Karen D. McCloskey

Kit Positive Cells In The Guinea Pig Bladder

PURPOSE We describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder. MATERIALS AND METHODS Bladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy. RESULTS Anti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active. CONCLUSIONS ICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.

Specialised pacemaking cells in the rabbit urethra.

1 Collagenase dispersal of strips of rabbit urethra yielded, in addition to normal spindle‐shaped smooth muscle cells, a small proportion of branched cells which resembled the interstitial cells of Cajal dispersed from canine colon. These were clearly distinguishable from smooth muscle in their appearance under the phase‐contrast microscope, their immunohistochemistry and their ultrastructure. They had abundant vimentin filaments but no myosin, a discontinuous basal lamina, sparse rough endoplasmic reticulum, many mitochondria and a well‐developed smooth endoplasmic reticulum. 2 Interstitial cells were non‐contractile but exhibited regular spontaneous depolarisations in current clamp. These could be increased in frequency by noradrenaline and blocked by perfusion with calcium‐free solution. In voltage clamp they showed abundant calcium‐activated chloride current and spontaneous transient inward currents which could be blocked by chloride channel blockers. 3 The majority of smooth muscle cells were vigorously contractile when stimulated but did not show spontaneous electrical activity in current clamp. In voltage clamp, smooth muscle cells showed very little calcium‐activated chloride current. 4 We conclude that there are specialised pacemaking cells in the rabbit urethra that may be responsible for initiating the slow waves recorded from smooth muscle cells in the intact syncitium.

Interstitial cells in the urinary bladder—localization and function†

This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC).

Mechanisms of Disease: specialized interstitial cells of the urinary tract--an assessment of current knowledge.

Scientists interested in the smooth muscles of the urinary tract, and their control, have recently been studying cells in the interstitium of tissues that express the c-kit antigen (Kit+ cells). These cells have morphologic features that are reminiscent of the well-described pacemaker cells in the gut, the interstitial cells of Cajal (ICC). The spontaneous contractile behavior of muscles in the urinary tract varies widely, and it is clear that urinary tract Kit+ interstitial cells cannot be playing an identical role to that played by the ICC in the gut. Nevertheless, there is increasing evidence that they do play a role in modulating the contractile behavior of adjacent smooth muscle, and might also be involved in mediating neural control. This review outlines the properties of ICC in the gut, and gives an account of the discovery of cells in the interstitium of the main components of the urinary tract. The physiologic properties of such cells and the functional implications of their presence are discussed, with particular reference to the bladder. In this organ, Kit+ cells are found under the lamina propria, where they might interact with the urothelium and with sensory nerves, and also between and within the smooth-muscle bundles. Confocal microscopy and calcium imaging are being used to assess the physiology of ICC and their interactions with smooth muscles. Differences in the numbers of ICC are seen in smooth muscle specimens obtained from patients with various pathologies; in particular, bladder overactivity is associated with increased numbers of these cells.

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels.

Abstract. Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.

Lamina propria: The functional center of the bladder?

The bladder mucosa consists of the urothelium, basement membrane, and lamina propria (LP). Although the urothelium has been given much attention, it may be regarded as one part of a signaling system involving another equally important component of the bladder mucosa, namely, the LP. The LP lies between the basement membrane of the mucosa and the detrusor muscle and is composed of an extracellular matrix containing several types of cells, including fibroblasts, adipocytes, interstitial cells, and afferent and efferent nerve endings. In addition, the LP contains a rich vascular network, lymphatic vessels, elastic fibers, and smooth muscle fascicles (muscularis mucosae). The roles of the LP and its components in bladder function have not been definitively established, though it has been suggested to be the capacitance layer of the bladder, determining bladder compliance and enabling adaptive changes to increasing volumes. However, the bladder LP may also serve as a communication center, with an important integrative role in signal transduction to the central nervous system (nociception, mechanosensation). The LP may also, by means of its different components, make it possible for the urothelium to transmit information to other components of the bladder wall, contributing to activation of the detrusor muscle. In addition, the LP may serve as a source for production of factors influencing the growth of both the overlying urothelium and the underlying detrusor muscle. Neurourol. Urodynam. 33:9–16, 2014.

Morphological expression of KIT positive interstitial cells of Cajal in human bladder.

Purpose We investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells. Materials and Methods Human bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy. Results Whole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall. Conclusions The human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Cholinergic-induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca2+ indicator fluo 4AM. ICC fired Ca2+ transients in response to stimulation by carbachol (1/10 μM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 μM), an M3 receptor antagonist, but not by the M2 receptor antagonist methoctramine (1 μM). The source of Ca2+ underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 μM) or Ni2+ (30–100 μM) to block Ca2+ channels or the removal of external Ca2+ reduced the amplitude of the carbachol transients. Application of ryanodine (30 μM) or tetracaine (100 μM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 μM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 μM) caused a significant reduction and Xestospongin C (1 μM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca2+ indicator showed distinctively different patterns of spontaneous Ca2+ events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca2+ transients.

KCNQ Currents and Their Contribution to Resting Membrane Potential and the Excitability of Interstitial Cells of Cajal From the Guinea Pig Bladder

Purpose The presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed. Materials and Methods Enzymatically dispersed interstitial cells of Cajal were patch clamped with K+ filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential. Results Cells were stepped from −60 to 40 mV to evoke voltage dependent currents using a modified K+ pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and Kadenosine triphosphate currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma®) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to −60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity. Conclusions This study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild-type and W/Wv mice

Background and purpose:  W/Wv and wild‐type murine bladders were studied to determine whether the W/Wv phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC).