Cellular mechanism of the voltage-dependent change in slow potentials generated in circular smooth muscle of the guinea-pig gastric corpus.

The cellular mechanism of the voltage-dependent properties of slow potentials were investigated in single bundles of circular smooth muscle isolated from the gastric corpus of guinea-pig using conventional microelectrode recordings. Hyperpolarization of the membrane by current injection decreased the frequency and increased the amplitude of slow potentials linearly. At potentials negative of -80 mV, slow potential generation was abolished and a periodic generation of clustered unitary potentials was evident. Application of cyclopiazonic acid (CPA, 20 microM) or thapsigargin (1 microM; inhibitors of Ca(2+)-ATPase), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 0.1 microM; mitochondrial protonophore) or 2-aminoethoxydiphenyl borate (2-APB, 20 microM; inhibitor of IP(3) receptor-mediated Ca(2+) release) depolarized the membrane and reduced or inhibited the amplitude and frequency of slow potentials: repolarization of the membrane to the resting level by current injection resulted in a recovery of the amplitude of slow potentials in the presence of CPA or CCCP, but not 2-APB. The slow potentials abolished by thapsigargin did not recover upon membrane repolarization. The altered frequency of slow potentials by 2-APB, CPA or CCCP was not reversed by membrane repolarization to control potentials. Depolarization of the membrane by about 10 mV with high-potassium solution also reduced the amplitude and increased the frequency of slow potentials in a manner restored by repolarization to control potentials upon current injection, suggesting that membrane depolarization did not affect the voltage dependency of pacemaker activity. The results indicate that in corpus circular muscles the voltage dependency of the frequency and amplitude of slow potentials requires a functional Ca(2+) store and mitochondria.

Effects of imatinib mesylate on spontaneous electrical and mechanical activity in smooth muscle of the guinea-pig stomach.

BACKGROUND AND PURPOSE: Effects of imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, on spontaneous activity of interstitial cells of Cajal (ICC) and smooth muscles in the stomach were investigated. EXPERIMENTAL APPROACH: Effects of imatinib on spontaneous electrical and mechanical activity were investigated by measuring changes in the membrane potential and tension recorded from smooth muscles of the guinea-pig stomach. Its effects on spontaneous changes in intracellular concentration of Ca(2+) ([Ca(2+)](i)) (Ca(2+) transients) were also examined in fura-2-loaded preparations. KEY RESULTS: Imatinib (1-10 microM) suppressed spontaneous contractions and Ca(2+) transients. Simultaneous recordings of electrical and mechanical activity demonstrated that imatinib (1 microM) reduced the amplitude of spontaneous contractions without suppressing corresponding slow waves. In the presence of nifedipine (1 microM), imatinib (10 microM) reduced the duration of slow waves and follower potentials in the antrum and accelerated their generation, but had little affect on their amplitude. In contrast, imatinib reduced the amplitude of antral slow potentials and slow waves in the corpus. CONCLUSIONS AND IMPLICATIONS: Imatinib may suppress spontaneous contractions of gastric smooth muscles by inhibiting pathways that increase [Ca(2+)](i) in smooth muscles rather than by specifically inhibiting the activity of ICC. A high concentration of imatinib (10 microM) reduced the duration of slow waves or follower potentials in the antrum, which reflect activity of ICC distributed in the myenteric layers (ICC-MY), and suppressed antral slow potentials or corporal slow waves, which reflect activity of ICC within the muscle bundles (ICC-IM), presumably by inhibiting intracellular Ca(2+) handling.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4. RESULTS: In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate. CONCLUSIONS: ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.

Evolving mechanisms of action of alverine citrate on phasic smooth muscles.

BACKGROUND AND PURPOSE: We have investigated the mechanisms underlying the paradoxical ability of the antispasmodic, alverine, to enhance spontaneous activity in smooth muscles while suppressing evoked activity. EXPERIMENTAL APPROACH: The effects of alverine on spontaneous and induced contractile activity were examined in preliminary experiments with various smooth muscles. More detailed effects were also investigated by recording membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and tension from single-bundle detrusor smooth muscle (DSM) of the guinea-pig urinary bladder. KEY RESULTS: Alverine (10 microM) increased the frequency and amplitude of spontaneous action potentials, transient increases in [Ca2+]i and associated contractions. Alverine also decreased action potential rate of decay, suggesting inhibition of L-type Ca channel inactivation. Charybdotoxin (50 nM) but neither cyclopiazonic acid (10 microM) nor Bay K 8644 (10 microM) attenuated alverine-induced enhancement of spontaneous contractions. Alverine suppressed contractions produced by high K (40 mM) or ACh (10 microM), without affecting electrical responses and with little suppression of increases in [Ca2+]i. This feature was very similar to that of the effects of the Rho kinase inhibitor Y-27632 (10 microM). CONCLUSIONS AND IMPLICATIONS: Alverine may increase Ca influx during action potentials due to inhibition of the inactivation of L-type Ca channels, but may also suppress evoked activity by inhibiting the sensitivity of contractile proteins to Ca2+. The proportional contribution of Ca-dependent and Ca-independent contractions in DSM may differ between spontaneous and evoked activity, necessitating further investigations into the interactions between these pathways for assessing the therapeutic potential of alverine to treat DSM dysfunction.

Mechanisms of PTHrP-induced inhibition of smooth muscle contractility in the guinea pig gastric antrum.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) that causes hypercalcemia of malignancy appears to function as an endogenous smooth muscle relaxant. For example, PTHrP released upon bladder wall distension relaxes detrusor smooth muscle to accommodate urine. Here, we explored mechanisms underlying PTHrP-induced suppression of the smooth muscle contractility in the gastric antrum that also undergoes a passive distension. METHODS: Effects of PTHrP on phasic contractions and electrical slow waves in the antral smooth muscle of the guinea pig stomach were studied using isometric tension and intracellular microelectrode recordings, respectively. Fluorescent immunohistochemistry was also carried out to identify the distribution of PTH/PTHrP receptors. KEY RESULTS: Parathyroid hormone-related protein (1-100 nM) reduced the amplitude of phasic contractions and the basal tension. Nω -nitro-l-arginine (L-NA, 100 µM), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 10 µM), a guanylate cyclase inhibitor, diminished the PTHrP (10 nM)-induced reduction in the amplitude of phasic contractions. SQ22536 (300 µM), an adenylate cyclase inhibitor, attenuated the PTHrP-induced reduction in basal tension. The combination of ODQ (10 µM) and SQ22536 (300 µM) inhibited the PTHrP-induced reductions in both phasic contractions and basal tension. PTHrP (100 nM) had no inhibitory effect on the electrical slow waves in the antral smooth muscle. PTH/PTHrP receptors were expressed in cell bodies of PGP9.5-positive neurons in the myenteric plexus. CONCLUSIONS & INFERENCES: Parathyroid hormone-related protein exerts its inhibitory actions on the antral smooth muscle via both nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and cyclic adenosine monophosphate (AMP) pathways. Thus, PTHrP may act as an endogenous relaxant of the gastric antrum employing the two complementary signaling pathways to ensure the adaptive relaxation of stomach.

Autonomic and sensory nerve modulation of peristalsis in the upper urinary tract.

The primary function of the upper urinary tract is to propel urine and various water-soluble toxic compounds from the kidneys to the bladder for storage and evacuation to maintain body ionic balance and contribute to the regulation of blood volume and pressure. The mechanism by which the upper urinary tract propels urine has long been considered to be myogenic in origin as peristaltic contractions in vivo and in vitro (pyeloureteric peristalsis) propagate in a manner little affected by drugs that block nerve conduction or the sympathetic and parasympathetic transmission. However, it is now well established that the release of intrinsic prostaglandins and neuropeptides from primary sensory nerves (PSNs) helps to maintain pyeloureteric peristalsis. Electrical field stimulation of PSNs evokes species-specific positive inotropic and chronotropic effects that have been attributed to release of excitatory tachykinins superimposed on negative inotropic and chronotropic effects associated with the release of calcitonin gene related peptide (CGRP), a rise in cellular cyclic-adenosine monophosphate (cAMP) and a protein kinase A-dependent activation of glibenclamide-sensitive ATP-dependent K+ (KATP) channels. This review summarises the existing evidence of the nervous control of the upper urinary tract and recent evidence suggesting that the autonomic innervation may indirectly modulate pyeloureteric peristalsis via the activation of PSN nicotinic receptors and via the modulation of KV7 channels located on interstitial cells within the renal pelvis wall.

Endothelium-dependent relaxation and hyperpolarization of canine coronary artery smooth muscles in relation to the electrogenic Na-K pump.

1 In the smooth muscle cells of canine coronary artery, acetylcholine (ACh) produced a transient, endothelium-dependent hyperpolarization of the membrane. A similar hyperpolarization was also elicited by exposure to Krebs solution after incubation of the artery in K-free solution for 30 min. 2 A hyperpolarization of reproducible amplitude was generated when ACh was applied at intervals greater than 30 min. Repetitive application of ACh at 15 min intervals caused a successive reduction in the amplitude of hyperpolarization. 3 The reduction in the amplitude of relaxation during five successive applications of ACh at 15 min intervals was less than 10% of the first relaxation. 4 The ACh-induced hyperpolarization was blocked by atropine but not by ouabain, whereas the K-free induced hyperpolarization was blocked by ouabain. In low Na (Li-substituted) solution, ACh still induced a hyperpolarization but the K-free induced hyperpolarization was absent. 5 In coronary artery precontracted by high-K solution, ACh produced an endothelium-dependent relaxation, without membrane hyperpolarization. The associated relaxation was resistant to ouabain but sensitive to atropine. 6 It is concluded that in the canine coronary artery, the electrogenic Na-K pump does not contribute to the endothelium-dependent hyperpolarization or relaxation. The results are consistent with the release of two different inhibitory factors from the vascular endothelium.

Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra.

1. Intracellular microelectrode recordings were made from circular smooth muscle of rabbit urethra. 2. The smooth muscle of urethra was spontaneously active exhibiting large, regularly occurring depolarizations, termed slow waves (SWs), 1-3 s in duration, up to 40 mV in amplitude and generated every 3-15 s and small irregularly occurring events (or summations there of) termed spontaneous transient depolarizations (STDs) of < 1 s in duration. 3. The SWs and STDs were not sensitive to 10(-6) M atropine, 10(-6) M phentolamine, 10(-5) M guanethidine or 10(-6) M tetrodotoxin, indicating that they were myogenic in origin. 4. Application of 3 x 10(-6) M nifedipine or 5 x 10(-5) M GdCl3 did not inhibit the generation of SWs or STDs, indicating that activation of L-type Ca2+ channels and non-selective cation channels are not essential for their generation. However, the duration of SWs but not STDs was reduced by nifedipine, indicating L-type Ca2+ channels contribute to the plateau-like potential of SWs. 5. Application of low chloride solution (6.4 mM), niflumic acid (10(-5) - 10(-4) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 10(-4) -5 x 10(-4) M) inhibited the generation of SWs and STDs, suggesting an involvement of chloride channels. 6. Application of nominally Ca2+ free solution, 5 x 10(-5) M BAPTA-AM, 10(-5) M cyclopiazonic acid, 10(-2) M caffeine or 10(-3) M procaine inhibited the generation of SWs and STDs, indicating that Ca2+ released from intracellular stores was required for the generation of SWs and STDs. 7. Exogenously applied noradrenaline (10(-7) - 10(-5) M) increased the frequency of SWs through stimulation of alpha-adrenoceptors which was inhibited by sodium nitroprusside (SNP, 10(-4) M). SNP also reduced the frequency of SWs without altering the membrane potential, an effect mimicked by 8-bromocyclic GMP (10(-3) M) indicating that SNP acted by elevating the production of cyclic GMP. 8. It is concluded that smooth muscle cells of the rabbit urethra exhibit SWs and STDs which are likely to be induced by stimulation of Ca(2+)-activated chloride channels evoked by release of Ca2+ from intracellular stores.

Modulators of internal Ca2+ stores and the spontaneous electrical and contractile activity of the guinea-pig renal pelvis.

1. The role of internal Ca(2+) stores in the generation of the rhythmic electrical and contractile activity in the guinea-pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. 2. Ryanodine (30 microM) transiently increased contraction amplitude, while caffeine (0.5 - 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2-aminoethoxy-diphenylborate (2-APB 60 microM), xestospongin C (1 microM), U73122 (5 microM) and neomycin (4 mM), blockers of IP(3)-dependent release from Ca(2+) stores. 3. 60 mM K(+) saline-evoked contractions were reduced by caffeine (1 mM), U73122 (5 microM) and neomycin (4 mM), but little affected by ryanodine or 2-APB (60 microM). 4. Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6+/-1.0 min(-1)) in small urothelium-denuded strips of proximal renal pelvis. 5. Action potential discharge was blocked in 75 and 35% of cells by 2-APB (60 microM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. 6. Cyclopiazonic acid (CPA 10 microM for 10 - 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. 7. We conclude that the action potential in caffeine-sensitive cells and the shoulder component of caffeine-insensitive action potential arise from the entry of Ca(2+) through Ca(2+) channels. The inhibitory actions of modulators of internal Ca(2+) release were partially explained by a blockade of Ca(2+) entry.

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.

Spectrophotometric determination of ruthenium with 8-quinolinol.

A spectrophotometric method is described for the determination of 2-80 mug of ruthenium. The method involves oxidation of ruthenium to ruthenate, addition of 8-quinolinol, adjustment of the pH to 4-6.5, digestion of the complex formed at 85 degrees for 30 min, extraction with chloroform, and measurement of absorbance at 430 nm. Almost all other metals and excess of reagent are removed by washing the extract. About 98 % of (106)Ru tracer was recovered.

Cesium-137 and mercury contamination in lake sediments.

The contribution of fluvial discharge and global fallout of 137Cs and mercury to sedimentation fluxes in Lake Shinji and Lake Nakaumi, Japan, was studied. The fluvial flux through soil erosion accounted for 11 to 27% of accumulated 137Cs in the sediments in the 1950's and 1960's, which were the periods of the most extensive atmospheric input, and for 90 to 100% in the 1990's. The vertical profiles of mercury concentrations in the lake sediments studied showed a maximum between 1959 and 1963, which was originated mostly from the extensive use of mercury-agrochemicals in paddyfields of the watershed in the past. These findings are representative examples of long-term contamination of lake sediments caused by the contaminated ground soil erosion.

Calcium responses in subserosal interstitial cells of the guinea-pig proximal colon.

BACKGROUND: In the subserosal layer between the longitudinal muscle layer and mesothelium, heterogeneous populations of interstitial cells are distributed. As the distribution of nerve elements in this layer is sparse as compared with the nerve plexus layer or tunica muscularis, there may be unique communication among subserosal interstitial cells (SSICs). This study aimed to explore functional properties of SSICs. METHODS: In subserosal preparations of the guinea-pig proximal colon, changes in intracellular Ca(2+) ([Ca(2+) ]i ) were visualized using Fluo-4 Ca(2+) imaging. Immunohistochemistry was also performed to identify the SSICs exhibiting Ca(2+) transients. KEY RESULTS: A majority of SSICs responded to adenosine triphosphate (ATP, 10 µM) by increasing [Ca(2+) ]i , but remained quiescent during the application of acetylcholine (10 µM). ATP-induced Ca(2+) responses were mimicked by adenosine 5'-diphosphate (10 µM), MRS2365 (10 nM) but not α, ß-methylene ATP (10 µM) or uridine triphosphate (10 µM), and could be reproduced in Ca(2+) -free solution, suggesting that ATP acts via P2Y receptors, most likely P2Y1 subtype, but not P2X receptors. Live staining of the same preparations after Ca(2+) imaging indicated the ATP-sensitive SSICs were not positive for c-Kit antibody, a specific marker for gastrointestinal interstitial cells of Cajal (ICC). Immunohistochemistry identified vimentin (mesenchymal cell marker)+/Kit- and SK3 (fibroblast-like cell (FLC) marker)+/Kit- cells that had a similar morphology to the ATP-sensitive SSICs in Ca(2+) imaging. CONCLUSIONS & INFERENCES: A majority of the SSICs in the guinea-pig proximal colon, presumably FLC, are capable of responding to ATP and thus may contribute to smooth muscle relaxation upon stimulation with ATP released from non-neuronal cells.

Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis.

BACKGROUND AND PURPOSE: The modulation of the spontaneous electrical and Ca(2+) signals underlying pyeloureteric peristalsis upon nicotinic receptor activation located on primary sensory afferents (PSAs) was investigated in the mouse renal pelvis. EXPERIMENTAL APPROACH: Contractile activity was followed using video microscopy, electrical and Ca(2+) signals in typical and atypical smooth muscle cells (TSMCs and ASMCs) within the renal pelvis were recorded separately using intracellular microelectrodes and Fluo-4 Ca(2+) imaging. KEY RESULTS: Nicotine and carbachol (CCh; 1-100 µM) transiently reduced the frequency and increased the amplitude of spontaneous phasic contractions in a manner unaffected by muscarininc antagonists, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) and pirenzipine (10 nM) or L-NAME (L-Nω-nitroarginine methyl ester; 200 µM), inhibitor of NO synthesis, but blocked by the nicotinic antagonist, hexamethonium or capsaicin, depletor of PSA neuropeptides. These negative chronotropic and delayed positive inotropic effects of CCh on TSMC contractions, action potentials and Ca(2+) transients were inhibited by glibenclamide (Glib; 1 µM), blocker of ATP-dependent K (KATP) channels. Nicotinic receptor-evoked inhibition of the spontaneous Ca(2+) transients in ASMCs was prevented by capsaicin but not Glib. In contrast, the negative inotropic and chronotropic effects of the non-selective COX inhibitor indomethacin were not prevented by Glib. CONCLUSIONS AND IMPLICATIONS: The negative chronotropic effect of nicotinic receptor activation results from the release of calcitonin gene-related peptide (CGRP) from PSAs, which suppresses Ca(2+) signalling in ASMCs. PSA-released CGRP also evokes a transient hyperpolarization in TSMCs upon the opening of KATP channels, which reduces contraction propagation but promotes the recruitment of TSMC Ca(2+) channels that underlie the delayed positive inotropic effects of CCh.

A survey of commonalities relevant to function and dysfunction in pelvic and sexual organs.

Micturition, defecation and sexual function are all programmed through spinal reflexes that are under descending control from higher centres. Interaction between these reflexes can clearly be perceived, and evidence is accumulating the dysfunction in one reflex is often associated with dysfunction in another. In this article, we describe some of the basic properties and neural control of the smooth muscles mediating the reflexes, reviewing the common features that underlie these reflex functions, and what changes may be responsible for dysfunction. We propose that autonomic control within the pelvis predisposes pelvic and sexual organs to crosstalk, with the consequence that diseases and conditions of the pelvis are subject to convergence on a functional level. It should be expected that disturbance of the function of one system will inevitably impact adjacent systems.

Factors modifying the frequency of spontaneous activity in gastric muscle.

The cellular mechanisms that determine the frequency of spontaneous activity were investigated in gastric smooth muscles isolated from the guinea-pig. Intact antral muscle generated slow waves periodically; the interval between slow waves was decreased exponentially by depolarization of the membrane to reach a steady interval value of about 7 s. Isolated circular muscle bundles produced slow potentials spontaneously or were evoked by depolarizing current stimuli. Evoked slow potentials appeared in an all-or-none fashion, with a refractory period of approximately 2-3 s. Low concentrations of chemicals that modify intracellular signalling revealed that the refractory period was causally related to the activity of protein kinase C (PKC). Activation of PKC increased and inhibition of PKC activity decreased the frequency of slow potentials. Chemicals that inhibit mitochondrial functions reduced the frequency of slow waves. Inhibition of internal Ca(2+)-store activity decreased the amplitude, but not the frequency of slow potentials, suggesting that the amplitude is causally related to Ca(2+) release from the internal store. The results suggest that changes in [Ca(2+)](i) caused by the activity of mitochondria may play a key role in determining the frequency of spontaneous activity in gastric pacemaker cells.

Neuroeffector transmission to different layers of smooth muscle in the rat penile bulb.

1. Using intracellular recording techniques, two distinct layers of smooth muscle were identified in the rat penile bulb. The inner muscle layer (parenchyma) exhibited spontaneous action potentials, while the outer sheet (sac) was electrically quiescent. 2. In the parenchyma, transmural stimulation initiated non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (IJPs) which were abolished by Nomeganitro-L-arginine (LNA) or 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The amplitude of IJPs was reduced by ouabain, dinitrophenol or decreasing the extracellular potassium concentration ([K+]o) but not by several K+ channel blockers. 3. The parenchyma also received an excitatory innervation mediated by alpha-adrenoceptors which caused a contraction that was not associated with a membrane potential change. 4. In the sac, transmural stimulation initiated two component excitatory junction potentials (EJPs) mediated by alpha-adrenoceptors and associated action potentials. The initial component was more dramatically suppressed than the secondary component by caffeine, ryanodine or cyclopiazonic acid (CPA). Lowering of the extracellular chloride concentration ([Cl-]o) selectively inhibited the rapid component of EJPs, while niflumic acid was less potent. 5. These results suggest that IJPs in the parenchyma result from the release of NO which stimulates sodium pump activity following the activation of guanylate cyclase. In the sac, the activation of alpha-adrenoceptors initiates EJPs by releasing Ca2+ from intracellular stores which activates Ca2+-activated channels.

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 microns) 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 microM), or substance P (0.1 microM), 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 microM). 3. In the presence of 50 microM 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 microns) 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 microM). The response produced by substance P was also reduced by 50 nM charybdotoxin: again the residual response was sensitive to 0.1 microM apamin. The hyperpolarizations produced by either ACh or substance P were insensitive to glibenclamide (10 microM) 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.

Altered electrical properties of bladder smooth muscle in streptozotocin-induced diabetic rats.

OBJECTIVE: To examine the alterations in the electrical properties of bladder smooth muscle in rats with diabetes mellitus (DM) induced by streptozotocin. MATERIALS AND METHODS: The study comprised 34 control rats and 20 with DM induced experimentally by injection with streptozotocin. At 10-15 weeks after the induction of DM, control and treated rats were killed, the bladder removed and the electrical responses of membranes from the detrusor smooth muscle recorded using intracellular microelectrodes. RESULTS: The resting membrane potential of the smooth muscle remained unaltered by DM but the frequency of spontaneous spike discharges sensitive to nicardipine was decreased. Transmural nerve stimulation elicited a purinergic excitatory junction potential (EJP) in muscles from both types of rat, but the threshold intensity of stimulation required to evoke an EJP was higher in muscle from the diabetic rat. Furthermore, EJPs large enough to trigger a nicardipine-sensitive spike potential could be elicited easily in muscles from the control rat, but not in muscles from diabetic rats. Stimulation of muscarinic receptors with exogenous acetylcholine (ACh) caused greater depolarization in muscle from diabetic rats. Stimulation of purinergic receptors with alpha, beta-methylene ATP caused a similar depolarization in both groups of rats. Application of potassium-free solution or ouabain depolarized the membrane by about 9 mV or 5 mV, respectively, in muscles from both groups of rat. Removal of potassium-free solution produced an ouabain-sensitive transient hyperpolarization which was larger in muscle from control rats, indicating that the potency of the Na-K pump was weakened in rats with DM. CONCLUSION: In the detrusor smooth muscle of rats with DM, there were fewer spontaneous spike discharges, supersensitivity of post-junctional muscarinic receptors, reduced potency of the post-junctional Na-K pump and a decrease in the release of neurotransmitter, possibly due to the impairment of prejunctional activity.

Effects of Y-26763, a novel K-channel opener, on electrical responses of smooth muscles in the guinea pig bladder.

PURPOSE: The effects of Y-26763, a novel K-channel opener, on spontaneous and carbachol-induced electrical responses of bladder smooth muscle in guinea pig were studied. MATERIALS AND METHODS: Intracellular microelectrodes were used to record membrane electrical responses from the detrusor smooth muscle of guinea pig. RESULTS: In most cells studied, the membranes were spontaneously active with continuous or periodic bursts of spike discharge which were sensitive to nicardipine, a Ca++ -antagonist. Y-26763 inhibited the spike discharges with or without hyperpolarization of the membrane; the spike inhibition appeared at lower concentrations (>10(-8 M.) than the hyperpolarization (>10(-7) M.). The effects of Y-26763 were antagonized by glibenclamide, which suggested an involvement of ATP-sensitive K-channels. Carbachol increased the discharge frequency of spikes, with (>10(-6)M.) or without (10(-7) M.) depolarization of the membrane. Y-26763 (>10(-7) M.) inhibited the spike generation but not the depolarization induced by carbachol (10(-6)M.) in a concentration-dependent manner. CONCLUSIONS: Y-26763 may directly prevent electrical excitability of bladder smooth muscles by inhibiting spike discharges and also by hyperpolarizing the membrane. The results suggest that this K-channel opener could have clinical applications for the treatment of unstable bladders.