Spontaneous activity and electrical coupling in human detrusor smooth muscle: implications for detrusor overactivity?

SPONTANEOUS ACTIVITY AND ELECTRICAL COUPLING IN HUMAN DETRUSOR SMOOTH MUSCLE: IMPLICATIONS FOR DETRUSOR OVERACTIVITY? CHRISTOPHER H. FRY, GUI-PING SUI, NICHOLAS J. SEVERS,

AND

CHANGHAO WU

ABSTRACT Large uncontrollable detrusor contractions, decreased compliance that increases luminal pressures during filling, and detrusor underactivity are all examples of abnormal bladder function. Studies of the nervous control of lower urinary tract function and measurement of the cellular properties of the component tissues of the bladder wall have been performed to deepen our knowledge of these problems. The resultant data have suggested that lower urinary tract smooth muscle should not be regarded solely as a collection of independent cellular contractile units that are each activated by separate neural inputs, but also as a syncytium of cells. Although this syncytial arrangement may not be as well developed as in other tissues, it should impose a new layer of activity that will affect overall bladder function. Recent studies have addressed this issue through investigation of spontaneous contractile activity, the cellular basis of syncytial function, and their normal and abnormal functional consequences. The results suggest that individual detrusor cells possess membrane properties that may lead to spontaneous activity fluctuations, which can affect adjacent cells and, thus, produce multicellular aberrant responses. It remains unclear whether these responses manifest themselves as dysfunctional activity in the whole bladder defects because the extent of local multicellular abnormalities is not known at present. The data do imply that myogenic defects can contribute to abnormal bladder function and, thus, suggest several new targeted drug models that should be explored. UROLOGY 63 (Suppl 3A): 3–10, 2004. © 2004 Elsevier Inc.

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bnormal bladder mechanical function may manifest itself in a number of ways, including large involuntary detrusor contractions associated with the overactive bladder, a reduction in compliance that increases luminal pressures during filling, and detrusor underactivity. There are 2 predominant approaches that have been pursued to understand the bases of these problems: (1) studies of neural control of lower urinary tract function, and (2) measurement of the cellular properties of the component tissues of the bladder wall—principally, the detrusor smooth muscle. These approaches are not mutually exclusive in the search for a cause for bladder dysfunction, and in many cases, a combination of abnormal nervous system From the Institute of Urology & Nephrology, London, United Kingdom; and National Heart & Lung Institute, Faculty of Medicine, Imperial College, London, United Kingdom The work described in this review was performed during tenure of a joint grant from the Medical Research Council, London, United Kingdom; and Pfizer Ltd, Sandwich, United Kingdom Reprint requests: Christopher H. Fry, DSc, Institute of Urology & Nephrology, 48 Riding House Street, London W1W 7EY, United Kingdom. E-mail: [email protected] © 2004 ELSEVIER INC. ALL RIGHTS RESERVED

function and smooth muscle contractile activity will be present. Studies of smooth muscle function are often “reductionist” in that all muscle cells are considered as essentially similar, so that regulating the activity of an individual cell will extrapolate to manipulating the function of the whole organ regardless of multicellular factors that also may modulate organ function. Conversely, an investigation of nervous control of the lower urinary tract may consider the muscular end organs as “black boxes” and ignore local interactions within the smooth muscle mass, which may influence the outcome of nervous system activity. Evidence is accumulating, however, that lower urinary tract smooth muscle—particularly detrusor smooth muscle— should not be regarded solely as a collection of independent cellular contractile units each activated by separate neural inputs but also as a syncytium of cells. Although this syncytial arrangement may not be as well developed as in tissues (for example, myocardium), such a property should impose a new layer of activity that will determine overall function of the bladder. The present review 0090-4295/04/$30.00 doi:10.1016/j.urology.2003.11.005 3

attempts to address this concept by investigating such phenomena as spontaneous contractile activity, the cellular basis of syncytial function, and some normal and abnormal functional consequences. SPONTANEOUS CONTRACTILE ACTIVITY IN THE DETRUSOR Spontaneous contractions are observed in vitro from detrusor preparations1 and in vivo from cystometrograms of animal and human bladders.2,3 Such activity is increased in vitro using human tissue from patients with detrusor overactivity,4,5 and in in vivo measurements in animal models of detrusor overactivity caused by outflow tract obstruction.2,6 –9 It has been proposed that such contractions contribute to detrusor overactivity in patients with overactive bladders, but it remains to be unequivocally shown that in vitro spontaneous activity is related to whole bladder phenomena. However, conditions that increase spontaneous activity in isolated preparations also enhance bladder wall stiffness.10 This would raise background wall tension so that superimposed spontaneous contractions would more readily manifest themselves as significant uncoordinated bladder activity. Spontaneous contractions are caused by factors different from those that initiate nerve-mediated responses caused by activation by motor nerves. These contractions are insensitive to neurotoxins, but they are attenuated by increased extracellular Mg2⫹ and by Ca2⫹ antagonists.11,12 In addition, spontaneous contractions occur in decentralized whole bladders and are enhanced in animal models of outflow obstruction that show bladder overactivity.6 These observations therefore support the hypothesis that the origin of spontaneous contractions is myogenic. Furthermore, agents that modulate spontaneous activity also affect bladder function. For example, adenosine triphosphate (ATP)– dependent K⫹ channel openers, such as pinacidil and cromakalim, effectively suppress detrusor overactivity,7 whereas Ca2⫹ antagonists, such as nifedipine, have been found to inhibit detrusor overactivity in a rat model of outflow obstruction.13 In addition, intravesical instillation of the cholinergic antagonist atropine is effective in ⬎40% of neuropathic detrusor overactivity but in ⬍20% of idiopathic instability.14 This also suggests a distinct nonneural, myogenic process contributing to idiopathic detrusor overactivity. Ca2ⴙ CHANNELS, CELLULAR Ca2ⴙ HOMEOSTASIS, AND OSCILLATORY MEMBRANE BEHAVIOR The detrusor cell has a high density of Ca2⫹ channels. An inward flux of Ca2⫹, however, does 4

not appear to play a significant role in the initiation of contractions—at least in human detrusor muscle from bladders without evidence of detrusor overactivity— because this is mediated largely by acetylcholine and release of Ca2⫹ from intracellular stores.15 It has been proposed that membrane Ca2⫹ channels are responsible for maintaining the Ca2⫹ content of these stores through a membrane feedback mechanism with K⫹ channels (Figure 1A). In this scheme, an increase of intracellular Ca2⫹ ([Ca2⫹]i) is associated with a brief membrane hyperpolarization, followed by a transient reduction of [Ca2⫹]i to a value below the resting level accompanied by depolarization.16 In Figure 1B, accompanying voltage-clamp records show an increase of spontaneous outward currents during the Ca2⫹ release phase and a quiescence during the period when [Ca2⫹]i is low. By way of explanation, an increase of [Ca2⫹]i activates K⫹ channels— probably the so-called large conductance (BK) K⫹ channels—which explains the hyperpolarization and increase in outward current activity. The reduction in [Ca2⫹]i as Ca2⫹ is sequestered by intracellular stores and lost from the cell through membrane ion exchangers, such as sodium–Ca2⫹ exchange,17 reduces K⫹ channel activity and, hence, depolarizes the membrane. This scheme (by no means unique to detrusor muscle) can achieve a number of purposes. The hyperpolarization that accompanies the increase in [Ca2⫹]i further limits Ca2⫹ entry into the cell through Ca2⫹ channels by reducing their open-probability and thus prevents an excessive increase of intracellular Ca2⫹. Conversely, the depolarization associated with a low [Ca2⫹]i facilitates Ca2⫹ entry by increasing the open probability of Ca2⫹ channels, thus replenishing that which is lost from the cell. Such interplay between inward and outward currents may manifest itself as oscillatory changes of membrane potential if there is a significant temporal lag between the 2 components of this feedback system. The resting potential of an isolated detrusor cell exhibits constant fluctuations over a considerable range of membrane potentials (Figure 2). Moreover, such fluctuations are not completely random. Analysis of these fluctuations by Fast Fourier Transforms (PC Clamp 8, Axon Instruments, Union City, CA) shows that they occur at a preferential frequency in any given cell and provides further evidence for a cyclical feedback process. However, there are many questions that remain. Which ion channels in particular contribute to this oscillatory activity? Could such behavior contribute to spontaneous mechanical activity in detrusor tissue? Could such activity contribute to abnormal detrusor function? Both L- and T-type Ca2⫹ channels are present in isolated guinea pig and human detrusor cells. The UROLOGY 63 (Supplement 3A), March 2004

FIGURE 1. Membrane feedback mechanism between inward and outward currents. (A) Simultaneous recording of intracellular Ca2⫹ ([Ca2⫹]i) and membrane potential (Em) in an isolated human detrusor myocyte during exposure to caffeine 10 mmol/L during the period indicated. The scheme on the right illustrates the proposed interrelation between inward and outward currents. (B) A similar experiment in which [Ca2⫹]i and membrane current are recorded simultaneously on exposure to caffeine 10 mmol/L; the cell was voltage-clamped at ⫺60 mV throughout. (Reprinted with permission from J Physiol.16)

FIGURE 2. Spontaneous fluctuations of membrane potential. Membrane potential was recorded in an isolated human detrusor myocyte with a patch electrode filled with a high-potassium solution designed to mimic composition of the intracellular compartment. (Right) Vertical bars show activation ranges of the T- and L-type Ca2⫹ currents. UROLOGY 63 (Supplement 3A), March 2004

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demonstration of T-type channels is significant because they are activated over a more negative range of membrane potentials than are L-type channels. Figure 2 shows the activation ranges for the 2 currents with respect to the actual ranges of membrane potentials recorded. Thus, if membrane potential oscillations are due to interplay between inward Ca2⫹ current and outward K⫹ current, the involvement of T-type Ca2⫹ channels could be significant to the maintenance of this phenomenon. This possibility is strengthened by the type of observation made in Figure 3A, which shows a voltage-clamp record from a detrusor cell depolarized to ⫺40 mV, which is too negative to activate L-type Ca2⫹ current, but sufficient to activate T-type current. In these records, the small inward current is followed by transient outward currents, demonstrating that T-type channels, at least in part, are able to contribute to this suggested oscillatory mechanism. It may be proposed that activation of T-type Ca2⫹ channels at more negative potentials can depolarize the cell sufficiently to activate L-type Ca2⫹ channels and that both components will subsequently activate outward currents. It should be stressed that although the larger Ltype Ca2⫹ current eventually dominates any such oscillatory processes, smaller T-type Ca2⫹ currents have an important role in initiating such events. If such a scheme contributes to altered mechanical activity in the form of spontaneous contractions, modulation of T-type channel activity offers a route to manipulate spontaneous detrusor activity. This may offer a novel route to alter detrusor function in a more targeted direction. This is, of course, provided that an effect relatively specific to the bladder (for instance, over the cardiovascular system) may be devised. Low Ni2⫹ concentrations may differentiate between L- and T-type Ca2⫹ channel activity (Figure 3B). NiCl2 (100 ␮mol/L) effectively abolishes inward current elicited at moderate depolarizations when L-type current would not be activated. At more positive potentials when the larger L-type Ca2⫹ current is activated, conventional L-type antagonists, such as verapamil and nifedipine, attenuate the inward current. Low Ni2⫹, therefore, offers a means to investigate the role of T-type Ca2⫹ channels in determining detrusor function. Such concentrations of Ni2⫹ have been shown18 to attenuate the frequency and magnitude of spontaneous mechanical activity in isolated preparations (Figure 3C). A future investigative step would be to determine whether such involvement of T-type Ca2⫹ channel activity can explain increased detrusor activity in detrusor overactivity. 6

INTERCELLULAR COMMUNICATION AND GAP JUNCTIONS Although the study of isolated smooth muscle cells reveals much about the mechanisms that regulate their electromechanical functions, measurement of intercellular interactions is necessary to understand more effectively the corresponding properties of multicellular preparations and possibly even that of the whole organ. The detrusor is arranged into muscle bundles, and it has been speculated that there are larger functional units.19 If this proves to be the case, a degree of coordination would be expected between component cells. This is, in part, because of coordinated nervous system excitation of individual muscle cells, but the ability of muscle cells to act as a functional syncytium would assist such coordination. Furthermore, if the syncytial structure were to become deranged, it could contribute to abnormal detrusor activity on a large scale. There has been much discussion in the past as to whether detrusor smooth muscle cells are functionally connected. Although mechanical adherens junctions may be demonstrated in the detrusor, connection of the sarcoplasmic compartments of adjacent cells to form a functional syncytium could only be via gap junctions. However, most electron microscopical studies have not demonstrated gap junctions in detrusor muscle from normally functioning bladders.20 This has led to the proposal that gap junctions might facilitate the generation of detrusor overactivity. Results of electrophysiologic experiments using microelectrodes are in accordance with electron microscopical data showing that current injection into a cell through an microelectrode could not be measured over a distance of a few cell lengths, which implies that current could not spread from cell to cell.21 However, electrophysiologic methods that used large intracellular currents provided support for intercellular electrical communication, although the degree of coupling was significantly less than that in well-coupled tissues, such as myocardium.22,23 The discrepancy between the different experimental approaches was therefore technical and suggested that the smaller currents passed from microelectrodes might be insufficient to readily cross the relatively poor electrical coupling between cells. Another approach to demonstrate the presence, or otherwise, of gap junctions in the detrusor is to locate connexins using either immunohistochemistry or blotting techniques. Connexins are the proteins that form the gap junction pore lining and are found as a number of subtypes. Those in gap junctions of well-coupled functional syncytia, such as ventricular myocardium, are of the subtype UROLOGY 63 (Supplement 3A), March 2004

FIGURE 3. Inward and outward currents and spontaneous activity in the human detrusor. (A) Ionic currents recorded during a step depolarization from a holding potential of ⫺80 to ⫺40 mV in an isolated human detrusor myocyte. High-potassium filling solution was used in the patch electrode. (B) Ionic currents recorded from a holding potential of ⫺80 to either ⫺30 or ⫹10 mV. In each panel, 2 superimposed traces are shown: the upper pair was obtained in the absence and presence of NiCl2 100 ␮mol/L, and the lower pair in the absence and presence of nifedipine 5 ␮mol/L. (C) The effect of NiCl2 100 ␮mol/L on the frequency (Freq) and magnitude of spontaneous contractions in isolated unstimulated guinea pig muscle strips.

connexin 43 (Cx43). Initial observations in bladder preparations were unable to localize Cx43 in detrusor bundles, except in a small number of sites UROLOGY 63 (Supplement 3A), March 2004

between bundles (Figure 4A). A strong signal, however, was found in a layer of suburothelial cells that were proposed to be myofibroblasts24 and 7

FIGURE 4. Identification of connexins in the human detrusor. Connexins are observed in the confocal sections (red areas), and collagen (green strands). (A) Connexin 43 in the human detrusor. Some labeling is identified (white arrowheads) mainly around the detrusor muscle bundle and sparse labeling (arrow) in the bundle. (B) Connexin 45 in the human detrusor is also identified (white arrows). The more numerous labels are shown within the muscle bundle. Scale: bars 25 ␮m. (Reprinted with permission from BJU Int.25)

hence was not a failure of the labeling technique. The sparse signal between the detrusor bundles also appears to originate from myofibroblasts or fibroblasts. However, another connexin subtype, connexin 45 (Cx45), was localized to the detrusor tissue itself25 and therefore suggests that gap junction coupling is possible between adjacent cells (Figure 4B). The magnitude of electrical coupling, however, would be expected to be less than in myocardium because (1) the unit conductance of gap junctions (ie, the ability to pass electrical current) formed by Cx45 is smaller than that of Cx43,26 and (2) the immunofluorescence spots of Cx45 were relatively small and infrequent, indicating equally small, sparse gap junctions. These observations are consistent with quantitative electrophysiologic estimates of gap junction conductance per unit volume in the detrusor versus myocardium, whereby the value in the detrusor is several times smaller than that in the myocardium.22 GAP JUNCTION COUPLING AND PATHOLOGIC IMPLICATIONS Previous electron microscopic studies proposed that gap junction coupling was enhanced in detrusor muscle from patients with detrusor overactiv8

ity.27 However, quantitative measurement of intercellular resistance—a measure of gap junction coupling resistance—suggests the opposite effect. Detrusor preparations from normal and overactive detrusor were subjected to an impedance analysis, whereby it is possible to measure the resistance of gap junctions in a unit amount of tissue. With tissue from overactive detrusor, gap junction coupling was reduced (ie, resistance was increased) by about 50% compared with a control group of tissue patients with normal detrusor function.25 The consequences of changes to gap junction resistance and the implications for abnormal detrusor and whole bladder function are issues worth considering. Detrusor muscle is an electrically active tissue, although in the human detrusor from normal bladders, action potentials are not responsible for the initiation of contraction as they are in striated muscles, for example. Ion channels can mediate intracellular Ca2⫹ regulation, and changes of membrane potential occur as regular fluctuations or small spikes; with the detrusor from pathologic bladders (as well as in most animal bladders), release of ATP as a secondary transmitter causes depolarization directly.28 The spread of electrical activity between contiguous cells, therefore, engenders particular properties to the tissue UROLOGY 63 (Supplement 3A), March 2004

FIGURE 5. Schematic representation of current flow in an electrically coupled system of cells. The diagrams show a linear array of several cells coupled by low, intermediate, and very high resistances in the 3 arrays. The dark arrow represents the flow of current in the intracellular space from a source on the left-hand side. The thickness of the arrow represents the current density in a particular cell, and the 2 parallel lines show the current density required to elicit regenerative electrical activity.

that complement cellular activity. The extent and velocity of the spread of electrical activity in a network of cells depends on the shape of the signal itself and the passive electrical properties of the tissue—in particular, the magnitude of gap junction resistance. A variety of conditions is illustrated in Figure 5, where gap junction resistance increases gradually. An increase in gap junction resistance limits the extent of electrical current flow in the network and thus reduces conduction velocity of a regenerative signal (an action potential) and decreases the extent of spread of other electrical activity. The mathematics of this biophysical treatment is known as cable theory and has been intensively studied.29 Perhaps less intuitively, an increase in gap junction resistance also tends to increase the excitability of tissue in the local region where such activity is initiated as the electrical current is dissipated less extensively. In cardiac muscle, it has been shown that this can lead to an increased likelihood of reentrant arrhythmias being initiated and sustained.30 In the detrusor, similar activity would also lead to local uncoordinated electrical and, hence, mechanical activity. The magnitude of changes to gap junction resistance, with respect to other electrical properties of myocardium, is crucial in determining whether reentrant pathways can be generated. Although extensive measurements have been obtained in myocardium, similar studies are absent in the detrusor; thus, the propensity of such phenomena to develop cannot be estimated at present. These recent observations, however, demonstrate 2 important facts: (1) detrusor tissue can behave as a functional syncytium so that electrical information can spread throughout a region of the smooth muscle mass, and (2) changes in the electrical properties of UROLOGY 63 (Supplement 3A), March 2004

detrusor occur in conditions associated with detrusor overactivity. It remains to be determined whether the extent of these changes contributes to abnormal detrusor mechanical function. CONCLUSION Abnormal detrusor activity is likely to have a number of causes that originate in defects to the cellular, multicellular, and nervous system mechanisms that determine normal function. In this review, it has been argued that individual detrusor cells have membrane properties that could produce spontaneous fluctuations of activity, which, if sufficiently large, may spread to adjacent cells and hence generate multicellular aberrant responses. Whether these responses result in whole bladder defects, however, remains to be answered because the extent of local multicellular abnormalities has not been determined. The important factor, however, is that there is evidence that myogenic defects can contribute on a macroscopic scale to abnormal detrusor function; this offers a number of novel targeted drug models that should be exploited. REFERENCES 1. Sibley GN: A comparison of spontaneous and nervemediated activity in bladder muscle from man, pig and rabbit. J Physiol 35: 431–443, 1984. 2. Persson K, Pandita RK, Waldeck K, et al: Angiotensin II and bladder obstruction in the rat: influence on hypertrophic growth and contractility. Am J Physiol 271: R1186 –R1192, 1996. 3. Robertson AS: Behaviour of the human bladder during natural filling: the Newcastle experience of ambulatory monitoring and conventional artificial filling cystometry. Scand J Urol Nephrol 201(suppl): 19 –24, 1999. 4. Kinder RB, and Mundy AR: Pathophysiology of idiopathic detrusor instability and detrusor hyper-reflexia: an in 9

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