Hikaru Hashitani

Role of interstitial cells and gap junctions in the transmission of spontaneous Ca2+ signals in detrusor smooth muscles of the guinea-pig urinary bladder

To investigate mechanisms underlying the transmission of spontaneous Ca2+ signals in the bladder, changes in intracellular concentrations of Ca2+ ([Ca2+]i) were visualized in isolated detrusor smooth muscle bundles of the guinea‐pig urinary bladder loaded with a fluorescent Ca2+ indicator, fura‐PE3 or fluo‐4. Spontaneous increases in [Ca2+]i (Ca2+ transients) preferentially originated along the boundary of muscle bundles and then spread to the other boundary (Ca2+ waves). The synchronicity of Ca2+ waves across the bundles was disrupted by 18β‐glycyrrhetinic acid (18β‐GA, 40 μm), carbenoxolone (30 μm) or 2‐aminoethoxydiphenylborate (2‐APB, 50–100 μm), while CPA (10 μm), ryanodine (100 μm), xestospongin C (3 μm) and U‐73122 (10 μm) had no effect. Intracellular recordings using two independent microelectrodes demonstrated that 2‐APB (100 μm) blocked electrical coupling between detrusor smooth muscle cells. Nifedipine (10 μm) but not nominal Ca2+‐free solution diminished the synchronicity of Ca2+ waves before preventing their generation. Staining for c‐kit identified interstitial cells (IC) located along both boundaries of muscle bundles. IC were also scattered amongst smooth muscle cells and were more dominantly distributed in connective tissue between muscle bundles. IC generated nifedipine‐resistant spontaneous Ca2+ transients, which occurred independently of those of smooth muscles. In conclusion, the propagation of Ca2+ transients in the bladder appears to be exclusively mediated by the spread of action potentials through gap junctions being facilitated by the regenerative nature of L‐type Ca2+ channels, without significant contribution of intracellular Ca2+ stores. IC in the bladder may modulate the transmission of Ca2+ transients originating from smooth muscle cells rather than being the pacemaker of spontaneous activity.

Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.

1 The origin and propagation of waves of spontaneous excitation in bundles of smooth muscle of the guinea‐pig bladder were examined using intracellular recording techniques and visualization of the changes in the intracellular calcium concentration ([Ca2+]i). 2 Bladder smooth muscle cells exhibited spontaneous transient increases in [Ca2+]i which originated along a boundary of each smooth muscle bundle and then spread to the other boundary with a conduction velocity of 2.0 mm s−1. 3 Spontaneous increases in [Ca2+]i were always preceded by action potentials. Nifedipine (10 μM) abolished increases in both [Ca2+]i and action potentials. Caffeine (10 mM), ryanodine (50 μM) and cyclopiazonic acid (10 μM) reduced the amplitude of the associated increases in [Ca2+]i without preventing the generation of action potentials. 4 Spontaneous action potentials had conduction velocities of 40 mm s−1 in the axial direction and 1.3 mm s−1 in the transverse direction. The electrical length constants of the bundles of muscle were 425 μm in the axial direction and 12.5 μm in the transverse direction. 5 Neurobiotin, injected into an impaled smooth muscle cell, spread more readily to neighbouring cells located in the axial direction than those located in the transverse direction. The spread of neurobiotin was inhibited by 18β‐glycyrrhetinic acid (18β‐GA, 40 μM), a gap junction blocker. 6 Immunohistochemistry for Connexin 43 showed abundant punctate staining on the smooth muscle cell membranes. 7 These results suggested that spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary probably through gap junctions.

K+ channels which contribute to the acetylcholine-induced hyperpolarization in smooth muscle of the guinea-pig submucosal arteriole.

1 Membrane potentials were recorded from submucosal arterioles (diameter, 30–80 μm) of the guinea‐pig small intestine, using conventional microelectrode techniques. In control solution the resting membrane potential was about −73 mV, and the addition of 0.5 mm Ba2+ depolarized the membrane to about −43 mV. 2 ACh (10 nm to 10 μM), or substance P (0.1 μM), caused a membrane hyperpolarization in preparations which had been depolarized by Ba2+ but not in control preparations. ACh produced a sustained hyperpolarization, whereas substance P produced a transient hyperpolarization, without being affected by either nitroarginine (0.1 mm) or indomethacin (10 μM). 3 In the presence of 50 μM BAPTA (acetoxymethyl ester form), the membrane potentials were not altered in the control solution or in the presence of Ba2+, but Ba2+ caused a smooth depolarization of the membrane. Following this procedure, both ACh and substance P caused membrane depolarization instead of hyperpolarization, suggesting that the ACh‐ and substance P‐induced hyperpolarization in arteriolar smooth muscle are intracellular [Ca2+] dependent. 4 In short segments (200–500 μM) of arteriole, the time constant of electrotonic potentials produced by passing current pulses through the recording electrode was about 75 ms. The addition of Ba2+ increased both the input resistance and the time constant. 5 The hyperpolarizations produced by ACh or substance P were associated with a reduction in the amplitude and the time constant of electrotonic potential. 6 The reversal potential for the ACh‐induced hyperpolarization, estimated from the current‐voltage relationship, was about −86 mV, a value close to the equilibrium potential for K+. 7 In the presence of 50 nM charybdotoxin the hyperpolarization produced by ACh became transient and was reduced in amplitude: the residual response was further reduced by apamin (0.1 μM). The response produced by substance P was also reduced by 50 nM charybdotoxin: again the residual response was sensitive to 0.1 μM apamin. The hyper‐polarizations produced by either ACh or substance P were insensitive to glibenclamide (10 μM) and 4‐aminopyridine (1 mm). 8 It is suggested that in submucosal arterioles of the guinea‐pig ileum, ACh‐ or substance P‐induced hyperpolarizations of smooth muscle result from activation of both charybdotoxin‐sensitive and apamin‐sensitive K+ channels, with the former being predominant. The results are discussed in relation to the possible involvement of one or more endothelium‐dependent hyperpolarizing factors in ACh‐ and substance P‐induced hyperpolarization.

Interaction between interstitial cells and smooth muscles in the lower urinary tract and penis

Smooth muscles in the lower urinary tract and corporal tissue exhibit spontaneous contractile activity which depends on L‐type Ca2+ channels. The mechanism underlying this activity is spontaneous electrical activity which shows varied form and property between these tissues. Recent studies revealed that interstitial cells (ICs) are widely distributed in the genitourinary system, and suggested their involvement in spontaneous muscle activity. ICs in the system are not a simple analogy of interstitial cells of Cajal (ICC) in the gut, which act as electrical pacemaker, but represent variability amongst tissues which may account for individual characteristics of each organ. In the bladder and corporal tissue, where smooth muscle cells are capable of generating spontaneous electrical activity, ICs may modulate smooth muscle activity. ICs in corporal tissue release prostaglandins via cyclooxygenase‐2 (COX‐2) activity and reinforce not only spontaneous but also nerve‐mediated α‐adrenergic contractions. In the bladder, their fundamental role in the integration of signals between populations of cells has been proposed, and thus changes in ICs may contribute to an overactive bladder, a pathological condition which results from increased excitability in detrusor smooth muscles. In the urethra, ICs may act as electrical pacemakers as do ICC. However, overall contractility of urethral smooth muscles does not necessarily rely on pacemaking of ICs, and thus some population of smooth muscles may also have their own excitability.

Spontaneous electrical and Ca2+ signals in typical and atypical smooth muscle cells and interstitial cell of Cajal-like cells of mouse renal pelvis

Electrical rhythmicity in the renal pelvis provides the fundamental drive for the peristaltic contractions that propel urine from the kidney to bladder for storage until micturition. Although atypical smooth muscles (ASMCs) within the most proximal regions of the renal pelvis have long been implicated as the pacemaker cells, the presence of a sparsely distributed population of rhythmically active Kit‐positive interstitial cells of Cajal‐like cells (ICC‐LCs) have confounded our understanding of pelviureteric peristalsis. We have recorded the electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and ICC‐LCs using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo‐4. Nifedipine (1–10 μm)‐sensitive driven action potentials and Ca2+ waves (frequency 6–15 min−1) propagated through the TSMC layer at a velocity of 1–2 mm s−1. High frequency (10–40 min−1) Ca2+ transients and spontaneous transient depolarizations (STDs) were recorded in ASMCs in the absence or presence of 1 μm nifedipine. ICC‐LCs displayed low frequency (1–3 min−1) Ca2+ transients which we speculated arose from cells that displayed action potentials with long plateaus (2–5 s). Neither electrical activity propagated over distances > 50 μm. In 1 μm nifedipine, ASMCs or ICC‐LCs separated by < 30 μm displayed some synchronicity in their Ca2+ transient discharge suggesting that they may well be acting as ‘point sources’ of excitation to the TSMC layer. We speculate that ASMCs act as the primary pacemaker in the renal pelvis while ICC‐LCs play a supportive role, but can take over pacemaking in the absence of the proximal pacemaker drive.

Potassium channels activated in the endothelium-dependent hyperpolarization in guinea-pig coronary artery

1 Properties of the endothelium‐dependent hyperpolarization evoked by acetylcholine (ACh) in smooth muscle of the guinea‐pig coronary artery were investigated using conventional microelectrode techniques. 2 ACh hyperpolarized the membrane in an endothelium‐dependent manner. The hyperpolarization comprised two components: an initial and a slow hyperpolarization. The former appeared during application of ACh, while the latter occurred after withdrawal of ACh. 3 Indomethacin and diclofenac, inhibitors of the enzyme cyclo‐oxygenase, blocked only the slow hyperpolarization, indicating that this potential was produced by endothelial prostanoids. 4 Clotrimazole and SKF 525a, known inhibitors of the enzyme cytochrome P450, inhibited both the initial and the slow hyperpolarizations, suggesting that these chemicals acted as non‐selective inhibitors of arachidonic acid metabolism. Inhibition of the lipoxygenase pathway of arachidonic acid metabolism by nordihydroguaiaretic acid had no effect on either component of the hyperpolarization. 5 The slow hyperpolarization was inhibited by 4‐aminopyridine (4‐AP; 10−4‐10−3 M) and glibenclamide (10−6 M). The initial hyperpolarization was greatly inhibited by charybdotoxin (CTX; 5 × 10−8 M) and partially inhibited by apamin (10−7 M), but was not inhibited by glibenclamide (10−5 M). Ba2+ (10−4 M) depolarized the membrane and increased the amplitude of both components of the ACh‐induced hyperpolarization. 6 Hyperpolarizations produced by Y‐26763, a K+ channel opener, were inhibited by glibenclamide, but not by 4‐AP. 7 The results indicate that the slow hyperpolarization is produced by endothelial prostanoids through activation of 4‐AP‐sensitive K+ channels (possibly delayed rectifier type). The initial hyperpolarization is produced mainly through activation of CTX‐sensitive K+ channels (possibly Ca2+‐sensitive type).

Electrical and mechanical responses produced by nerve stimulation in detrusor smooth muscle of the guinea-pig

In smooth muscles of the guinea-pig bladder, intramural nerve stimulation generated an excitatory junctional potential (e.j.p.), action potential and twitch contraction. Nicardipine inhibited the action potential but not the e.j.p. The e.j.p. amplitude was reduced by suramin, or desensitization of the ATP receptor with receptor agonists. The amplitude of the twitch contraction was reduced by atropine, and the remainder was blocked by nicardipine. In the presence of maximally effective concentrations of atropine, the threshold concentration of acetylcholine required to produce contraction was about 10(-7) M, whereas acetylcholine concentrations greater than 10(-6) M were required to cause depolarization. It is concluded that nerve stimulation releases acetylcholine and ATP, and the former produces contraction without change in the membrane potential, while the latter generates the e.j.p. which triggers an action potential and thus elicits contractions.

Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis

Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs.

Role of K+ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder

PURPOSE We investigated the functional role of K(+) channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. MATERIALS AND METHODS The effects of different K(+) channels blockers on spontaneous changes in membrane potential and intracellular Ca(2+) dynamics were examined using intracellular recording techniques and Ca(2+) imaging with fluo-4 fluorescence, respectively. RESULTS Detrusor smooth muscle generated spontaneous action potentials and Ca(2+) transients. Iberiotoxin (0.1 microM), charybdotoxin (0.1 microM) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 microM) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 microM) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca(2+) transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca(2+) waves. Transmural nerve stimulations invariably initiated synchronous Ca(2+) transients within and across muscle bundles. Charybdotoxin (0.1 microM) increased the amplitude of spontaneous Ca(2+) transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca(2+) transients in muscle bundles. CONCLUSIONS These results suggest that large and intermediate conductance Ca(2+) activated K(+) channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K(+) channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability.

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Interstitial cells of Cajal‐like cells (ICC‐LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC‐LCs, spontaneous changes in [Ca2+]i (Ca2+ transients) were visualized in fluo‐4 loaded preparations of rabbit urethral smooth muscle. ICC‐LCs were sparsely distributed, rather than forming an extensive network. Ca2+ transients in ICC‐LCs had a lower frequency and a longer half‐width than those of USMCs. ICC‐LCs often exhibited Ca2+ transients synchronously with each other, but did not often show a close temporal relationship with Ca2+ transients in USMCs. Nicardipine (1 μm) suppressed Ca2+ transients in USMCs but not in ICC‐LCs. Ca2+ transients in ICC‐LCs were abolished by cyclopiazonic acid (10 μm), ryanodine (50 μm) and caffeine (10 mm) or by removing extracellular Ca2+, and inhibited by 2‐aminoethoxydiphenyl borate (50 μm) and 3‐morpholino‐sydnonimine (SIN‐1; 10 μm), but facilitated by increasing extracellular Ca2+ or phenylephrine (1–10 μm). These results indicated that Ca2+ transients in urethral ICC‐LCs in situ rely on both Ca2+ release from intracellular Ca2+ stores and Ca2+ influx through non‐L‐type Ca2+ channel pathways. ICC‐LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.