Action Potentials In Smooth Muscle Research Articles

Characterization of alternating neurogenic motor patterns in mouse colon.

Colonic motor complexes (CMCs) have been widely recorded in the large intestine of vertebrates. We have investigated whether in the smooth muscle, a single unified pattern of electrical activity, or different patterns of electrical activity give rise to the different neurogenic patterns of motility underlying CMCs in vitro. To study differences of the CMCs between proximal and distal colon, we used a novel combination of techniques to simultaneously record muscle diameter and force at multiple sites along the whole mouse colon ex vivo. In addition, electrical activity of smooth muscle was recorded by suction electrodes. Two distinct types of CMCs were distinguished; CMCs that propagated along the entire colon (complete CMC) and CMCs which were restricted to the proximal colon (incomplete CMC). The two types of CMC often occurred in the same preparations. Incomplete CMCs had longer bursts of smooth muscle action potentials than complete CMCs and propagated more slowly. Interestingly, both types of CMC were associated with similar frequency bursts of smooth muscle action potentials at ~2.4Hz. In the most proximal colon, an additional firing frequency was detected close to ~7Hz generating multiple peaks within each CMC. We report distinct characteristics underlying complete and incomplete CMCs in isolated mouse colon. Recognizing these distinct patterns of motility will be important for future interpretation of analysis of murine colonic motility recordings. The identification of alternating patterns of motor activity in proximal colon, but not distal colon may reflect specific neural mechanisms for fecal pellet formation.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s−1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

Effects of K+ channel openers on spontaneous action potentials in detrusor smooth muscle of the guinea-pig urinary bladder

The modulation of spontaneous excitability in detrusor smooth muscle (DSM) upon the pharmacological activation of different populations of K+ channels was investigated. Effects of distinct K+ channel openers on spontaneous action potentials in DSM of the guinea-pig bladder were examined using intracellular microelectrode techniques. NS1619 (10μM), a large conductance Ca2+-activated K+ (BK) channel opener, transiently increased action potential frequency and then prevented their generation without hyperpolarizing the membrane in a manner sensitive to iberiotoxin (IbTX, 100nM). A higher concentration of NS1619 (30μM) hyperpolarized the membrane and abolished action potential firing. NS309 (10μM) and SKA31 (100μM), small conductance Ca2+-activated K+ (SK) channel openers, dramatically increased the duration of the after-hyperpolarization and then abolished action potential firing in an apamin (100nM)-sensitive manner. Flupirtine (10μM), a Kv7 channel opener, inhibited action potential firing without hyperpolarizing the membrane in a manner sensitive to XE991 (10μM), a Kv7 channel blocker. BRL37344 (10μM), a β3-adrenceptor agonist, or rolipram (10nM), a phosphodiesterase 4 inhibitor, also inhibited action potential firing. A higher concentration of rolipram (100nM) hyperpolarized the DSM and abolished the action potentials. IbTX (100nM) prevented the rolipram-induced blockade of action potentials but not the hyperpolarization. BK and Kv7 channels appear to predominantly contribute to the stabilization of DSM excitability. Spare SK channels could be pharmacologically activated to suppress DSM excitability. BK channels appear to be involved in the cyclic AMP-induced inhibition of action potentials but not the membrane hyperpolarization.

(2R,3S,2'' R,3''R)-manniflavanone, a new gastrointestinal smooth muscle L-type calcium channel inhibitor, which underlies the spasmolytic properties of Garcinia buchananii stem bark extract.

Garcinia buchananii Baker stem bark extract (GBB) is a traditional medication of diarrhea and dysentery in sub-Saharan Africa. It is believed that GBB causes gastrointestinal smooth muscle relaxation. The aim of this study was to determine whether GBB has spasmolytic actions and identify compounds underlying these actions. Calcium (Ca2+) imaging was used to analyze the effect of GBB on Ca2+ flashes and Ca2+ waves in guinea pig gallbladder and distal colon smooth muscle. Intracellular microelectrode recording was used to determine the effect of GBB, six fractions of GBB, M1–5 and M7, and (2R,3S,2”R,3”R)-manniflavanone, a compound isolated from M3 on action potentials in gallbladder smooth muscle. The technique was also used to analyze the effect of GBB, M3, and (2R,3S,2”R,3”R)-manniflavanone on action potentials in the circular muscle of mouse and guinea pig distal colons, and the effect of GBB and (2R,3S,2”R,3”R)-manniflavanone on slow waves in porcine ileum. GBB inhibited Ca2+ flashes and Ca2+ waves. GBB, M3 and (2R,3S,2”R,3”R)-manniflavanone inhibited action potentials. L-type Ca2+ channel activator Bay K 8644 increased the discharge of action potentials in mouse colon but did not trigger or increase action potentials in the presence of GBB and (2R,3S,2”R,3”R)-manniflavanone. GBB and (2R,3S,2”R,3”R)-manniflavanone inhibited action potentials in the presence of Bay K 8644. GBB and (2R,3S,2”R,3”R)-manniflavanone reduced the amplitude but did not alter the frequency of slow waves in the porcine ileum. In conclusion, GBB and (2R,3S,2”R,3”R)-manniflavanone relax smooth muscle by inhibiting L-type Ca2+ channels, thus have potential for use as therapies of gastrointestinal smooth muscle spasms, and arrhythmias.

Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

Members of the voltage-gated K(+) (K(V)) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits K(V)2.1, K(V)2.2, K(V)4.2, and K(V)2.1/9.3 channels. The objective of this study was to investigate whether K(V) channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 microM). This indicates that, under conditions of sustained membrane depolarization, the K(V) channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3, but not K(V)4.2 channel subunits. Protein expression of K(V)2.1 and K(V)2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that K(V)2.1 and K(V)2.2, but not K(V)4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.

Voltage‐gated K+ (Kv) currents sensitive to stromatoxin‐1 in Guinea pig urinary bladder smooth muscle

Members of the voltage‐gated K+ (Kv) channel superfamily have been suggested to mediate the resting membrane potential and the repolarization phase of the urinary bladder smooth muscle (UBSM) action potential. Here, we tested the hypothesis that stromatoxin‐1 (ScTx1)‐sensitive Kv channels are expressed and control the membrane excitability in Guinea pig UBSM cells. Freshly isolated UBSM cells were studied using RT‐PCR, immunocytochemistry and the perforated mode of the whole cell patch‐clamp technique. In all patch‐clamp experiments 100 nM paxilline, a specific BK channel inhibitor, was present in the recording solutions to separated the KV currents from the BK currents. Whole‐cell current was elicited by voltage‐step depolarizations to potentials between ‐70 to +30 mV. Application of 100 nM ScTx1 inhibited about 60% of the remaining whole‐cell current (n=5; P<0.05) and the effect was more pronounced at positive voltages. We have further identified the molecular fingerprints of the ScTx1‐sensitive Kv currents. RT‐PCR experiments on isolated cells revealed mRNA expression for Kv2.1 and Kv9.3 family members (n=3). The immunocytochemical experiments on freshly isolated single UBSM cells confirmed the expression of the Kv2.1 channel protein (n=3). Our data revealed that ScTx1‐sensitive Kv2.1 channels are expressed and serve to control cell excitability in Guinea pig UBSM cells. Supported by DK070909 to GVP

Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.

Action potential repolarization enabled by Ca++ channel deactivation in PSpice simulation of smooth muscle propagation.

BackgroundPreviously, only the rising phase of the action potential (AP) in cardiac muscle and smooth muscle could be simulated due to the instability of PSpice upon insertion of a second black box (BB) into the K+ leg of the basic membrane unit. This restriction was acceptable because only the transmission of excitation from one cell to the next was investigated.MethodsIn the current work, the repolarization of the AP was accomplished by inserting a second BB into the Ca++ leg of the basic membrane unit. Repolarization of the AP was produced, not through an activation of the K+ channel conductance, but rather through a mimicking of the deactivation of the Ca++ channel conductance. Propagation of complete APs was studied in a chain (strand) of 10 smooth muscle cells, in which various numbers of gap-junction (gj) channels (assumed to be 100 pS each) were inserted across the cell junctions.ResultsThe shunt resistance across the junctions produced by the gj-channels (Rgj) was varied from 100,000 MΩ (0 gj-channels) to 10,000 MΩ (1 gj-channel), to 1,000 MΩ (10 channels), to 100 MΩ (100 channels), to 10 MΩ (1000 channels), and to 1.0 MΩ (10,000 channels). Velocity of propagation (θ, in cm/sec) was calculated from the measured total propagation time (TPT, the time difference between when the AP rising phase of the first cell and the last cell crossed -20 mV), assuming a constant cell length of 200 μm. When there were no gj-channels, or only one, the transmission of excitation between cells was produced by the electric field (EF), i.e., the negative junctional cleft potential, that is generated in the narrow junctional clefts (e.g., 100 A) when the prejunctional membrane fires an AP (a fraction of a millisecond before the adjacent surface membrane). There were significant end-effects at the termination of the strand, such that the last cell (cell #10) failed to fire, or fired after a prolonged delay. This end-effect was abolished when the strand termination resistance (Rbt) was increased from 1.0 KΩ to 600 MΩ. When there were 1000 or 10,000 gj-channels, the transmission of excitation was produced by local-circuit current flow from one cell to the next through the gj-channels.DiscussionIn summary, it is now possible to simulate complete APs in smooth muscle cells that could propagate along a single chain of 10 cells, even when there were no gj-channels between the cells.

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.

Propagated repolarization of simulated action potentials in cardiac muscle and smooth muscle

BackgroundPropagation of repolarization is a phenomenon that occurs in cardiac muscle. We wanted to test whether this phenomenon would also occur in our model of simulated action potentials (APs) of cardiac muscle (CM) and smooth muscle (SM) generated with the PSpice program.MethodsA linear chain of 5 cells was used, with intracellular stimulation of cell #1 for the antegrade propagation and of cell #5 for the retrograde propagation. The hyperpolarizing stimulus parameters applied for termination of the AP in cell #5 were varied over a wide range in order to generate strength / duration (S/D) curves. Because it was not possible to insert a second "black box" (voltage-controlled current source) into the basic units representing segments of excitable membrane that would allow the cells to respond to small hyperpolarizing voltages, gap-junction (g.j.) channels had to be inserted between the cells, represented by inserting a resistor (Rgj) across the four cell junctions.ResultsApplication of sufficient hyperpolarizing current to cell #5 to bring its membrane potential (Vm) to within the range of the sigmoidal curve of the Na+ conductance (CM) or Ca++ conductance (SM) terminated the AP in cell #5 in an all-or-none fashion. If there were no g.j. channels (Rgj = ∞), then only cell #5 repolarized to its stable resting potential (RP; -80 mV for CM and -55 mV for SM). The positive junctional cleft potential (VJC) produced only a small hyperpolarization of cell #4. However, if many g.j. channels were inserted, more hyperpolarizing current was required (for a constant duration) to repolarize cell #5, but repolarization then propagated into cells 4, 3, 2, and 1. When duration of the pulses was varied, a typical S/D curve, characteristic of excitable membranes, was produced. The chronaxie measured from the S/D curve was about 1.0 ms, similar to that obtained for muscle membranes.ConclusionsThese experiments demonstrate that normal antegrade propagation of excitation can occur in the complete absence of g.j. channels, and therefore no low-resistance pathways between cells, by the electric field (negative VJC) developed in the narrow junctional clefts. Because it was not possible to insert a second black-box into the basic units that would allow the cells to respond to small hyperpolarizing voltages, only cell #5 (the cell injected with hyperpolarizing pulses) repolarized in an all-or-none manner. But addition of many g.j. channels allowed repolarization to propagate in a retrograde direction over all 5 cells.

Effect of Variation in Membrane Excitability on Propagation Velocity of Simulated Action Potentials for Cardiac Muscle and Smooth Muscle in the Electric Field Model for Cell-to-Cell Transmission of Excitation

We have previously published several studies on the propagation of simulated action potentials (APs) of cardiac muscle and smooth muscle using the PSpice program. Those studies were done on single chains of five to ten cells in length to examine longitudinal propagation between the cells, either not connected by gap-junction (g.j) channels or connected by various numbers of channels. In addition, transverse propagation was examined between parallel chains (two to five chains) not connected by g.j. channels. In all those studies, the myocardial cells and smooth muscle cells (SMCs) were unintentionally somewhat hyperexcitable by virtue of the values inserted into the GTABLEs of the PSpice program. Because transmission of excitation from cell to cell occurred very well in the absence of g.j. channels, by virtue of the electric field (EF) generated in the narrow junctional clefts (negative cleft potential V/sub JC/), the present study was carried out, in which the cells were made hypo-excitable by altering the GTABLE values. Three levels of excitability of the cardiac cells and SMC were examined: 1) high; 2) intermediate; and 3) low. It was found that propagation of excitation, both longitudinally and transversely, can occur by the EF mechanism alone, even when the excitability of the cells was low. Therefore, the EF mechanism alone can account for propagation of excitation in cardiac muscles and smooth muscles that do not possess gap junctions. In those cases in which gap junctions do exist and are functioning, the EF mechanism would act in parallel and thereby increase the safety factor for conduction.

Calcium imaging of gut activity.

The major cell types regulating gut motility include enteric neurones, interstitial cells of Cajal (ICC) and their effector smooth muscle cells. These cells are arranged conveniently in nested layers through the gut wall. Our knowledge of how many of these cells in each layer are integrated to produce the various patterns of motility is largely unknown. So far, much of our knowledge of gut motility has usually been obtained by examining point sources of activity (e.g. intracellular recordings from enteric neurones, ICC and smooth muscle cells), rather than the spread of activity through these spatially distributed nerve and ICC networks, or smooth muscle syncitia. Our understanding of how these cells are integrated to produce gut movements would be greatly enhanced if we could image the activity in many of these cells in each layer, or many cells in several layers, simultaneously. Calcium (Ca2+) is a major signalling and regulatory molecule in most cells. In fact, electrical excitability in enteric neurones, ICC and smooth muscle is associated with robust rises in intracellular Ca2+ that long outlast the electrical events (e.g. action potentials in neurones and smooth muscle) that gave rise to them. These prolonged Ca2+ responses, together with the development of several high quality Ca2+ indicators, has provided a unique opportunity to image many cells in intact tissues simultaneously using ICCD video-rate cameras along with conventional microscopy. However, confocal microscopy has also been used, and has several advantages over the above systems. These include reduced photo-toxicity and bleaching and the elimination of out of focus light from different layers within the tissue. So far, despite some limitations with the calcium imaging techniques, the spread of activity through the two layers of smooth muscle, ICC networks and myenteric neurones in intact preparations, or cultured myenteric neuronal networks, is beginning to yield exciting new data about how these different cells interact and process information.

Combined electric field and gap junctions on propagation of action potentials in cardiac muscle and smooth muscle in pspice simulation

Propagation of action potentials in cardiac muscle and smooth muscle were simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of 6 cells (cardiac muscle) or 10 cells (smooth muscle) either not connected (control) or connected by low-resistance tunnels (gap-junction connexons). A significant negative cleft potential (V jv ) develops in the narrow junctional cleft when the pre-JM fires. V jc depolarizes the postjunctional membrane (post-JM) to threshold by a patch-clamp action. With few connecting tunnels, cell-to-cell transmission by the EF mechanism was facilitated. With many tunnels, propagation was dominated by the low-resistance mechanism, and propagation velocity (θ) became very fast and nonphysiological. In conclusion, when the 2 mechanisms for cell-to-cell transfer of excitation were combined, the two mechanisms facilitated each other in a synergistic manner. When there were many connecting tunnels, the tunnel mechanism was dominant.

Effect of external resistance on propagation of action potentials in cardiac muscle and visceral smooth muscle in PSpice simulation

Propagation of action potentials in cardiac muscle and visceral smooth muscle were simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of six cells (cardiac muscle) or ten cells (smooth muscle) not connected by low-resistance tunnels (gap-junction connexons). The entire surface membrane of each cell fired nearly simultaneously, and nearly all the propagation time was spent at the cell junctions, thus, giving a staircase-shaped propagation profile. The junctional delay time was about 0.2-0.5 msec in cardiac muscle and 2–6 msec in smooth muscle. A significant negative cleft potential develops in the narrow junctional clefts, and its magnitude depends on several factors, including the radial cleft resistance ( R jc ). The cleft potential ( V C ) depolarizes the postjunctional membrane (post-JM) to threshold by a patch-clamp action. Therefore, one mechanism for the transfer of excitation from one cell to the next is by the electric field (EF) that is generated in the junctional cleft when the pre-JM fires. When the external resistance ( R o ) was increased over a very wide range (e.g., the radial ( r or ) and longitudinal ( r ol ) components increased over 10 6 times) (from 1 KΩ to 1000 MΩ), propagation still occurred. At R jc values between 5 MΩ and 40 MΩ, the propagation velocity (θ) actually increased in cardiac muscle. At lower R jc : values (1, 0.1, 0.01MΩ), propagation was not only facilitated at high R o values, but also enabled because partial or complete block occurred at the lower R o values. Similar results were found for smooth muscle. These data underscore the fact that propagation, in this simulation model, does not occur by means of local-circuit current. Instead, transmission across the junctional cleft occurs by the EF in the cleft.

Glycyrrhetinic derivatives inhibit hyperpolarization in endothelial cells of guinea pig and rat arteries.

Glycyrrhetinic acid (GA) derivatives have been used to implicate gap junctions in vasorelaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). The aim of this study was to assess whether GA compounds affect endothelial cell hyperpolarization. Membrane potentials were recorded from dye-identified endothelial and smooth muscle cells of guinea pig coronary and rat mesenteric arteries. GA derivatives had varied effects on the resting membrane potential: depolarization, hyperpolarization, or no effect, depending on the artery. 18alpha-GA (50 microM) had a small variable effect on ACh-induced hyperpolarizations in endothelial cells. 18beta-GA (30 microM) and carbenoxolone (100 microM) significantly reduced ACh-induced hyperpolarizations in both endothelial and smooth muscle cells. Smooth muscle action potentials in rat tail arteries were smaller and slower in the presence of 18beta-GA. Nerve-induced excitatory junction potentials were inhibited by 18beta-GA and carbenoxolone, whereas the time course of their decay initially increased and then decreased. In conclusion, the GA compounds had a range of effects. Their inhibition of the EDHF hyperpolarization and relaxation in the smooth muscle may stem from the inhibition of endothelial cell hyperpolarization.

BK channels play an important role as a negative feedback mechanism in the regulation of spontaneous rhythmic contraction of urinary bladder smooth muscles

Smooth muscles of urinary bladder wall exhibit spontaneous rhythmic contraction which is myogenic in origin. Although the precise mechanism responsible for the generation of this mechanical activity remains to be established, it can be related closely to the action potential (AP) in urinary bladder smooth muscle (UBSM) cell, and may be the fundamental constituent to determine urinary bladder physiological functions to store and micturate urine. In the present study, possible roles of voltage-dependent and Ca(2+)-sensitive K+ (BK) channels, highly expressed in UBSM cells, were examined in the regulation of spontaneous UBSM contraction with reference to the generation of AP. Iberiotoxin (IbTx), a selective BK channel blocker, strongly increased mechanical activity and AP generation in guinea-pig UBSM. In contrast, BK channel openers (NS-1619, niflumic acid; estradiol, tamoxifen: BK channel alpha- and beta-subunit activators, respectively) significantly diminished AP generation and spontaneous mechanical activity. The present study indicates that BK channels play the primary role as a negative feedback element to limit extracellular Ca2+ influx through affecting AP configurations in the generation of UBSM contraction. BK channel openers including beta-subunit activators may be a potentially useful therapeutic remedy for the treatment of urinary bladder dysfunctions such as frequent urination.

Propagating contractions of the circular muscle evoked by slow stretch in flat sheets of guinea-pig ileum.

Flat sheet preparations of guinea-pig ileum were stretched circumferentially and the propagation of circular muscle contractions along the preparation was investigated. Slow stretch, at 100 microm s-1, of a 50-mm long flat sheet of intestine, evoked circular muscle contraction orally, which propagated, without decrement, for up to 30 mm. This occurred despite circular muscle shortening being prevented, and in the absence of propulsion of contents. Thus, propagation in this flat sheet preparation could not explained on the basis of neuro-mechanical interactions, as previously proposed. Irrespective of the length of preparations, contraction amplitude decreased significantly in the most aboral 10-15 mm of intestine. This was not due to descending inhibitory pathways, but was associated with interruption of ascending excitatory pathways near the aboral end. Slow waves were not detected in circular muscle cells in any preparation (n=8). Smooth muscle action potentials evoked in circular muscle cells, in the presence of tetrodotoxin (TTX, 0.6 micromol L-1), did not propagate for more than 1 mm in the longitudinal axis. Propagation of circular muscle activity, evoked by slow stretch of flat sheet preparations, reveals the presence of a mechanism other than myogenic spread or the neuro-mechanical interactions previously proposed to account for propagation; the nature of this mechanism remains to be determined.

K+ currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels.

Membrane currents attributed to endothelium-derived hyperpolarizing factor (EDHF) were recorded in short segments of submucosal arterioles of guinea-pigs using single microelectrode voltage clamp. The functional responses of arterioles and human subcutaneous, rat hepatic and guinea-pig coronary arteries were also assessed as changes in membrane potential recorded simultaneously with contractile activity. The current-voltage (I-V) relationship for the conductance due to EDHF displayed outward rectification with little voltage dependence. Components of the current were blocked by charybdotoxin (30-60 nM) and apamin (0.25-0.50 microM), which also blocked hyperpolarization and prevented EDHF-induced relaxation. The EDHF-induced current was insensitive to Ba2+ (20-100 microM) and/or ouabain (1 microM to 1 mM). In human subcutaneous arteries and guinea-pig coronary arteries and submucosal arterioles, the EDHF-induced responses were insensitive to Ba2+ and/or ouabain. Increasing [K+]o to 11-21 mM evoked depolarization under conditions in which EDHF evoked hyperpolarization. Responses to ACh, sympathetic nerve stimulation and action potentials were indistinguishable between dye-labelled smooth muscle and endothelial cells in arterioles. Action potentials in identified endothelial cells were always associated with constriction of the arterioles. 18beta-Glycyrrhetinic acid (30 microM) and carbenoxolone (100 microM) depolarized endothelial cells by 31 +/- 6 mV (n = 7 animals) and 33 +/- 4 mV (n = 5), respectively, inhibited action potentials in smooth muscle and endothelial cells and reduced the ACh-induced hyperpolarization of endothelial cells by 56 and 58 %, respectively. Thus, activation of outwardly rectifying K+ channels underlies the hyperpolarization and relaxation due to EDHF. These channels have properties similar to those of intermediate conductance (IKCa) and small conductance (SKCa) Ca2+-activated K+ channels. Strong electrical coupling between endothelial and smooth muscle cells implies that these two layers function as a single electrical syncytium. The non-specific effects of glycyrrhetinic acid precludes its use as an indicator of the involvement of gap junctions in EDHF-attributed responses. These conclusions are likely to apply to a variety of blood vessels including those of humans.

Initiation of peristalsis by circumferential stretch of flat sheets of guinea-pig ileum.

1. Segments of isolated guinea-pig intestine, 12 mm long, were distended slowly by intraluminal fluid infusion or by mechanical stretch as either a tube or flat sheet. In all cases, at a constant threshold length, a sudden, large amplitude contraction of the circular muscle occurred orally, corresponding to the initiation of peristalsis. 2. Circumferential stretch of flat sheet preparations evoked graded contractions of the longitudinal muscle (the 'preparatory phase'), which were maintained during circular muscle contraction. This suggests that the lengthening reported during the emptying phase of peristalsis is due to mechanical interactions. 3. The threshold for peristalsis was lower with more rapid stretches and was also lower in long preparations (25 mm) compared with short preparations (5-10 mm), indicating that ascending excitatory pathways play a significant role in triggering peristalsis. 4. Stretching a preparation beyond the threshold for peristalsis evoked contractions of increasing amplitude; thus peristalsis is graded above its threshold. However, during suprathreshold stretch maintained at a constant length, contractions of the circular muscle quickly declined in amplitude and frequency. 5. Circular muscle cells had a resting membrane potential approximately 6 mV more negative than the threshold for action potentials. During slow circumferential stretch, subthreshold graded excitatory motor input to the circular muscle occurred, prior to the initiation of peristalsis. However, peristalsis was initiated by a discrete large excitatory junction potential (12 +/- 2 mV) which evoked bursts of smooth muscle action potentials and which probably arose from synchronized firing of ascending excitatory neuronal pathways.

Tetrodotoxin-sensitive action potentials in smooth muscle of mouse vas deferens

Action potentials were recorded during impalements of some but not all smooth muscle cells of mouse vas deferens in response to both nerve stimulation and intracellular current injection. They were resistant to blockade by nifedipine (0.1–1.0 μM) but were blocked by tetrodotoxin (TTX, 0.2–1.0 μM) when this was added in the presence of nifedipine. It is suggested that voltage-dependent sodium (Na +) channels are present in mouse vas deferens that function to amplify calcium (Ca 2+) influx through voltage-dependent Ca 2+ channels.