The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca²⁺ from intracellular stores and activation of Ca²⁺-activated Cl⁻ channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca²⁺ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na⁺/Ca²⁺ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca²⁺ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca²⁺ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca²⁺ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na⁺/Ca²⁺ exchange inhibitor, CGP-37157 inhibited Ca²⁺ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca²⁺ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca²⁺ (Ca_V3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced Ca_V3.2 currents. CCCP blocked Ca_V3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca²⁺ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.     

Effects of transient receptor potential channel blockers on pacemaker activity in interstitial cells of Cajal from mouse small intestine

The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract and transient receptor potential melastatin type 7 (TRPM7) is a candidate for pacemaker channels. The effect of the 5-lipoxygenase (5-LOX) inhibitors NDGA, AA861, MK886 and zileuton on pacemaking activity of ICCs was examined using the whole cell patch clamp technique. NDGA and AA861 decreased the amplitude of pacemaker potentials in ICC clusters, but the resting membrane potentials displayed little change, respectively. Also, perfusing NDGA and AA861 into the bath reduced both inward current and outward current in TRPM7-like current in single ICC, respectively. But, they had no effects on Ca²⁺ activated Cl⁻ currents. The 5-LOX inhibitors MK886 and zileuton were, however, ineffective in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC, respectively. A specific TRPC3 inhibitor, pyrazole compound (Pyr3), and a specific TRPM4 inhibitor, 9-phenanthrol, had no effects in pacemaker potentials in ICC clusters and in TRPM7-like current in single ICC. These results suggest that, among the tested 5-LOX inhibitors, NDGA and AA861 modulate the pacemaker activities of the ICCs, and that the TRPM7 channel can affect intestinal motility.     

A biophysically based mathematical model of unitary potential activity in interstitial cells of Cajal

Unitary potential (UP) depolarizations are the basic intracellular events responsible for pacemaker activity in interstitial cells of Cajal (ICCs), and are generated at intracellular sites termed “pacemaker units”. In this study, we present a mathematical model of the transmembrane ion flows and intracellular Ca²⁺ dynamics from a single ICC pacemaker unit acting at near-resting membrane potential. This model quantitatively formalizes the framework of a novel ICC pacemaking mechanism that has recently been proposed. Model simulations produce spontaneously rhythmic UP depolarizations with an amplitude of ∼3 mV at a frequency of 0.05 Hz. The model predicts that the main inward currents, carried by a Ca²⁺-inhibited nonselective cation conductance, are activated by depletion of sub-plasma-membrane [Ca²⁺] caused by sarcoendoplasmic reticulum calcium ATPase Ca²⁺ sequestration. Furthermore, pacemaker activity predicted by our model persists under simulated voltage clamp and is independent of [IP₃] oscillations. The model presented here provides a basis to quantitatively analyze UP depolarizations and the biophysical mechanisms underlying their production.     

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca²⁺ activated Cl⁻ channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720- P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.     

Use of a microelectrode array to record extracellular pacemaker potentials from the gastrointestinal tracts of the ICR mouse and house musk shrew (Suncus murinus)

BackgroundThe rhythmic contraction and relaxation of smooth muscles in the gastrointestinal (GI) tract is governed by pacemaker electrical potentials, also termed slow waves, which are calcium currents generated by interstitial cells of Cajal (ICCs). Malfunction of pacemaker rhythms contributes to a number of clinically challenging gastrointestinal motility disorders.MethodA microelectrode array (MEA) was used to record slow waves in vitro from intact GI tissues freshly isolated from the ICR mouse and Suncus murinus. The effects of temperature, extracellular calcium and potassium concentrations on pacemaker potentials were quantified using spatiotemporal metrics.ResultsPacemaker frequency decreased from the duodenum to the ileum in the mouse, but this phenomenon was less significant in Suncus murinus. In both the mouse and Suncus murinus, the stomach had a much lower pacemaker frequency than the intestine. Propagation velocity and amplitude were highest in the proximal intestine. Temperature significantly increased pacemaker frequency in the intestinal tissues of both species. Removal of Ca²⁺ from the medium inhibited pacemaker potential and increasing the Ca²⁺ concentration increased pacemaker frequency in the mouse ileum. Increasing K⁺ concentration decreased pacemaker frequency in the absence of nifedipine.ConclusionsThe MEA allows efficient investigation of gut pacemaker frequency and propagation.     

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca²⁺-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of–70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M₃ receptor antagonist, but not by methotramine, a muscarinic M₂ receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na⁺-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca²⁺. In recording of intracellular Ca²⁺ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca²⁺ concentrations with increasing of Ca²⁺ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M₃ receptors by a G-protein dependent intracellular Ca²⁺ release mechanism.     

Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization

This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP₃R)-operated Ca²⁺ stores interact as coupled oscillators to produce near-synchronous Ca²⁺ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP₃, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca²⁺ transients and/or intracellular Ca²⁺ waves. Larger near-synchronous Ca²⁺ transients then occurred leading to global synchronous Ca²⁺ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca²⁺ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca²⁺ transients and resultant action potentials, leaving only asynchronous Ca²⁺ transients and local Ca²⁺ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca²⁺ release from IP₃R-operated Ca²⁺ stores, which causes depolarization; 2), L-type Ca²⁺ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca²⁺ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca²⁺ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca²⁺ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca²⁺ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca²⁺ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca²⁺ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells.     

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca²⁺-free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-β-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca²⁺ influx, phospholipase C activation, and Ca²⁺ release from internal storage in a G protein-independent and protein kinase C-independent manner.     

Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca²⁺ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca²⁺ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca²⁺ transient wave fronts propagated through the ICC-MY networks at 2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca²⁺ transients in LM but had no visible effect on the transients in ICC-MY. -Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca²⁺ entry (Ni²⁺ and mibefradil) and elevated external K⁺ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca²⁺ transients in ICC-MY. These data show that global Ca²⁺ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca²⁺ handling mechanisms and entrained by voltage-dependent Ca²⁺ entry and coupling of cells via gap junctions. Ca²⁺ signaling; slow waves; gastrointestinal motility     

Biophysically Based Mathematical Modeling of Interstitial Cells of Cajal Slow Wave Activity Generated from a Discrete Unitary Potential Basis

Spontaneously rhythmic pacemaker activity produced by interstitial cells of Cajal (ICC) is the result of the entrainment of unitary potential depolarizations generated at intracellular sites termed pacemaker units. In this study, we present a mathematical modeling framework that quantitatively represents the transmembrane ion flows and intracellular Ca²⁺ dynamics from a single ICC operating over the physiological membrane potential range. The mathematical model presented here extends our recently developed biophysically based pacemaker unit modeling framework by including mechanisms necessary for coordinating unitary potential events, such as a T-Type Ca²⁺ current, V_m-dependent K⁺ currents, and global Ca²⁺ diffusion. Model simulations produce spontaneously rhythmic slow wave depolarizations with an amplitude of 65 mV at a frequency of 17.4 cpm. Our model predicts that activity at the spatial scale of the pacemaker unit is fundamental for ICC slow wave generation, and Ca²⁺ influx from activation of the T-Type Ca²⁺ current is required for unitary potential entrainment. These results suggest that intracellular Ca²⁺ levels, particularly in the region local to the mitochondria and endoplasmic reticulum, significantly influence pacing frequency and synchronization of pacemaker unit discharge. Moreover, numerical investigations show that our ICC model is capable of qualitatively replicating a wide range of experimental observations.     

Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice

Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and pharmacological actions in various organs. However, little is known about the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker potentials by ginsenoside in the interstitial cells of Cajal (ICCs) using the whole-cell patch clamp technique in the current clamp mode. Among ginsenosides, we investigated the effects of ginsenoside Rb1, Rg3 and Rf. While externally applied Rb1 and Rg3 had no effects on pacemaker potentials, Rf caused membrane depolarization. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker potentials and inhibited the Rf-induced membrane depolarization. Membrane depolarization induced by Rf was not inhibited by intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca²⁺-free solution, thapsigargin, a Ca²⁺-ATPase inhibitor of the endoplasmic reticulum, U-73122, a phospholipase C inhibitor, or 2-APB, an IP3 receptor inhibitor, abolished the generation of pacemaker potentials and suppressed Rfinduced actions. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block Rf-induced effects on pacemaker potentials. These results suggest that ginsenoside Rf modulates the pacemaker activities of ICCs and therby regulates intestinal motility.     

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca²⁺. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca²⁺ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca²⁺ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.     

Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 µM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca²⁺ channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (dV/dt_max) of pacemaker potentials and slow waves in a dose-dependent manner (1–30 µM). Mibefradil (30 µM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 µM), an ATP-sensitive K⁺ channel opener, hyperpolarized ICC-MY and increased the amplitude and dV/dt_max without affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and dV/dt_max of slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 µM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca²⁺ channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca²⁺ entry during upstroke depolarization. pacemaker activity; slow waves; gastrointestinal motility; calcium channel     

Ionic requirements for bursting activity in lobster stomatogastric neurons

Summary The ionic requirements for bursting activity have been investigated in the electrically coupled PD-AB cells group of the Stomatogastric ganglion in the lobster.The passive electrical properties and coupling parameters have been determined in either current or voltage clamp conditions. In voltage clamped cells, the current displayed slow inward transients with superimposed fast transients corresponding respectively to the slow waves and spikes of the coupled undamped cells. The amplitude and frequency of the slow transients were reduced upon hyperpolarization.Cyclic conductance changes were observed with short current pulses, the coupling ratio also changes cyclically being lower during the bursts and slowly increasing during the interburst interval.The slow wave amplitude increased in low K-saline. The post-burst hyperpolarization but not the top level of the wave behaved like a potassium electrode for [K]_o higher than 10 mM/l.TEA at low concentration (1 to 5 mM/l) increased the slow wave amplitude by lifting its top level by 10 to 20 mV. The post-burst hyperpolarization remained almost unchanged and its K-dependence was not altered by TEA.Low Na-saline reduced the slow wave amplitude (6 to 11 mV per decade). The Na-dependence increased in the presence of TEA. Slow waves devoid of spikes persisted in 10% Na saline containing TEA. 10^–9 M/1 TTX blocked the spikes. 10^–7 M/1 TTX blocked the slow waves.Mg-free saline had no effect on the slow wave. In Ca-free saline the cells depolarized and the bursting activity tended to vanish. Repolarization with current led to long lasting slow waves deprived of post-burst hyperpolarization. The bursting ceased when EDTA was added to the Ca-free saline.Cobalt (up to 10 mM/l) was similar to Ca-free saline in its effects; lengthening of the wave and blockage of the repolarization. Replacing Ba for Ca produced large (up to 70 mV) slow waves which were reduced by Co and Ca.Bistable states were observed in various experimental conditions. It is concluded that the slow waves are produced by activation of sodium and calcium currents. The amplitude of the slow wave is modulated by the simultaneous activation of a TEA-sensitive K current. The repolarization is caused by increased K current activated by the inward calcium current. The slow pacemaker potential in the interburst interval corresponds to the slow disappearance of the K current.This work was supported by N.I.H. grant no. 09322, NSF grant no. 00250, and a Guggenheim Foundation Fellowship to A.D.S. and by the CNRS and a DGRST grant no. 16501891 to M. Gola. We are grateful to Stuart Thompson and Felix Strumwasser for helpful comments and to Barbara McLean for technical assistance.     

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF''s importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).     

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (V_ex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs⁺ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (I_ex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs⁺ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs⁺ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs⁺-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca²⁺]_o (8.1 mM), and are reduced by low Ca²⁺ (0.45 mM), Cd²⁺ (0.2 mM) and Ni²⁺ (2 mM). Ni²⁺ also abolishes the tail current that follows steps more positive than E_Ca. We conclude that Cs⁺ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca²⁺-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.     

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P_1,3,4,5 agonist) and SEW 2871 (an S1P₁ agonist) had no effects on pacemaker activity. Suramin (an S1P₃ antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P₂ antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P₂ in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH₂-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca²⁺]_i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P₂ via regulation of external and internal Ca²⁺ and mitogenactivated protein kinase activation.     

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca²⁺ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K⁺-, Na⁺-free pipette solution, and bathed with K⁺-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca²⁺ current. Trials with depolarized holding potential, Ca²⁺ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na⁺ current (I_Na). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late I_Na may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I_Na.     

Ryanodine inhibits calcium transients evoked by action potentials in a subpopulation of rat dorsal root ganglion neurons

Effects of ryanodine on calcium transients evoked by depolarization of external membrane under voltage clamp conditions or by a train of action potentials under current clamp conditions were studied on isolated dorsal root ganglion neurons of newborn rats. In 70% neurons tested, ryanodine, a blocker of Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum, significantly decreased the amplitude of calcium transients. The data obtained indicate that the Ca²⁺-induced Ca²⁺ release plays an important role for calcium signal generation in a subpopulation of sensory neurons.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 420–422, November–December, 1994.