Potentiation of Ca(2+)-activated secretory activity by a cAMP-mediated mechanism in avian salt gland cells

1994 ◽  
Vol 267 (1) ◽  
pp. C255-C265 ◽  
Author(s):  
S. C. Martin ◽  
J. Thompson ◽  
T. J. Shuttleworth
Keyword(s):  
Membrane Potential ◽  
Driving Force ◽  
Secretory Activity ◽  
Salt Gland ◽  
Receptor Activation ◽  
Equilibrium Potential ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Cl Secretion ◽  
K Current

In the avian salt gland carbachol (CCh) evokes oscillations in K+ and Cl- current that are sufficient to fully activate secretory activity. Employing the perforated patch-clamp technique, we demonstrate that beta-adrenergic receptor activation stimulates a sustained adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- current with no increase in K+ current. This evokes only a modest increase in secretory activity. However, application of isoproterenol in the presence of a threshold dose of CCh results in maximal secretory activity. Membrane potential measurements demonstrate that isoproterenol stimulates a sustained membrane depolarization from approximately -45 mV to the Cl- equilibrium potential (ECl), whereas CCh evokes oscillations in membrane potential to levels more negative than ECl, representing a mixture of K+ and Cl- conductances. We conclude that, in agreement with current models of fluid secretion, maximal stimulation can only be achieved with simultaneous activation of both K+ and Cl- currents. Because isoproterenol fails to stimulate a K+ current, Cl- secretion is reduced as the driving force for Cl- secretion is dissipated. However, if a driving force is imposed by increasing K+ channel activity (by coadministering CCh), Cl- efflux is sustained. These results could provide a basis for the marked potentiation of Ca(2+)-mediated secretion by agonists that increase cAMP seen in in vivo studies of salivary glands and other exocrine tissues.

scholarly journals Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish.

1976 ◽  
Vol 67 (6) ◽  
pp. 621-638 ◽  
Author(s):  
S Hagiwara ◽  
S Miyazaki ◽  
N P Rosenthal
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Time Dependent ◽  
Equilibrium Potential ◽  
K Channel ◽  
Egg Cell ◽  
Dimensionless Number ◽  
First Order Kinetics ◽  
K Current ◽  
Kinetics Of

The kinetics of the membrane current during the anomalous or inward-going rectification of the K current in the egg cell membrane of the starfish Mediaster aequalis were analyzed by voltage clamp. The rectification has instantaneous and time-dependent components. The time-dependent increase in the K conductance for the negative voltage pulse as well as the decrease in the conductance for the positive pulse follows first-order kinetics. The steady-state conductance increases as the membrane potential becomes more negative and reaches the saturation value at about -40 mV more negative than the K equilibrium potential, V(K). The entire K conductance can be expressed by g(K).n; g g(K) represents the component for the time-independent conductance which depends on V-V(K) and [K+]o, and n is a dimensionless number (1 is greater than or equal to n is greater than or equal to 0) and determined by two rate constants which depend only on V-V(K). Cs+ does not carry any significant current through the K channel but blocks the channel at low concentration in the external medium. The blocking effect increases as the membrane potential is made more negative and the potential-dependent blocking by the external Cs+ also has instantaneous and time-dependent components.

scholarly journals Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.

1993 ◽  
Vol 102 (4) ◽  
pp. 667-692 ◽  
Author(s):  
E Hamada ◽  
T Nakajima ◽  
S Ota ◽  
A Terano ◽  
M Omata ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Epithelial Cell ◽  
Cell Line ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Induced Response ◽  
Epithelial Cell Line ◽  
Bathing Solution ◽  
Pipette Solution ◽  
K Current

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Modulation of membrane currents by cyclic AMP in cleavage-arrested Drosophila neurons.

1996 ◽  
Vol 199 (3) ◽  
pp. 537-548
Author(s):  
W B Alshuaib ◽  
L Byerly
Keyword(s):  
Cyclic Amp ◽  
Membrane Currents ◽  
K Channel ◽  
Neuronal Membrane ◽  
Patch Clamp Technique ◽  
Large Variability ◽  
Whole Cell ◽  
Intracellular Environment ◽  
K Current ◽  
Channel Currents

A number of Drosophila learning mutants have defective intracellular second-messenger systems. In an effort to develop techniques that will allow direct measurement of the effects of these mutations on whole-cell neuronal membrane currents, the perforated-patch whole-cell (PPWC) technique has been applied to cleavage-arrested cultured embryonic Drosophila neurons. This technique permits the measurement of membrane currents without disturbing the intracellular environment. As a result of the maintenance of the intracellular environment, Drosophila neuron currents are found to be much more stable than when measured using the conventional whole-cell (CWC) patch-clamp technique. Ca2+ channel currents, which typically 'wash out' within a few minutes of the beginning of CWC recording, are stable for the duration of the seal (tens of minutes) when measured using the PPWC technique. Since the learning mutations dunce and rutabaga disrupt cyclic AMP signalling, the action of externally applied dibutyryl cyclic AMP (db-cAMP) and theophylline on Ca2+ and K+ channel currents were studied. db-cAMP and theophylline enhanced the Ba2+ current, carried by Ca2+ channels, but had no effect on the K+ current in the cleavage-arrested neurons. However, the large variability and reduction in density of Ba2+ and K+ currents raise questions about the suitability of using these cleavage-arrested cells as models for Drosophila neurons.

Diverse current and voltage responses to baclofen in an identified molluscan photoreceptor

1995 ◽  
Vol 74 (2) ◽  
pp. 506-518 ◽  
Author(s):  
L. D. Matzel ◽  
I. A. Muzzio ◽  
R. F. Rogers
Keyword(s):  
Voltage Clamp ◽  
Action Potentials ◽  
Gabab Receptor ◽  
Outward Current ◽  
Gabab Receptors ◽  
Equilibrium Potential ◽  
K Channel ◽  
Electrode Voltage ◽  
Voltage Dependent ◽  
K Current

1. gamma-Aminobuturic acid-B (GABAB) receptors play a role in the mediation of slow inhibitory postsynaptic potentials in mammalian as well as some nonmammalian species. In identified photoreceptors from the marine mollusc Hermissenda, recent evidence has suggested that GABA, as well as the GABAB receptor agonist baclofen, might simultaneously modulate multiple conductances on the postsynaptic membrane. Here, using intracellular current-clamp and single-electrode voltage-clamp techniques, we have characterized responses to baclofen in the B photoreceptors of the Hermissenda eye. 2. Microapplication of baclofen (12.5–62.5 microM) to the terminal branches of the B photoreceptors induced a slow, concentration-dependent hyperpolarization (approximately 3–8 mV) that was accompanied by a cessation of spontaneous action potentials and a positive shift in firing threshold. Both the hyperpolarization and the shift in spike threshold in response to baclofen were attenuated largely by the K+ channel blocker tetraethylammonium chloride (TEA; 50 mM). 3. Bath application of baclofen (100 microM) decreased the amplitude, duration, and the afterhyperpolarization (AHP) of evoked action potentials. Although baclofen's effect on spike duration and amplitude persisted in the absence of extracellular Ca2+, the reduction of the AHP by baclofen was eliminated, suggesting that multiple conductances mediated the baclofen-induced modification of the action potential. 4. Using a single-electrode voltage-clamp technique, microapplication of baclofen to the terminal branches of the B photoreceptor produced a slow, net outward current (< 0.5 nA) that reversed near the equilibrium potential for K+ and shifted to more positive potentials when extracellular K+ was increased, in approximate agreement with the Nernst equation for K+. 5. Baclofen induced an increase in amplitude of the nonvoltage dependent leak conductance (IL), and the increase was blocked by TEA. The baclofen-induced increase of IL was accompanied by an increase in amplitude and a negative shift in the voltage dependence of a slow, steeply voltage-dependent K+ current (IK), which displays selective sensitivity to TEA but does not normally contribute to leak conductance. The amplitude and steady-state inactivation of a fast, transient K+ current, as well as the amplitude of an inwardly rectifying K+ current were unaffected by baclofen. 6. Both the rate of activation as well as the amplitude of a voltage-dependent Ca2+ current (ICa) were reduced by baclofen. The reduction of ICa resulted in a concomitant suppression of a Ca(2+)-dependent K+ current (IK-Ca) that was sufficient to account for the reduction of the AHP after evoked action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of intracellular calcium by membrane potential in human melanoma cells

1993 ◽  
Vol 265 (6) ◽  
pp. C1501-C1510 ◽  
Author(s):  
B. Nilius ◽  
G. Schwarz ◽  
G. Droogmans
Keyword(s):  
Cell Proliferation ◽  
Membrane Potential ◽  
Intracellular Calcium ◽  
Reversal Potential ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Human Melanoma Cells

The modulation of intracellular calcium ([Ca2+]i) by the membrane potential was investigated in human melanoma cells by combining the nystatin-perforated patch-clamp technique with Ca2+ measurements. Voltage steps to -100 mV induced a rise in [Ca2+]i and a creeping inward current. These effects were absent in Ca(2+)-free solution and could be blocked by Ni2+ or La3+. Voltage ramps revealed a close correlation between [Ca2+]i and voltage, with the strongest voltage dependence around the resting potential. Long-lasting tail currents, closely correlated with the rise in [Ca2+]i and a reversal potential close to the K+ equilibrium potential, occurred if the membrane potential was clamped back to 0 mV. They were absent if intracellular K+ was replaced by Cs+ and blocked by extracellular tetraethylammonium (5 mM), Ba2+ (1 mM), or a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue. These observations are discussed in relation to cell proliferation. The enhanced expression of K+ channels during cell proliferation provides a positive-feedback mechanism resulting in long-term changes in [Ca2+]i required for the G1-S transition in the cell cycle.

Acute reductions in PO2 depolarize pulmonary artery endothelial cells and decrease [Ca2+]i

1994 ◽  
Vol 266 (4) ◽  
pp. H1416-H1421 ◽  
Author(s):  
T. Stevens ◽  
D. N. Cornfield ◽  
I. F. McMurtry ◽  
D. M. Rodman
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Pulmonary Artery ◽  
Driving Force ◽  
Primary Cultures ◽  
K Channel ◽  
Channel Blockers ◽  
Electrochemical Gradient ◽  
Similar Reduction ◽  
Fura 2

Whereas pulmonary artery endothelial cells (PAECs) are sensitive to oxygen, neither the effect of an acute reduction in PO2 on PAEC membrane potential nor its effect on intracellular free Ca2+ ([Ca2+]i) is known. We hypothesized that in confluent primary cultures of PAECs, an acute decrease in PO2 would depolarize the cell membrane, inhibit Ca2+ influx, and reduce [Ca2+]i. To test this hypothesis, the membrane-sensitive fluorophore bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4, 1 microM) and [Ca2+]i-sensitive probe fura 2 (3 microM) were used. A decrease in PO2 from 125 to 35 mmHg caused membrane depolarization and a 60 +/- 8% (data are means +/- SE) reduction in Ca2+ influx, estimated by manganese quenching of fura 2 fluorescence. While basal [Ca2+]i was 79 +/- 5 nM in normoxic cells, it decreased to 31 +/- 2 nM after 15 min of hypoxia. Decreasing the electrochemical gradient for Ca2+ entry with either low extracellular Ca2+, the K+ channel blockers tetraethylammonium or charybdotoxin, or blockade of Ca2+ entry with lanthanum decreased [Ca2+]i by 54-71% of that observed during an acute reduction in PO2. These results demonstrate that an acute reduction in PO2 1) depolarizes PAECs, 2) reduces Ca2+ influx, and 3) decreases [Ca2+]i, and that a similar reduction in [Ca2+]i was observed with interventions designed to reduce the electrochemical driving force for Ca2+ entry.

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29

1997 ◽  
Vol 273 (4) ◽  
pp. C1186-C1193 ◽  
Author(s):  
P. Sand ◽  
T. Svenberg ◽  
B. Rydqvist
Keyword(s):  
Membrane Potential ◽  
Reversal Potential ◽  
Tumor Cell Line ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Control Solution ◽  
Whole Cell ◽  
Calcium Dependent ◽  
High K ◽  
Ht 29

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells ( n = 23) depolarized close to the equilibrium potential for Cl−([Formula: see text]; −48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). In substitution experiments (22 mM Cl− in bath solution,[Formula: see text]= 0 mV), the reversal potential changed from −41.6 ± 2.2 mV (means ± SE, n = 9) to −3.2 ± 2.0 mV (means ± SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 ± 3.7 mV (means ± SE, n = 11) with control solution in the pipette and at 46.2 ± 5.3 mV (means ± SE, n = 10) with a 15 mM Cl− solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly ( P ≤ 0.05, n = 8). In inside-out patches, calcium-dependent Cl−channels could be demonstrated with a conductance of 19 pS ( n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl−channels and a K+ permeability.

An outwardly rectifying K+ current active near resting potential in human retinal pigment epithelial cells

1995 ◽  
Vol 269 (1) ◽  
pp. C179-C187 ◽  
Author(s):  
B. A. Hughes ◽  
M. Takahira ◽  
Y. Segawa
Keyword(s):  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Outward Current ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
K Current

Currents in freshly dissociated adult human retinal pigment epithelial (RPE) cells were studied using the perforated patch-clamp technique. The zero-current potential (V0) averaged -48.9 +/- 7.7 mV (n = 50). Depolarizing voltage pulses from -70 mV evoked an outward current that activated with first-order kinetics and that did not inactivate during prolonged depolarizations. Repolarizing the membrane potential produced tail currents that reversed near the K+ equilibrium potential, indicating that the sustained outward current was carried mainly by K+. The outwardly rectifying K+ conductance (gK) had an activation threshold voltage near -60 mV and was half-maximal at -37 mV. Approximately 25% of gK was active at the average V0. The K+ current was nearly completely blocked by 2 mM Ba2+ but was relatively insensitive to 20 mM tetraethylammonium. The kinetics, voltage dependence, and blocker sensitivity of this current clearly distinguish it from delayed rectifier K+ currents previously identified in RPE cells. We conclude that the sustained outward K+ current may help establish the resting potential of the apical and/or basolateral membranes and may also participate in K+ transport across the RPE.

Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

2002 ◽  
Vol 282 (6) ◽  
pp. C1396-C1403 ◽  
Author(s):  
Atsushi Inanobe ◽  
Akikazu Fujita ◽  
Minoru Ito ◽  
Hitonobu Tomoike ◽  
Kiyoshi Inageda ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Granule Cells ◽  
Pdz Domain ◽  
Postsynaptic Membrane ◽  
Equilibrium Potential ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Kir Channel ◽  
Anchoring Proteins

Classical inwardly rectifying K+ channels (Kir2.0) are responsible for maintaining the resting membrane potential near the K+ equilibrium potential in various cells, including neurons. Although Kir2.3 is known to be expressed abundantly in the forebrain, its precise localization has not been identified. Using an antibody specific to Kir2.3, we examined the subcellular localization of Kir2.3 in mouse brain. Kir2.3 immunoreactivity was detected in a granular pattern in restricted areas of the brain, including the olfactory bulb (OB). Immunoelectron microscopy of the OB revealed that Kir2.3 immunoreactivity was specifically clustered on the postsynaptic membrane of asymmetric synapses between granule cells and mitral/tufted cells. The immunoprecipitants for Kir2.3 obtained from brain contained PSD-95 and chapsyn-110, PDZ domain-containing anchoring proteins. In vitro binding assay further revealed that the COOH-terminal end of Kir2.3 is responsible for the association with these anchoring proteins. Therefore, the Kir channel may be involved in formation of the resting membrane potential of the spines and, thus, would affect the response of N-methyl-d-aspartic acid receptor channels at the excitatory postsynaptic membrane.

scholarly journals Coupled Ion Movement Underlies Rectification in an Inward-Rectifier K+ Channel

1998 ◽  
Vol 112 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Maria Spassova ◽  
Zhe Lu
Keyword(s):  
Voltage Dependence ◽  
Inward Rectifier ◽  
Membrane Voltage ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
K Channel ◽  
Channel Blockade ◽  
Electrical Field ◽  
K Current ◽  
Internal Pore

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zδ values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zδ), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.

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