also compatible with a single component in cultured cells but a dual component in freshly isolated cells. The action potential was completely abolished in cultured cells by L-type Ca2+ channel blockers but incompletely so in freshly isolated cells. Outward current depended strongly on previous inward current, suggesting a predominant Ca2+ dependent outward current. CONCLUSIONS: In freshly isolated guinea pig cells T and L-type Ca2+ current is present but T-type current is absent in confluent cultures.
Animals↗
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.
Action Potentials↗
Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle.
The role of Na(+)/Ca(2+) exchange in regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in isolated smooth muscle cells from the guinea pig urinary bladder was investigated. Incremental reduction of extracellular Na(+) concentration resulted in a graded rise of [Ca(2+)](i); 50-100 microM strophanthidin also increased [Ca(2+)](i). A small outward current accompanied the rise of [Ca(2+)](i) in low-Na(+) solutions (17.1 +/- 1.8 pA in 29.4 mM Na(+)). The quantity of Ca(2+) influx through the exchanger was estimated from the charge carried by the outward current and was approximately 30 times that which is necessary to account for the rise of [Ca(2+)](i), after correction was made for intracellular Ca(2+) buffering. Ca(2+) influx through the exchanger was able to load intracellular Ca(2+) stores. It is concluded that the level of resting [Ca(2+)](i) is not determined by the exchanger, and under resting conditions (membrane potential -50 to -60 mV), there is little net flux through the exchanger. However, a small rise of intracellular Na(+) concentration would be sufficient to generate significant net Ca(2+) influx.
Animals↗
A new approach to recording the electromyographic activity of detrusor smooth muscle.
PURPOSE: Extracellular recordings from the whole intact mammalian bladder of the electrical events leading to contraction of the organ have been elusive for almost 50 years despite the widespread potential applications of such a technique. The principal problem is the need to isolate the small real signals reflecting membrane depolarization from the large electromechanical artifact generated as the organ contracts. In this preliminary study we determined whether electrical signals may be isolated and verified as biological using extracellular bipolar reversible suction electrodes. MATERIALS AND METHODS: Six whole excised guinea pig bladders were mounted in an especially constructed organ bath. Electrical activity resulting from nerve stimulation of the organ was recorded using a novel 10 bipolar Pt/PtCl suction electrode simultaneously with changes in intravesical pressure. Mechanical and pharmacological control experiments were performed to determine the true origin of these signals. RESULTS: A predominantly biphasic electrical signal of a mean amplitude plus or minus standard deviation of 647 plus or minus 301 microV. and a mean duration of 293 plus or minus 51 milliseconds was consistently recorded from the serosal surface of all guinea pig bladders. In all cases the electrical signal and mechanical response to stimulation were completely abolished by 1 microM. tetrodotoxin. The signal always preceded any change in intravesical pressure. It was sensitive to changes in the CaCl2 concentration of the superfusate, abolished by purinergic but not cholinergic neuromuscular blockade and independent of electromechanical artifact. CONCLUSIONS: In this preliminary report we describe a novel technique by which nerve mediated detrusor electrical activity leading to contraction of the whole intact guinea pig bladder may be isolated from artifact and verified as real. We hope that development of this technique may enable its application to the in situ human bladder. However, to our knowledge whether electromyographic activity may be recorded from human detrusor remains to be determined.
Animals↗
The actions of metabolic inhibition on human detrusor smooth muscle contractility from stable and unstable bladders.
OBJECTIVES: To determine the important cellular site(s) of action of a brief exposure to NaCN (chosen to reduce mitochondrial respiration and hence mimic cellular hypoxia) on the mechanical properties and regulation of intracellular [Ca2+] in human detrusor smooth muscle. Using muscle samples obtained from patients with stable and unstable bladders, to determine whether the unstable bladder is associated with changes in the functional properties of detrusor muscle under these circumstances. Materials and methods Experiments were conducted in vitro on muscle strips or isolated cells. Isometric tension was recorded in muscle strips during electrical stimulation or exposure to agonists. Intracellular [Ca2+] and [H+] were measured by epifluorescence microscopy, and cell autofluorescence measured as an index of mitochondrial function. RESULTS: There were no differences in the responses to electrical stimulation and varying concentrations of carbachol in muscle strips from stable and unstable bladders. NaCN (2 mmol/L) reduced the contraction induced by carbachol (10 micromol/L) by a mean (SD) of 43 (16)% and 56 (15)% in the two groups; the reduction in the unstable was significantly less than in the stable group. NaCN similarly reduced the response to 10 mmol/L caffeine, but had no effect on the KCl-induced contraction. NaCN significantly increased the resting sarcoplasmic [Ca2+] and attenuated the calcium transients evoked by carbachol and caffeine, but again had no effect on the KCl-induced transient. The reduction of the carbachol calcium transient was also less in cells from unstable bladders than in those from stable bladders. There was no effect of NaCN on intracellular pH, except for a brief, transient alkalosis. CONCLUSIONS: NaCN reduces both the contraction and Ca-transient to carbachol by reducing Ca2+ accumulation by intracellular stores, because the carbachol- and caffeine-evoked responses were similar. Any effect on transmembrane Ca2+ flux was minimal because there was no effect on KCl-induced responses. The greater resilience of tissue from unstable bladders to acute cellular hypoxia may reflect some adaptation acquired in vivo.
Adult↗
The actions of altered osmolarity on guinea-pig detrusor smooth muscle contractility and intracellular calcium.
The study measured the effects in vitro of changing extracellular osmolarity on the contractility of detrusor smooth muscle strips. The data were interpreted in the context of separate measurements from isolated cells of alterations to the intracellular [Ca2+], [Ca2+]i. Increased osmolarity (300-700 mosmol l-1) reduced phasic contractions but increased resting tension regardless of whether sucrose, LiCl or NaCl were used as osmolytes. [Ca2+]i was decreased slightly only when NaCl increased osmolarity, otherwise it was unchanged. The contractile effects may be explained by tissue shrinkage and reduction of detrusor excitability. Lowered osmolarity (300-64 mosmol l-1) decreased phasic contractions but increased resting tension and [Ca2+]i. The raised resting tension was due solely to low osmolarity and was independent of changes to [Na], [Cl] or ionic strength. The rise of [Ca2+]i was due partly to Ca2+ influx through Na(+)-Ca2+ exchange but a fraction was independent of extracellular Ca, unaffected by Gd3+, and persisted in the presence of caffeine. By contrast, reduction of phasic tension was due mainly to the reduced ionic strength, not osmolarity. The results do not support the presence of functional stretch-activated channels and suggest only a minor role for Na(+)-Ca2+ exchange under these conditions. However, they do suggest an intracellular source of Ca2+, which is independent of the sarcoplasmic reticulum.
Animals↗
The physicochemical basis of urinary catheter encrustation.
OBJECTIVES: To determine the relationship between urinary pH and Ca2+ solubility in urine samples from patients who experienced either frequent ('blockers') or infrequent ('nonblockers') catheter blockage by crystalline deposits of divalent cation salts. MATERIALS AND METHODS: Fresh urine samples from 'blockers' and 'nonblockers' were collected and the ionic calcium concentration ([Ca2+ ]) measured using a Ca2+-selective electrode whilst the urinary pH was increased in 0.25 increments between 4.75 and 9.00. The pH at which crystallization occurred (nucleation) was determined and crystal composition analysed. RESULTS: The mean (sd) voided urinary pH of catheter 'blockers' was significantly more alkaline than that from 'nonblockers', at 7.63 (0.64) and 5.97 (0.80), respectively (P=0. 001). The nucleation pH of catheter 'blockers' was significantly more acid than in 'nonblockers', at 7.43 (0.73) and 6.45 (0.65), respectively (P=0.005). Urine from 'blockers' had significantly more Ca phosphate and Mg ammonium phosphate crystals. 'Blockers' were further divided into two subsets with and without urease-based urinary tract infection; both showed a decrease in the nucleation pH. CONCLUSION: In the urine from 'nonblockers' there is a wide safety margin between voided and nucleation pHs; this margin was less in the urine from 'blockers'. This reduction in the safety margin arises partly because the voided pH in those with a urinary tract infection is more alkaline. However, the decrease in the nucleation pH also suggests that a fundamental property of urine is altered, which reduces Ca2+ solubility at more neutral pH values. The long-term goal is to increase the nucleation pH of catheter 'blockers' and increase the margin of safety.
Calcium↗
Bladder instability and detrusor smooth muscle function.
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Animals↗
A quantitative study of atropine-resistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders.
PURPOSE: The objective of the study was to quantify in vitro the magnitude of atropine-resistant contractions using human detrusor samples and to determine the cellular processes underlying these contractions. MATERIALS AND METHODS: Isometric contractile responses were measured in isolated strips of human detrusor muscle obtained from patients with i) stable, ii) unstable or iii) obstructed bladders. Preparations were electrically stimulated or exposed to carbachol and ATP in the superfusate. RESULTS: Force-frequency curves were shifted to the right in samples from unstable and obstructed bladders. These same tissue groups also showed significant atropine-resistant contractions which were abolished by the neurotoxin TTX, or the non-hydrolysable ATP analog, alpha,beta-methylene ATP, suggesting that these contractions were mediated by neurally released ATP. Sub-division of the patient group with unstable bladders demonstrated that those with neuropathic instability did not show atropine-resistance, whereas those with idiopathic instability or secondary instability after obstruction did show atropine-resistant contractions. The potency of carbachol in generating a contracture was significantly greater than ATP (mean EC50 0.65 microM and 151 microM respectively) however, for each agonist there was no difference in potency between the three patient groups. Direct muscle excitability was similar in all three patient groups. CONCLUSIONS: It is concluded that purinergic, atropine-resistant contractions are present in some types of dysfunctional bladder, and these are not caused by a differential sensitivity of the muscle to ATP and cholinergic agonists.
Adenosine Triphosphate↗
A comparison of the mode of action of ATP and carbachol on isolated human detrusor smooth muscle.
PURPOSE: The objectives of the study were: i) to examine the ability of carbachol and ATP to raise intracellular [Ca2+] in isolated detrusor myocytes; ii) to determine the origin of the intracellular Ca2+ and iii) to address the question of whether the appearance of purinergic contractions in detrusor from unstable and obstructed human bladders is reflected in the sensitivity of the cell to the two agonists. MATERIALS AND METHODS: Intracellular Ca2+ transients generated by extracellular ATP and carbachol were recorded from isolated human detrusor myocytes. Cells were dissociated by collagenase disruption of the biopsy. Intracellular Ca2+ was measured by epifluorescence microscopy using Fura-2 and electrophysiological recordings were made with patch electrodes. RESULTS: In cells from stable bladder biopsies the half-maximal concentrations (EC50) for ATP and carbachol to generate Ca2+ transients were 0.10 and 0.25 microM respectively. With cells from unstable bladders the EC50 values for both agonists and the magnitude of the Ca2+ transients were not significantly different from those obtained in cells from normal bladders. The transient in ATP was preceded by a transient depolarisation generated by a large inward current. The carbachol-Ca2+ transient was independent of changes to membrane potential, except in a subset of cells where complex membrane potential changes followed the rise of intracellular Ca2+. The ATP-Ca2+ transient was partially abolished by nicardipine and completely abolished by zero-Ca solutions, the carbachol-Ca2+ transient was unaffected by nicardipine and less completely attenuated by zero-Ca solutions. Prior exposure to caffeine suggested that the carbachol-Ca2+ transient, but not the ATP-Ca2+ transient, originated from intracellular stores. CONCLUSIONS: It is concluded that both agonists are equipotent in increasing intracellular Ca2+, but by different routes. The generation of purinergic contractions in detrusor from unstable bladder is not due to altered sensitivities of the detrusor myocyte to ATP or cholinergic agonists.
Adenosine Triphosphate↗
Visco-elastic properties of isolated detrusor smooth muscle.
The generation of force by the contractile apparatus and the modulation of that force by the intra- and extracellular matrix are the initial steps in the production of bladder wall tension. The biomechanical components contributing to the observed rise in bladder wall tension may be studied in isolated detrusor tissue and attempts can be made to isolate these. The problem is to determine whether clinically observed alterations in detrusor function are due to changes in the contractile apparatus or in the surrounding matrix. This review discusses the viscoelastic properties of detrusor muscle, concentrates upon the influence of bladder outflow obstruction on the mechanical properties of the detrusor in an attempt to understand changes in contractile function.
Animals↗
Measurement of intercellular electrical coupling in guinea-pig detrusor smooth muscle.
PURPOSE: The electrical impedance of detrusor smooth muscle strips to alternating current has been measured to calculate the resistance of the intracellular pathway, in particular gap junction resistance. Values have been compared with myocardium, which is electrically well-coupled. MATERIALS AND METHODS: Alternating current was passed along the intracellular pathway of muscle strips by creating a high extracellular resistance around the preparation. The data were analyzed in terms of an equivalent circuit consisting of an intracellular and extracellular pathway. RESULTS: Intracellular resistance was divided into two series components, a cytoplasmic resistance and a gap junction resistance. Detrusor intracellular resistance was about three times that of myocardium. The greater value was attributed to a larger gap-junction resistance. Superfusion of detrusor strips with an isosmotic solution of 50% sucrose, 50% Tyrode's increased both cytoplasm and gap junction resistances. CONCLUSIONS: Gap-junction resistance is larger in detrusor compared with myocardium. However, significant electrical current can still pass between adjacent detrusor cells. Calculation of the space constant however shows that functionally detrusor is electrically well-coupled because of the high membrane resistance. The functional consequences of these findings are discussed.
Animals↗
The effects of extracellular and intracellular pH on intracellular Ca2+ regulation in guinea-pig detrusor smooth muscle.
1. Intracellular pH (pHi) and intracellular [Ca2+] ([Ca2+]i) were measured during changes to superfusate PCO2 and/or [NaHCO3]. Changes to superfusate PCO2 produced sustained changes to pHi and [Ca2+]i, while changes to [NaHCO3] altered only extracellular pH (pHo). 2. Carbachol or caffeine induced a transient rise of [Ca2+]i due to Ca2+ release from an intracellular store. This Ca2+ transient was reduced by extracellular acidosis, but increased by intracellular acidosis. Alkalosis in either compartment produced opposite effects to acidosis. Changes to the Ca2+ transient mirrored those to phasic tension previously reported in this preparation. 3. A raised superfusate [K+] also induced a Ca2+ transient, due to transmembrane influx of Ca2+. This transient was depressed by extracellular acidosis, but unaffected by changes to pHi. The L-type Ca2+ current was similarly affected by changes to pHo, but not by alteration of pHi. 4. The results suggest that extracellular acidosis depresses the Ca2+ transient by reducing transmembrane influx through the L-type Ca2+ channel. The increase in the carbachol- and caffeine-induced Ca2+ transients by intracellular acidosis is due to enhancement of Ca2+ uptake into intracellular stores as a result of a raised resting [Ca2+]i.
Animals↗
Electrophysiological properties of the bladder.
The electrophysiological properties of detrusor smooth muscle are described, in particular with regard to their influence on the contractile properties of the tissue. The Ca2+ and K+ channel activities are most important in generating action potentials, but the role of several other ionic currents is described, including Cl-, Ca2+-activated, stretch-activated and ligand-gated channels. The variable appearance and functions of different ionic currents in disease states is discussed, as well as the question of whether electrical activity can transmit between adjacent smooth muscle cells. In addition, the precise role that electrophysiological phenomena play in the regulation of the contractile state of the smooth muscle cells, as well as the generation of bladder electromyograms, is discussed.
Action Potentials↗