Resting Potential

When a ureteral muscle cell is in a nonexcited or resting state, the electric potential difference across the cell membrane, transmembrane potential, is referred to as the resting membrane potential (RMP). The RMP is determined primarily by the distribution of potassium ions (K⁺) across the cell membrane and by the permeability of the membrane to K⁺ (Hendrickx et al, 1975). In the resting state, the K⁺ concentration on the inside of the cell is greater than that on the outside of the cell, that is, [K⁺]_i > [K⁺]_o, and the membrane is preferentially permeable to K⁺. Because of the tendency for the positively charged K⁺ ions to diffuse from the inside of the cell, where they are more concentrated, to the outside of the cell, where they are less concentrated, an electric gradient is created, with the inside of the cell membrane being more negative than the outside (Fig. 59–1A). The electric gradient that is formed tends to oppose the further movement of K⁺ outward across the cell membrane along its concentration gradient, and an equilibrium is reached.

Figure 59–1 Ionic basis for the resting membrane potential (RMP) in smooth muscle. In the resting state, the K⁺ concentration inside the cell is greater than the K⁺ concentration outside the cell, and the Na⁺ concentration outside the cell is greater than the Na⁺ concentration inside the cell. A, Electrochemical changes that would occur if the membrane were solely permeable to potassium. Potassium would diffuse from the inside of the cell, where it is more concentrated, to the outside of the cell, where it is less concentrated. The outward movement of the positively charged K⁺ ions would make the inside of the cell membrane negative with respect to the outside of the cell membrane. B, Electrochemical changes that would occur if the resting membrane were also permeable to sodium. An inward movement of Na⁺ along its concentration gradient would make the inside of the cell membrane less negative with respect to the outside of the cell membrane than is depicted in A. C, Pump mechanism for extruding Na⁺ from within the cell against concentration and electrochemical gradients. Inward movement of K⁺ is coupled with outward movement of Na⁺. This mechanism helps to maintain a steady state of ion distribution across the cell membrane and a stable RMP. ECF, extracellular fluid; ICF, intracellular fluid.

(A to C, From Weiss RM. Ureteral function. Urology 1978;12:114.)

If the membrane in the resting state were exclusively permeable to K⁺, the measured RMP of the ureteral smooth muscle cell would approximate −90 mV, the K⁺ equilibrium potential, as predicted by the Nernst equation:

where E_k is the potential difference attributable to the concentration difference of K⁺ across the cell membrane, R is the molar gas constant, T is the absolute temperature, n is the number of moles of K⁺, and F is the faraday constant (Nernst, 1908). However, in the ureter and in other smooth muscles, the RMP is considerably less than the K⁺ equilibrium potential, with values of −33 to −70 mV, the inside of the cell being negative relative to the outside (Kuriyama et al, 1967). Studies from single isolated ureteral cells show spontaneous transient hyperpolarizations, with the RMP transiently becoming more negative (Imaizumi et al, 1989). This phenomenon appears to be due to spontaneous release of calcium ions (Ca²⁺⁾ from the sarcoplasmic reticulum, with activation of tetraethylammonium (TEA) and charbydotoxin-sensitive Ca²⁺-dependent K⁺ channels (I_K(Ca)). Although the low resting potential of ureteral cells may be explained in part by a relatively small resting K⁺ conductance (Imaizumi et al, 1989), it also may be due to the contribution of other ions.

One such ion that could account for the relatively low RMP of the ureter and other smooth muscles is the sodium ion (Na⁺) (Kuriyama, 1963). In the resting state, the Na⁺ concentration on the outside of the cell membrane is greater than that on the inside, that is, [Na⁺]_o > [Na⁺]_i. If the resting membrane were somewhat permeable to Na⁺, both the concentration and the electric gradient would support an inward movement of Na⁺ across the cell membrane, with a resultant decrease in the electronegativity of the inner surface of the cell membrane (Fig. 59–1B).

If such an inward movement of Na⁺ went unchecked, the RMP would be expected to decrease to a level lower than that actually observed, and the concentration gradient for Na⁺ might become reversed. In order to maintain a steady-state ion distribution across the cell membrane with [K⁺]_o < [K⁺]_i and [Na⁺]_o > [Na⁺]_I, and to prevent the transmembrane potential from becoming lower than the measured ureteral RMP, an active mechanism capable of extruding Na⁺ from within the cell against a concentration and electrochemical gradient is required (Fig. 59–1C). Such an outward Na⁺ pump that is coupled with an inward movement of K⁺ derives its energy requirements from the dephosphorylation of ATP (Casteels, 1970). Na⁺/Ca²⁺ exchange also may play a role in Na⁺ extrusion, especially when the Na⁺ pump is inhibited (Aickin, 1987; Aickin et al, 1987; Lamont et al, 1998).

The dynamic processes illustrated in Figure 59–1 enable the ureter in its resting state to maintain a relatively low RMP. In addition to the mechanisms described, the distribution of chloride ions (Cl⁻) across the cell membrane and the relative permeability of the membrane to Cl⁻ may be factors in the maintenance of the RMP in the ureter and other smooth muscles (Kuriyama, 1963; Washizu, 1966).

Action Potential

The transmembrane potential of an inactive or resting ureteral cell remains stable until it is excited by an external stimulus—electric, mechanical (stretch), or chemical—or by conduction of electric activity (action potential) from an already excited adjacent cell. When a ureteral cell is stimulated, depolarization occurs, with the inside of the cell membrane becoming less negative than it was before stimulation. If a sufficient area of the cell membrane is depolarized rapidly enough to reach a critical level of transmembrane potential, referred to as the threshold potential, a regenerative depolarization, or action potential, is initiated.

The changes that occur are diagrammatically depicted in Figure 59–2. If a stimulus is very weak, as shown by arrow a, the transmembrane potential may remain unchanged. A slightly stronger, yet subthreshold, stimulus may result in an abortive displacement of the transmembrane potential, but not to such a degree that an action potential is generated (arrow b). If the stimulus is strong enough to decrease the transmembrane potential to the threshold potential, the cell becomes excited and develops an action potential (arrow c). The action potential, which is the primary event in the conduction of the peristaltic impulse, has the capability to act as the stimulus for excitation of adjacent quiescent cells and, through a complicated chain of events, gives rise to the ureteral contraction.

Figure 59–2 Response of ureteral transmembrane potential to stimuli. At arrow a, a weak stimulus is applied that does not alter the resting membrane potential (MP). At arrow b, a stimulus is applied that decreases the transmembrane potential but not to the level of the threshold potential TP (subthreshold stimulus). At arrow c, a stimulus is applied that decreases the transmembrane potential to TP, and an action potential is initiated (suprathreshold stimulus).

(From Weiss RM. Ureteral function. Urology 1978;12:114.)

When the ureteral cell is excited, its membrane loses its preferential permeability to K⁺ and becomes more permeable to Ca²⁺ ions that move inward across the cell membrane, primarily through fast L-type Ca²⁺ channels and give rise to the upstroke of the action potential (Fig. 59–3A) (Kobayashi, 1965; Kuriyama and Tomita, 1970; Imaizumi et al, 1989; Lang, 1989, 1990; Sui and Kao, 1997a, b; Smith et al, 2002). This channel is inhibited by the calcium channel blocker nifedipine and by cadmium ions (Cd²⁺), and it is potentiated by barium ions (Ba²⁺). As the positively charged Ca²⁺ ions move inward across the cell membrane, the inside of the membrane becomes less negative relative to the outside and may even become positive at the peak of the action potential, a state referred to as overshoot. Na⁺ ions also may play a role in the upstroke of the ureteral action potential (Kobayashi, 1964, 1965; Muraki et al, 1991). The rate of rise of the upstroke of the ureteral action potential is relatively slow, 1.2 ± 0.06 V/sec in the cat (Kobayashi, 1969). This compares with a 610 V/sec rate of rise in dog cardiac Purkinje fibers (Draper and Weidmann, 1951) and a 740 V/sec rate of rise in skeletal muscle (Ferroni and Blanchi, 1965). The slow rate of upstroke rise of the ureteral action potential accounts for the slow conduction velocity in the ureter.

Figure 59–3 Schematic representation of ionic currents in (A) nonpacemaker (solid line) and (B) pacemaker (dashed line) action potentials: (0) upstroke or depolarization phase; (2) plateau phase; (3) repolarization phase; and (4) resting potential of the nonpacemaker cell and spontaneous depolarization phase of the pacemaker cell. A spontaneous decrease in the transmembrane potential of pacemaker cells accounts for their spontaneous activity.

After reaching the peak of its action potential, the ureter maintains its potential for a period of time (plateau of the action potential) before the transmembrane potential returns to its resting level (repolarization) (Kuriyama et al, 1967). The plateau phase of the guinea pig action potential is superimposed with multiple oscillations, a phenomenon not observed in the rat, rabbit, or cat (Fig. 59–4) (Bozler, 1938). The plateau phase appears to depend on the persistence of an inward Ca²⁺ current and on Na⁺ influx through a voltage-dependent Na⁺ channel (see Fig. 59–3A) (Kuriyama and Tomita, 1970; Imaizumi et al, 1989; Sui and Kao, 1997b). Also involved in the plateau formation is the maintenance of depolarization by an inward calcium-dependent chloride current (I_Cl(Ca)) which is countered by outward voltage-gated (I₇₀) and Ca²⁺-activated K⁺ currents (I_(KCa)) (Smith et al, 2002). There are species differences in the ionic currents involved in the formation of the action potential, with the Ca²⁺-activated chloride current being present in the rat but not in the guinea pig ureter. The inward Cl⁻ current can be inhibited by niflumic acid and by Ba²⁺ (Smith et al, 2002). The oscillations on the plateau of the guinea pig action potential appear to depend on the repetitive activation of an inward Ca²⁺ current (Kuriyama and Tomita, 1970) and of a Ca²⁺-dependent outward K⁺ current (Imaizumi et al, 1989). Prolongation of the inward calcium current and the duration of the action potential correlate with an increased force of contraction (Burdyga and Wray, 1999b).

Figure 59–4 Intracellular recordings of ureteral action potentials (upper tracings) and isometric recordings of contractions (lower tracings) in response to electrical stimuli. Action potentials precede contractions. A, Guinea pig ureter; oscillations on the plateau of the action potential. B, Cat ureter; no oscillations on the plateau of the action potential.

(A and B, From Weiss RM. Ureteral function. Urology 1978;12:114.)

The activation of a Ca²⁺-dependent K⁺ current that is involved in repolarization is mainly due to Ca²⁺ release from the endoplasmic reticulum, which is triggered by the influx of extracellular Ca²⁺ through voltage-dependent Ca²⁺ channels. The increase in intracellular Ca²⁺ concentration during the upstroke and plateau of the action potential finally may activate the outward Ca²⁺-dependent K⁺ current (I_K(Ca)) to such a degree that repolarization occurs with return of the transmembrane potential to its resting level (see Fig. 59–3A) (Imaizumi et al, 1989; Sui and Kao, 1997c). This I_K(Ca) is sensitive to inhibition by tetraethylammonium (TEA). Based on this, TEA increases the amplitude and duration of in-vitro ureteral contractions (Floyd et al, 2008). A voltage-dependent, Ca²⁺ insensitive outward K⁺ current (I_TO) also appears to be involved in the repolarization (Lang, 1989; Imaizumi et al, 1990). These currents are TEA-insensitive and 4-aminopyridine (4-AP)–sensitive. In the rat, but not the guinea pig, ureter there is a late TEA-, Cd²⁺– and Ca²⁺-insensitive outward K⁺ current that also is involved in the repolarization process. The duration of the action potential in the cat ranges from 259 to 405 milliseconds (Kobayashi and Irisawa, 1964).

In summary, the RMP of the ureteral cell is approximately −33 to −70 mV and is determined primarily by the distribution of K⁺ ions across the cell membrane and the relatively selective permeability of the resting cell membrane to K⁺. When excited by a suprathreshold stimulus, the membrane becomes less permeable to K⁺ and more permeable to Ca²⁺, which moves inward across the cell membrane and provides the ionic mechanism for the development of the upstroke of the action potential. After reaching the peak of its action potential, the membrane maintains a depolarized state—plateau of the action potential—for a period of time before the membrane potential of the activated cell returns to its resting level (repolarization). The plateau appears to be related to a persistent inward Ca²⁺ current and to an influx of Na⁺. Repolarization of the membrane is related to a renewed increase in permeability to K⁺.

Pacemaker Potentials and Pacemaker Activity

Electric activity arises in a cell either spontaneously or in response to an external stimulus. If the activity arises spontaneously, the cell is referred to as a pacemaker cell. Pacemaker fibers differ from nonpacemaker fibers in that their transmembrane resting potential is lower (less negative) than that of nonpacemaker fibers (Lang and Zhang, 1996) and does not remain constant but, rather, undergoes a slow spontaneous depolarization (see Fig. 59–3B). If the spontaneously changing membrane potential reaches the threshold potential, the upstroke of an action potential occurs. The ionic conduction underlying pacemaker activity in the upper urinary tract is due to the opening and slow closure of voltage-activated L-type Ca²⁺ channels that are amplified by prostanoids (Santicioli et al, 1995a). This is opposed by the opening and closure of voltage and Ca²⁺-dependent K⁺ channels. It has been suggested that prostaglandins and excitatory tachykinins, released from sensory nerves, help maintain autorhythmicity in the upper urinary tract through maintenance of Ca²⁺ mobilization (Lang et al, 2002). Tetrodotoxin and blockers of the autonomic nervous system, both parasympathetic and sympathetic, have little effect on peristalsis, suggesting that autonomic neurotransmitters have a small role in maintaining pyeloureteral motility (Lang et al, 2001, 2002). Changes in the frequency of action potential development may result from a change in the level of the threshold potential, a change in the rate of slow spontaneous depolarization of the resting potential, or a change in the level of the resting potential.

Gosling and Dixon (1971, 1974) provided morphologic evidence of specialized pacemaker tissue in the proximal portion of the urinary collecting system and described species differences. In species with a multicalyceal system, such as the pig, sheep, and human, the “pacemaker cells” are located near the pelvicalyceal border (Dixon and Gosling, 1973). In species with a unicalyceal system, such as the dog, cat, rat, rabbit, and guinea pig, the “pacemaker cells” extend from the pelvicalyceal border to the ureteropelvic junction (UPJ). These atypical smooth muscle cells that give rise to pacemaker activity in the rat and guinea pig, in contrast to typical smooth muscle cells, have less than 40% of their cellular area occupied by contractile elements and demonstrate sparse immunoreactivity for smooth muscle and actin (Klemm et al, 1999; Lang et al 2001). These spindle-shaped cells are 90 to 230 µm in length, and their electrical activity consists of simple waveforms of alternating depolarizing and repolarizing phases that occur at a relatively rapid frequency of 8 to 15/min (Fig. 59–5A) (Tsuchida and Suzuki, 1992; Klemm et al, 1999). Pacemaker potentials have a lower resting membrane potential (RMP), a slower rate of rise, and a lower amplitude than action potentials recorded from nonpacemaker cells. In the guinea pig, these atypical, presumably pacemaker cells comprise greater than 80% of the cells at the pelvicalyceal junction, about 15% of the cells in the proximal renal pelvis, but are not present in the distal renal pelvis or ureter (Klemm et al, 1999). Electrical recordings correlate with histologic findings in that pacemaker potentials were not observed in the distal renal pelvis or ureter (Klemm et al, 1999).

Figure 59–5 Action potentials in the upper urinary tract of the guinea pig. A, Pacemaker potentials. B, Driven action potentials. C, Intermediate action potentials.

(Modified with permission from Klemm MF, Exintaris B, Lang RJ. Identification of the cells underlying pacemaker activity in the guinea pig upper urinary tract. J Physiol [Lond] 1999;519:867.)

Driven action potentials that fire at lower frequency (3 to 5/min) than pacemaker potentials are recorded from longer (150 to 400 µm) spindle-shaped typical smooth muscle cells (Fig. 59–5B) (Klemm et al, 1999). Most muscle cells of the ureter (100%), distal renal pelvis (97.5%), and proximal renal pelvis (83% are typical nonpacemaker smooth muscle cells with typical action potentials. Lang et al (1998) described fibroblast-like cells resembling the interstitial cells of Cajal (ICC), which serve as pacemaker cells in the intestine, in the proximal portion of the guinea pig renal pelvis. These “ICC-like” cells are irregularly shaped with oval nuclei and many branching interconnecting processes and contain numerous mitochondria, caveolae, and prominent endoplastic reticulum. The ICC-like cells in the guinea pig are not immunoreactive for α-smooth muscle actin, which is present in typical smooth muscle cells, or for c-KIT, a tyrosine kinase receptor that is expressed in intestinal ICC pacemaker cells (Klemm et al, 1999). The immunoreactivity to c-KIT appears to be species specific, because ICC-like cells that are immunoreactive to antibodies raised against the c-KIT proto-oncogene are present in the upper urinary tract of a number of mammals (Lang and Klemm, 2005; Metzger et al, 2005). Electrical recordings from these cells demonstrate action potentials with properties intermediate to the pacemaker and driven action potentials. These intermediate action potentials in the guinea pig have a single spike, a plateau without the superimposed spikes seen in driven action potentials and a rapid repolarization phase (Fig. 59–5C). Intermediate action potentials are noted in 11% to 17% of cells at the pelvicalyceal junction and the proximal and distal renal pelvis (Lang et al, 2002). These ICC-like cells in the upper urinary tract do not appear to be primary pacemaker cells but, rather, may provide for preferential conduction of electrical signals from pacemaker cells to typical smooth muscle cells of the renal pelvis and ureter (Klemm et al, 1999). In the mouse ureteropelvic junction, c-KIT–positive ICC-like cells have been identified that showed high-frequency spontaneous transient inward currents that often occurred in bursts to sum and produce long-lasting large inward currents (Lang et al, 2007b). It is postulated that in the absence of a proximal pacemaker drive these ICC-like cells could act as pacemaker cells and trigger contractions in adjacent smooth muscle cells in the ureteropelvic junction. Thus both atypical smooth muscle cells and ICC-like cells both may play a pacemaker role in the initiation and propagation of pyeloureteric peristalsis (Lang et al, 2006; Lang et al, 2007a).

c-KIT is a tyrosine kinase receptor that promotes cell migration and proliferation of melanoblasts, hematopoietic progenitors and primordial germ cells. Mice expressing mutant inactivating c-Kit alleles, lack intestinal interstitial cells of Cajal (ICC), have abnormal intestinal peristalsis, and develop bowel obstruction, showing that c-Kit is important in the development of pacemaker activity and peristalsis of the gut (Der-Silaphet et al, 1998). Pezzone et al (2003) identified c-Kit positive cells in the mouse ureter. They suggested that the difference from previous studies in the guinea pig upper urinary tract, in which c-Kit–positivity was not identified in ICC-like cells (Klemm et al, 1999), may be due to species differences, the c-Kit antibody used, and/or the fixation methods. c-Kit gene expression was noted to be upregulated in the embryonic murine ureter prior to its development of unidirectional peristaltic contractions (David et al, 2005). Incubation of isolated cultured embryonic murine ureters with antibodies that neutralize c-Kit activity altered ureteral morphology and inhibited unidirectional peristalsis. These data suggest that c-Kit–containing cells, which are probably ICC-like cells, have an important role in pyeloureteric peristalsis. c-Kit–positive cells have been identified in the human ureter (Metzger et al, 2004; van der Aa et al, 2004) and in the human UPJ (Solari et al, 2003). In the presence of obstruction, c-Kit–positive cells ICC-like cells at the UPJ were sparse or absent (Solari et al, 2003).

Bozler (1942), using small extracellular surface electrodes, demonstrated the characteristic slow spontaneous depolarization of pacemaker-type fibers in the proximal portion of the isolated ureter of a unicalyceal upper collecting system. In a multicalyceal kidney, Morita and associates (1981), using extracellular electrodes, recorded low-voltage potentials that appeared to be pacemaker potentials from the border of the pig minor calyces and the major calyx with the contraction rhythm varying between each calyx. Multiple pacemakers fire simultaneously as coupled oscillators, or individually, as pacemaker activity shifts from one site to another along the renal pelvis of the unicalyceal kidney or the pelvicalyceal border of the multicalyceal pig and sheep kidney (Golenhofen and Hannappel, 1973; Constantinou et al, 1977; Constantinou and Yamaguchi, 1981; Lammers et al, 1996).

Although the primary pacemaker for ureteral peristalsis is located in the proximal portion of the collecting system, other areas of the ureter may act as latent pacemakers. Under normal conditions, the latent pacemaker regions are dominated by activity arising at the primary pacemaker sites. When the latent pacemaker site is freed of its domination by the primary pacemaker, it, in turn, may act as a pacemaker. To demonstrate latent pacemaker sites, Shiratori and Kinoshita (1961) transected the in-vivo dog ureter at various levels. Before transection, peristaltic activity arose proximally from the primary pacemaker. When the ureter was transected at the UPJ, antiperistaltic waves of lower frequency than the previous normoperistaltic waves originated from the UVJ. Division of the ureter at the UVJ did not affect the normoperistaltic waves. After division of the midureter, the normoperistaltic waves in the upper segment remained unchanged, and the lower segment demonstrated antiperistaltic waves, which originated at the UVJ at a frequency less than that of the normoperistaltic waves in the upper segment. Thus cells at the UVJ of the dog may act as pacemaker cells when freed of control from the primary proximally located pacemaker. Latent pacemaker cells are present in all regions of the ureter (Imaizumi et al, 1989; Meini et al, 1995).

Propagation of Electric Activity

Excitable cells possess resistive and capacitative membrane properties similar to those of a cable or core conductor. The transverse resistance of the membrane is higher than the longitudinal resistance of the extracellular or intracellular fluid; this allows current resulting from a stimulus to propagate along the length of the fibers. The spread of current is referred to as electrotonic spread (Hoffman and Cranefield, 1960). The space constant (λ) determines the degree to which the electrotonic potential dissipates with increasing distance from an applied voltage. In a cable, this relation is expressed by

where X is the distance from the applied voltage, P is the displacement of the membrane potential at X, P_o is the displacement of the membrane potential at the site of the applied voltage, e is the base of the natural logarithm, and λ is the space constant. Thus the electrotonic potential decreases by 1/e in one space constant. The space constant of the guinea pig ureter measured by extracellular stimulation is 2.5 to 3 mm (Kuriyama et al, 1967).

The time constant τ_m is expressed by

where R is the membrane resistance and C is the membrane capacity. The time constant τ_m signifies that a small displacement of potential is decreased by 1/e of its value in one τ_m. The time constant of the guinea pig ureter measured by extracellular stimulation is 200 to 300 milliseconds (Kuriyama et al, 1967).

The ureter acts as a functional syncytium. Engelmann (1869, 1870) showed that stimulation of the ureter produces a contraction wave that propagates proximally and distally from the site of stimulation. Under normal conditions, electric activity arises proximally and is conducted distally from one muscle cell to another across areas of close cellular apposition referred to as intermediate junctions (Uehara and Burnstock, 1970; Libertino and Weiss, 1972). The similarity of these close cellular contacts to nexuses, which have been shown to be low-resistance pathways for cell-to-cell conduction in other smooth muscles (Barr et al, 1968), suggests that a similar mechanism for conduction may be present in the ureter. Gap junctions, consisting of groups of channels in the plasma membrane of adjacent smooth muscle cells, enable exchange of ions and small molecules and play a role in electrical coupling between adjacent cells and electromechanical coupling (Gabella, 1994; Santiciolli and Maggi, 2000). 18β-glycyrrhetinic acid, a gap junction inhibitor, inhibits cell-to-cell electrical coupling in the guinea pig renal pelvis and ureter and dissociates electrical and mechanical events (Santicioli and Maggi, 2000). Conduction velocity in the ureter is 2 to 6 cm/sec (Kobayashi, 1964; Kuriyama et al, 1967); it has been shown to vary with temperature, the time interval between stimuli (van Mastrigt et al, 1986), and the pressure within the ureter (Tsuchiya and Takei, 1990). This compares to conduction velocities ranging from 1.5 to 2 m/sec in cardiac Purkinje fibers (Rosen et al, 1981) and from 10 to 100 m/sec in the dorsal and ventral roots of the spinal cord (Biscoe et al, 1977). Conduction in the ureter is similar to that in cardiac tissue, even to the extent that the Wenckebach phenomenon (a partial conduction block) has been demonstrated in the ureter, as it has been in specialized cardiac fibers (Weiss et al, 1968).

Contractile Proteins

In skeletal muscle, Ca²⁺ appears to act as a derepressor. It is thought that in the relaxed state, a regulator system, consisting of the proteins troponin and tropomyosin, prevents the interaction of actin and myosin. In the relaxed state, troponin that is attached to tropomyosin is inactive, and tropomyosin prevents the interaction between actin and myosin. With activation, there is an increase in the sarcoplasmic Ca²⁺ concentration. The Ca²⁺ binds to troponin, producing a conformational change that results in the displacement of tropomyosin, thus allowing the interaction of actin and myosin and the development of a contraction.

In smooth muscle, on the other hand, Ca²⁺ appears to act as an activator. The most widely accepted theory suggests that phosphorylation of myosin is involved in the contractile process and that a troponin-like system does not constitute the primary regulatory mechanism, as it does in skeletal muscle. With excitation, there is a transient increase in the sarcoplasmic Ca²⁺ concentration from its steady-state concentration of 10⁻⁸ to 10⁻⁷ M to a concentration of 10⁻⁶ M or higher. At this higher concentration, Ca²⁺ forms an active complex with the Ca²⁺–binding protein calmodulin (Watterson et al, 1976; Cho et al, 1988). Calmodulin without Ca²⁺ is inactive (Fig. 59–7). The Ca²⁺–calmodulin complex activates a calmodulin-dependent enzyme, myosin light-chain kinase (see Fig. 59–7). The activated myosin light-chain kinase, in turn, catalyzes the phosphorylation of the 20,000-D light chain of myosin (Fig. 59–8). Phosphorylation of the myosin light chain allows actin to activate myosin Mg²⁺-ATPase activity, leading to hydrolysis of ATP and the development of smooth muscle tension or shortening (Fig. 59–9). Actin cannot activate the ATPase activity of the dephosphorylated myosin light chain.

Figure 59–7 Schematic representation of the contractile process in smooth muscle. Calmodulin is activated by Ca²⁺. The activated calcium-calmodulin complex activates the enzyme myosin light-chain kinase, which phosphorylates the light chain of myosin. Phosphorylation of myosin light-chain kinase decreases the rate of activation of the enzyme by the Ca²⁺-calmodulin complex.

Figure 59–8 Schematic representation of the contractile process in smooth muscle. The activated enzyme myosin light-chain kinase catalyzes the phosphorylation of myosin. Myosin must be phosphorylated in order for actin to activate myosin ATPase. ADP, adenosine diphosphate; ATP, adenosine triphosphate.

Figure 59–9 Schematic representation of the contractile process in smooth muscle. Actin activates ATPase activity of phosphorylated myosin. This allows interaction of actin and myosin with the development of a contraction. ADP, adenosine diphosphate; ATP, adenosine triphosphate.

When the Ca²⁺ concentration in the region of the contractile proteins is low, the myosin light-chain kinase is not active, because calmodulin requires Ca²⁺ to activate the enzyme. This prevents activation of the contractile apparatus, because the myosin light chain cannot be phosphorylated, a process that must precede tension development. Furthermore, a phosphatase dephosphorylates the myosin light chain, thus preventing actin activation of myosin ATPase activity, and relaxation results.

Evidence indicates that phosphorylation of the enzyme myosin light-chain kinase by a cAMP-dependent protein kinase decreases myosin light-chain kinase activity by decreasing the affinity of this enzyme for calmodulin (Adelstein et al, 1981).

Although Ca²⁺ is required for most smooth muscle contractile events, there is evidence in some smooth muscles, including in the rabbit and human bladder, that Ca²⁺-independent contractions can occur (Yoshimura and Yamaguchi, 1997). Carbachol, a muscarinic cholinergic agonist, and phorbol ester, which activate protein kinase C (PKC), can induce contraction in Ca²⁺-depleted bladder strips that can be inhibited by a PKC inhibitor, H7. It is suggested that activation of PKC, coupled with agonist stimulation of the muscarinic receptor, can induce a Ca²⁺-independent contraction.

Calcium and Excitation-Contraction Coupling

The mechanical event of ureteral peristalsis follows an electric event to which it is related. The Ca²⁺ involved in the ureteral contraction is derived from two main sources. Because smooth muscle cells have a very small diameter, the inward movement of extracellular Ca²⁺ into the cell through L-type Ca²⁺ channels during the upstroke of the action potential provides a significant source of sarcoplasmic Ca²⁺ (Brading et al, 1983; Hertle and Nawrath, 1989; Yoshida et al, 1992; Maggi et al, 1994a; Maggi and Giuliani, 1995; Floyd et al, 2008) (see Fig. 59–6). This inward movement across the cell membrane is the major source of calcium used for contraction in most smooth muscles. Na⁺-Ca²⁺ exchange, with an outward movement of Na⁺ and an inward movement of Ca²⁺, also plays a role in the ureteral contraction (Lamont et al, 1998). Furthermore, in response to an excitatory impulse, Ca²⁺ release from tightly bound storage sites (i.e., the endoplasmic or sarcoplasmic reticulum) also increases the Ca²⁺ concentration in the sarcoplasm (Burdyga et al, 1998; Burdyga and Wray, 1999a; Lang et al, 2002). Calcium may be released from the sarcoplasmic reticulum (SR) of smooth muscle by an inositol 1,4,5-trisphosphate (IP₃)–induced release mechanism or by Ca²⁺-induced Ca²⁺ release (CICR) (Somlyo and Somlyo, 1994). These processes appear to be species dependent. CICR, which involves ryanodine receptors, appears to be the sole mechanism for calcium release from the SR in the guinea pig ureter, whereas the SR stored in the rat ureter appears to be exclusively under the control of IP₃ receptors (Burdyga et al, 1995). IP₃ and ryanodine receptors are expressed in the human ureter (Floyd et al, 2008). The opening of caffeine-sensitive ryanodine receptors produces small local elevations of Ca²⁺ that are termed Ca²⁺ sparks (Nelson et al, 1995). In addition to providing a source of calcium for contraction, Ca²⁺ released from the SR activates Ca²⁺–sensitive surface membrane channels and modulate membrane excitability (Imaizumi et al, 1989; Carl et al, 1996). Both calcium-activated outward potassium currents (K_Ca), or STOCS (spontaneous transient outward currents), and calcium-activated inward chloride currents (Cl_Ca), or STICS (spontaneous transient inward currents), have been identified in smooth muscles. These currents affect membrane potential and thus affect calcium entry through L-type Ca²⁺ channels in the membrane. The Ca²⁺–activated chloride currents are present in rat but not guinea pig ureteral smooth muscle (Burdyga and Wray, 2002). Thus some Ca²⁺ released from SR results in contraction, but it has been shown that caffeine-induced release of SR Ca²⁺, in the form of Ca²⁺ sparks, increases outward potassium currents (K_Ca), or STOCS, with an inhibitory effect on action potentials and contractility (Borisova et al, 2007). At least in the guinea pig ureter, this increase in the outward potassium current hyperpolarizes the membrane and determines the refractory period, which is important in determining the frequency of ureteral peristalsis and may have a protective effect on the kidney (Burdyga and Wray, 2005).

Support for use of a dual source of Ca²⁺ in the ureter has been provided by Vereecken and coworkers (1975), who noted that it took approximately 45 minutes for spontaneous contractions of isolated guinea pig ureters to cease when the tissue was placed in a Ca²⁺-free medium. They interpreted this to indicate that some of the Ca²⁺ involved in the contractile process is derived from tightly bound intracellular stores. They also noted that recovery of the contractile response to electric stimuli was almost immediate when the tissue was returned to a physiologic solution containing a normal concentration of Ca²⁺. This suggests that free extracellular Ca²⁺ entering the cell during excitation also provides a source of Ca²⁺ for the contractile machinery. A similar conclusion was reached by Hong and associates (1985). There is, however, some evidence that Ca²⁺ release from the sarcoplasmic reticulum may not play a significant role in ureteral contractions, at least in the guinea pig (Maggi et al, 1994a, 1995, 1996), and some perturbations of contractility may be related to movements of ions other than Ca²⁺. The increase in developed force in the guinea pig ureter, with intracellular acidification and decrease with intracellular alkalinization, appear to result from modulation of outward K⁺ currents rather than effects on inward Ca²⁺ currents (Smith et al, 1998). That is, alkalinization (increasing intracellular pH) enhances outward K⁺ currents, and this reduces excitability; acidification has the opposite effect. Relaxation results from a decrease in the concentration of free sarcoplasmic Ca²⁺ in the region of the contractile proteins. The decrease in sarcoplasmic Ca²⁺ can result from the uptake of Ca²⁺ into intracellular storage sites (Maggi et al, 1994a, 1995) or from extrusion of Ca²⁺ from the cell (Burdyga and Magura, 1988).

In addition to the Ca²⁺– signaling cascade that affects contractility, there is a Rho/Rho-kinase signaling pathway that affects contractility by altering the Ca²⁺-sensitivity of the contractile system (Somlyo and Somlyo, 2003). The Rho-kinase pathway is involved in ureteral contractions in a number of species (Levent and Buyukafsar, 2004; Shabir et al, 2004; Hong et al, 2005). Rho-kinase inhibits myosin phosphatase by phosphorylation of its regulatory subunit, which prevents dephosphorylation of myosin light chain, which, in turn, leads to Ca²⁺ sensitization of the smooth muscle with a subsequent increase in contractility. Y-27632, an inhibitor of Rho-kinase, decreases both spontaneous and EFS-induced contractile responses of in vitro human ureteral segments (Hong et al, 2005). Y-27632 inhibits kinase activity by competing with ATP for binding to the Rho-kinases.

Urothelial Effects on Contractile Activity

Furchgott (1999) showed that the endothelium produced a factor that had a relaxing action on the smooth muscle layer of blood vessels. This factor was originally termed endothelium-derived relaxing factor (EDRF) and was subsequently shown to be nitric oxide (NO). Mastrangelo et al (2003) showed that the urothelium of rat ureter produced NO, which inhibited contractile responses of the rat ureter. Recently in the rat ureter, it was shown that the urothelium inhibited spontaneous contractions of isolated ureteral segments and that removal of the urothelium potentiated the stimulatory effects of neurokinin A, vasopressin, carbachol, bradykinin, and angiotensin II (Mastrangelo and Iselin, 2007). In intact ureteral segments, cyclooxygenase inhibitors potentiated the stimulatory effects of neurokinin A, vasopressin, carbachol, bradykinin, and angiotensin II. Cyclooxygenase inhibitors had no effect on the responses to these agents in urothelium-free ureters. These data suggest that the inhibitory effects of the urothelium on ureteral contractile events may involve the participation of a urothelial cyclooxygenase product such as prostacyclin.

Second Messengers

The functional response to a number of hormones, neurotransmitters, and other agents is mediated by means of second messengers. The agonist, or first messenger, interacts with a specific membrane-bound receptor (Alquist, 1948; Furchgott, 1964); the agonist-receptor complex then activates or inactivates an enzyme, which leads to alteration of a second messenger within the cell. These second messengers include cyclic AMP (cAMP), cyclic GMP (cGMP), Ca²⁺, inositol 1,4,5-trisphosphate (IP₃), and diacylglycerol (DG). They mediate the functional response to the agonist (first messenger) through a process that frequently involves protein phosphorylation.

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