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B K Fleischmann

Publications and source records attributed to B K Fleischmann.

35 records · Page 2Linked to original sources
PubMed1999

ATP-evoked Ca2+ transients and currents in murine thymocytes: possible role for P2X receptors in death by neglect.

The P2X family of ATP receptors (P2XR) have been implicated in thymocyte death in vitro and in vivo. We characterized ATP-evoked Ca2+ transients and membrane currents in thymocytes to better understand the role of P2XR during thymocyte development. ATP4-, but not UTP or GTP, activated a sustained non-selective cation current in voltage-clamped CD4- CD8- and CD4+ CD8+ thymocytes that was reversed by apyrase, which hydrolyzes ATP, and by the P2XR antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The more selective P2XR agonist alphabeta-methylene ATP activated a smaller rapidly decaying current in both thymocyte populations. Reverse transcription-PCR results indicate that P2X1, P2X2, P2X6, and/or P2X7 are expressed in thymocytes. Finally, we used PPADS to examine the role of P2XR during thymocyte development in situ. PPADS-treated thymi yielded significantly more thymocytes (38%), due to a selective increase in CD4+ CD8+ cells. Together these data suggest that one or more PPADS-sensitive P2XR (P2X1, P2X2, P2X7) are involved in thymocyte apoptosis, and we propose more specifically a role associated with death by neglect.

Adenosine Triphosphate↗
PubMed1999

Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture.

Three-dimensional cell aggregates (embryoid bodies, EBs) containing clusters of spontaneously beating cardiomyocytes were derived from permanent mouse embryonic stem (ES) cells. Extracellular recordings of the population action potentials of cardiomyocyte clusters were made using permanently mounted silver wire electrodes and microelectrode arrays integrated into the bottom of the culture dish. These techniques allowed long-term recordings (for up to several weeks) from individual EBs under cell culture conditions. The normal electrical activity consisted of regular spiking with a frequency of 0.5-5 Hz. However, most EBs (87%) spontaneously developed temporary or persistent complex activity patterns because of intermittent block of action potential propagation at narrow pathways connecting larger beating areas. Similar propagation blocks could also be reversibly induced in regularly spiking EBs by nimodipine (NDP). In addition to a slowing of pacemaker activity, NDP (20-200 nM) induced a stepwise decrease of the action potential frequency at the recording site. Perforated patch-clamp recordings from enzymatically isolated ES-cell-derived cardiomyocytes showed that similar activity patterns do not occur at the single-cell level. We suggest that this novel approach may provide a useful tool for in vitro studies of chronotropy and phenomena of propagation failure similar to AV block.

Action Potentials↗
PubMed1999

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition.

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.

Adenine↗
PubMed1999

Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system.

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis.

Animals↗
PubMed1999

Role of ATP-dependent K(+) channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes.

Single, murine embryonic stem cell-derived early stage cardiomyocytes dissociated from embryoid bodies expressed two inward rectifier K(+) channels, I(K1) and the ATP dependent K(+) current. I(K1) exhibited low density in early stage cardiomyocytes, but increased significantly in late stage cells. In contrast, the ATP dependent K(+) current was expressed at similar densities in early and late stage cardiomyocytes. This current was found to be involved in the determination of the membrane potential, since glibenclamide depolarized early cardiomyocytes and exerted a positive chronotropic effect. Some cardiomyocytes displayed a bursting behavior of action potentials, characterized by alternating periods with and without action potentials. During the phases without action potentials, the membrane potential was hyperpolarized, indicating the involvement of K(+) channels in the generation of this bursting behavior. Extracellular recording techniques were applied to spontaneously contracting areas of whole embryoid bodies. In 20% of these bursting behavior similar to that seen in the single cells was observed. In regularly beating embryoid bodies, bursting could be induced by reduction of substrates from the extracellular medium as well as by superfusion with the positive chronotropic agents Bay K 8644 or isoproterenol. Perfusion with substrate-reduced medium induced bursting behavior after a short latency, isoproterenol and Bay K 8644 resulted in a positive chronotropic response followed by bursting behavior with longer latencies. The spontaneous bursting was blocked by glibenclamide. These experimental results suggest that intermittent activation of ATP dependent K(+) channels underlies the bursting behavior observed in single cardiomyocytes and in the whole embryoid body. Conditions of metabolic stress lead to the rhythmic suppression of action potential generation. Our data indicate that ATP dependent K(+) channels play a prominent role in the cellular excitability of early cardiomyocytes.

2,4-Dinitrophenol↗
PubMed1998

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1998

Cardiac specific expression of the green fluorescent protein during early murine embryonic development.

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Actins↗
PubMed1997

Muscarinic activation and calcium permeation of nonselective cation currents in airway myocytes.

We examined the activation and Ca2+ permeation of nonselective cation channels in voltage-clamped (nystatin), fura 2-loaded equine tracheal myocytes at 35 degrees C. Methacholine (50 microM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) and a biphasic inward current consisting of a large, rapidly inactivating Ca(2+)-activated Cl current [ICl(Ca)] and a smaller, sustained nonselective cation current (Icat) ICl(Ca) but not Icat was activated by caffeine. Neither Icat nor the sustained rise in [Ca2+]i was blocked by nisoldipine, whereas both were rapidly blocked by Ni2+; Icat was determined to be Ca2+ permeant, since 1) a sustained elevation of [Ca2+]i occurred when Icat was activated, and blockade of Icat produced a rapid decline in [Ca2+]i; 2) increasing extracellular Ca2+ during Icat increased [Ca2+]i; 3) 110 mM extracellular Ca2+ shifted the reversal potential of Icat to 12 mV (Ca(2+)-to-Cs+ permeability ratio = 3.6); and 4) instantaneous voltage-clamp steps to negative potentials during Icat increased the current and [Ca2+]i, whereas depolarizing steps decreased the current and [Ca2+]i. The fraction of Icat carried by Ca2+ under physiological conditions was estimated to be 14% at -60 mV.

Animals↗
PubMed1997

Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes.

The role of Ca2+ influx through voltage-dependent Ca2+ channels and the inhibitory effects of methacholine on large-conductance Ca2+-activated K+ (K(Ca)) channels (maxi-K+ channels) were studied in voltage-clamped (nystatin), fura 2-loaded airway smooth muscle cells. Spontaneous transient outward currents (STOCs) were strongly coupled to voltage-dependent Ca2+ channel activity; activity was suppressed by nisoldipine and Cd2+ and increased by BAY K 8644 within seconds. Moreover, release of intracellular Ca2+ by caffeine or cyclopiazonic acid only partially suppressed STOCs, and the remainder were almost completely blocked by nisoldipine. Methacholine suppressed STOCs but also significantly decreased the mean outward current. Whole cell current inhibition was observed in the presence of 4-aminopyridine but not in the presence of charybdotoxin. Caffeine inhibited STOCs but macroscopic outward currents were not altered. In the continued presence of caffeine, methacholine abolished the remaining STOCs and decreased the mean K+ current. We conclude that STOCs are activated by influx of Ca2+ through plasmalemmal voltage-dependent Ca2+ channels, as well as by release of Ca2+ from intracellular stores, and muscarinic stimulation depresses the mean K(Ca) current via a pathway independent of the depletion of intracellular Ca2+ stores.

Animals↗
PubMed1997

M2 receptor activation of nonselective cation channels in smooth muscle cells: calcium and Gi/G(o) requirements.

Muscarinic stimulation of fura 2-loaded smooth muscle cells evoked a rapidly inactivating Ca(2+)-activated Cl- current [ICl(Ca)] and a sustained nonselective cation current (Icat) as well as a transient (delta Ca(tran)) and a sustained (delta Ca(sus)) elevation of cytosolic Ca2+ concentration ([Ca2+]i). Caffeine and inositol 1,4,5-trisphosphate induced delta Ca(tran) and ICl(Ca) but not Icat or delta Ca(sus). M2 receptor antagonism blocked muscarinic activation of Icat and delta Ca(sus) but not ICl(Ca) and delta Ca(tran). M3 antagonism blocked activation of ICl(Ca) and Icat and a rise in [Ca2+]i, but application of caffeine with methacholine restored Icat and delta Ca(sus). After depletion of intracellular Ca2+ stores, methacholine failed to induce Icat or a [Ca2+]i increase and, in pertussis toxin-treated cells, ICl(Ca) and delta Ca(tran) but not Icat or delta Ca(sus) were evoked. Anti-G alpha i-1/G alpha i-2 antibodies and anti-G alpha i-3/ G(o) alpha antibodies blocked Icat but did not affect ICl(Ca). Anti-Gq alpha/ G alpha 11 antibodies greatly inhibited ICl(Ca) but did not affect Icat. Activation of M2 receptors leads to the opening of nonselective cation channels through Gi/G(o) proteins in smooth muscle cells, resulting in a sustained rise in [Ca2+]i. Arise in [Ca2+]i is necessary but not sufficient for activation of nonselective cation channels.

Animals↗
PubMed1996

Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle.

Patch clamp and RNA-polymerase chain reaction methods were used to determine the expression of voltage-dependent potassium channel currents and mRNAs in human airway smooth muscle cells, and tension measurements were used to examine the functional role of specific potassium channel gene products in human bronchial smooth muscle. RNA from airway smooth muscle tissue revealed the presence of Kv1.2 (11 kilobases (kb)) and Kv1.5 (3.5 and 4.4 kb) transcripts, as well as Kv1.1 mRNA (9.5 kb), which has not previously been reported in smooth muscle; transcripts from other gene families were not detected. RNA-polymerase chain reaction from cultured human myocytes confirmed that the identified transcripts were expressed by smooth muscle cells. The available voltage-dependent potassium current in human airway myocytes was insensitive to charybdotoxin (200 nM) but blocked by 4-aminopyridine. Dendrotoxin (1-300 nM; inhibits Kv1.1 and Kv1.2 channels), charybdotoxin (10 nM to 1 microM; inhibits KCa and Kv1.2 channels), and glybenclamide (0.1-100 microM; inhibits KATP channels) had no effect on resting tone. Conversely, 4-aminopyridine increased resting tension with an EC50 (1.8 mM) equivalent to that observed for current inhibition (1.9 mM). Human airway myocytes express mRNA from several members of the Kv1 gene family; the channel that underlies the predominate voltage-dependent current and the regulation of basal tone appears to be Kv1.5.

Base Sequence↗
PubMed1996

Voltage-dependent calcium currents and cytosolic calcium in equine airway myocytes.

1. The relationship between voltage-dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura-2 AM-loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/- 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium-induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, (ii) increases in [Ca2+]i associated with I(Ca) were consistent with cytoplasmic buffering of calcium ions entering through voltage-dependent calcium channels (VDCCs) only, (iii) there was a fixed instantaneous relationship between transmembrane calcium flux (J(Ca)) and the change in cytosolic free calcium concentration (delta [Ca2+]i) during I(Ca), (iv) caffeine (8 mM) triggered 8-fold higher calcium transients than I(Ca), and (v) I(Ca) evoked following release of intracellular calcium by caffeine resulted in an equivalent delta[Ca2+]i-J(Ca) relationship. 3. The time constant (T) for the decay in [Ca2+]i was 8.6 +/- 1.5 s (n = 8) for single steps and 8.6 +/- 1.1 s (n = 13) following multiple steps that increased [Ca2+]i to much higher levels. Following application of caffeine (8 mM), however, [Ca2+]i decay was enhanced (T = 2.0 +/- 0.2 s, n = 3). The rate of [Ca2+]i decay was not voltage dependent, was not decreased in the absence of extracellular Na+ ions, and no pump current was detected. 4. We conclude that under near physiological conditions, neither CICR nor Na(+)-Ca2+ exchange play a substantial role in the regulation of I(Ca)-induced increases in [Ca2+]i, and that, even following release of intracellular calcium by caffeine, Na(+)-Ca2+ exchange does not play an appreciable role in the removal of calcium ions from the cytosol.

Animals↗
PubMed1995

Identification of Kv1.1 expression by murine CD4-CD8- thymocytes. A role for voltage-dependent K+ channels in murine thymocyte development.

The patch-clamp recording technique and RNA-polymerase chain reaction were used to identify the voltage-dependent K+ channels expressed by murine fetal and adult CD4-CD8- thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K+ channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 +/- 8% (n = 8 experiments), and CTX-treated thymi contained 74 +/- 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K+ channels are critical to thymocyte preclonal expansion and/or maturation.

Animals↗
PubMed1994

Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells.

Action potentials activate voltage-dependent calcium channels and attendant increases in cytosolic calcium concentration ([Ca2+]i) in many excitable cells. The role of these channels in the regulation of [Ca2+]i in nonspiking cells that do not depolarize to membrane potentials sufficient to activate a substantial fraction of the available current is less clear. Measurements of the peak activation and steady-state inactivation of L-type calcium currents have predicted the existence of a noninactivating current window over a voltage range where channel inactivation is incomplete. The degree to which such small currents might regulate [Ca2+]i, however, has not been established. Here we demonstrate a "calcium window" in nondialyzed, quiescent smooth muscle cells over a small voltage range near the resting membrane potential. Sustained depolarizations in this voltage range, but not to more positive potentials, resulted in sustained rises in calcium, despite the fact that macroscopic inward currents were < 2 pA. The calcium window corresponded well with the predicted window current determined under the same conditions; the peak of the calcium window occurred at -30 mV, with steady-state rises in [Ca2+]i in some cells at -50 mV. Steady-state rises in [Ca2+]i following depolarization were completely blocked by nisoldipine and were augmented and shifted to more negative potentials by BAY K8644. Voltage-dependent calcium channels thus regulate steady-state calcium levels in nonspiking cells over a voltage range where macroscopic currents are only barely detectable. This voltage range is bounded at negative potentials by calcium channel activation and at more positive potentials by channel inactivation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells.

1. In order to determine the physiological role of specific potassium currents in airway smooth muscle, potassium currents were measured in freshly dissociated ferret trachealis cells using the nystatin-permeabilized, whole-cell method, at 35 degrees C. 2. The magnitude of the outward currents was markedly increased as bath temperature was increased from 22 to 35 degrees C. This increase was primarily due to the increase in maximum potassium conductance (gK,max), although there was also a small leftward shift in the relationship between gK and voltage at higher temperatures. The maximum conductance and the kinetics of current activation and inactivation were also temperature dependent. At 35 degrees C, gating of the current was steeply voltage dependent between -40 and 0 mV. Current activation was well fitted by fourth-order kinetics; the mean time constants of activation (30 mV clamp step) were 1.09 +/- 0.17 and 1.96 +/- 0.27 ms at 35 and 22 degrees C, respectively. 3. Outward currents using the nystatin method were qualitatively similar to delayed rectifier currents recorded in dialysed cells with high calcium buffering capacity solutions. 4-Aminopyridine (4-AP; 2 mM), a specific blocker of delayed rectifier potassium channels in this tissue, inhibited over 80% of the outward current evoked by voltage-clamp steps to between -10 and +20 mV (n = 6). Less than 5% of the outward current was blocked over the same voltage range by charybdotoxin (100 nM; n = 15), a specific antagonist of large-conductance, calcium-activated potassium channels in this tissue. 4. The degree to which delayed rectifier and calcium-activated potassium conductances control resting membrane potential was examined in current-clamp experiments. The resting membrane potential of current clamped cells was -33.6 +/- 1.0 mV (n = 62). Application of 4-AP (2 mM) resulted in a 14.4 +/- 1.0 mV depolarization (n = 8) and an increase in input resistance. Charybdotoxin (100 nM) had no effect on resting membrane potential (n = 6). 5. Force measurements were made in isolated strips of trachealis muscle to determine the effect of pharmacological blockade of individual potassium conductances on resting tone. In the presence of tetrodotoxin (1 microM) and atropine (1 microM), 4-AP increased baseline tension in a dose-dependent manner, with an EC50 of 1.8 mM (n = 13); application of 5 mM 4-AP increased tone to 86.8 +/- 8.1% of that produced by 1 microM methacholine, and this tone was almost completely inhibited by nifedipine (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1993

Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques.

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗