cAMP modulation of a Ca2+-dependent K+ conductance in rat submandibular acinar cells

1997 ◽  
Vol 272 (3) ◽  
pp. G454-G462 ◽  
Author(s):  
T. Ishikawa
Keyword(s):  
Submandibular Gland ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Dependent Manner ◽  
K Channels ◽  
A 23187 ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Rat Submandibular Gland ◽  
In Cells

Rat submandibular acinar cells exhibit a tetraethylammonium (TEA)-insensitive, Ca2+-activated K+ conductance, which may play an important role in Ca2+-dependent fluid secretion by this gland (T. Ishikawa, M. Murakami, and Y. Seo. Pfluegers Arch. 428: 516-525, 1994; T. Ishikawa and M. Murakami. Pfluegers Arch. 429: 748-750, 1995). We have now investigated whether this conductance would be modified during the potentiation of Ca2+-activated fluid secretion by an adenosine 3',5'-cyclic monophosphate (cAMP)-mediated mechanism in the rat submandibular gland. In isolated, vascularly perfused rat submandibular gland, we found that the adenylate cyclase activator forskolin (10 and 30 microM) enhanced a transient initial phase of a biphasic fluid secretion induced by the Ca2+ ionophore A-23187 (3 microM). We also unexpectedly found that forskolin reduced a smaller sustained phase of the secretion. The dual effects of forskolin were mimicked by the application of the cocktail of 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and dibutyryl cAMP (DBcAMP) (0.1 and 0.5 mM). Basolateral K+ efflux studies showed that 1) forskolin significantly enhanced a large transient net K+ efflux induced by A-23187 in a dose-dependent manner; 2) the forskolin-induced enhancement was also mimicked by a cocktail of IBMX and DBcAMP; and 3) the A-23187-induced K+ efflux enhanced by these agents was not inhibited by TEA (5 or 10 mM), a concentration known to completely block Ca2+ voltage-dependent large-conductance K+ channels (maxi-K+ channels). With use of microspectrofluorometry with fura 2, we then showed that an increase in cytosolic Ca2+ concentration induced by the nonfluorescent, brominated derivative 4-bromo-A-23187 (3 microM) was not altered by forskolin (30 microM) in freshly isolated rat submandibular acinar cells. With use of whole cell patch-clamp techniques, we demonstrated that a TEA-insensitive K+ conductance induced by A-23187 in cells dialyzed with pipette solutions containing cAMP (0.1 mM) was significantly larger than that in cells treated with control pipette solutions. Taken together, these results indicate that potentiation of the Ca2+-activated fluid secretion by a cAMP-mediated mechanism is accompanied by potentiation of a TEA-insensitive, Ca2+-activated K+ conductance in this gland.

Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium

1990 ◽  
Vol 259 (1) ◽  
pp. C56-C68 ◽  
Author(s):  
Y. Segal ◽  
L. Reuss
Keyword(s):  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
K Channel ◽  
Dependent Manner ◽  
Gallbladder Epithelium ◽  
Concentration Dependent Manner ◽  
K Channels ◽  
Voltage Dependent ◽  
Channel Currents

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

2005 ◽  
Vol 289 (2) ◽  
pp. C425-C436 ◽  
Author(s):  
Bok Hee Choi ◽  
Jung-Ah Park ◽  
Kyung-Ryoul Kim ◽  
Ggot-Im Lee ◽  
Yong-Tae Lee ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cytochalasin B ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk− cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC50 of 4.2 μM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC50 of 1.4 μM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 μM/s and 7.5 s−1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 μM) also inhibited an ultrarapid delayed rectifier K+ current ( IK,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous IK,ur in a cytoskeleton-independent manner as an open-channel blocker.

Post-weaning xenohormone intake affects adult rat submandibular gland in a sex-dependent manner

Oral Diseases ◽  
2018 ◽  
Vol 24 (7) ◽  
pp. 1235-1246
Author(s):  
Wided Kouidhi ◽  
Raymond Bergès ◽  
Gaêtan Drouin ◽  
Catherine Desmetz ◽  
Jacques Auger ◽  
...  
Keyword(s):  
Submandibular Gland ◽  
Dependent Manner ◽  
Adult Rat ◽  
Rat Submandibular Gland

Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells

2007 ◽  
Vol 292 (6) ◽  
pp. R2380-R2390 ◽  
Author(s):  
Tetsuji Nakamoto ◽  
Alaka Srivastava ◽  
Victor G. Romanenko ◽  
Catherine E. Ovitt ◽  
Patricia Perez-Cornejo ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Ion Transport ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Specific Cell ◽  
Parotid Glands ◽  
Voltage Dependent ◽  
Intracellular Ca

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near −51 mV, while the intracellular [Cl−] was ∼62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active Cl− uptake mechanisms included a bumetanide-sensitive Na+-K+-2Cl− cotransporter and paired DIDS-sensitive Cl−/HCO3− and EIPA-sensitive Na+/H+ exchangers that correlated with expression of NKCC1, AE2, and NHE1 transcripts, respectively. Intracellular Ca2+ stimulated a niflumic acid-sensitive Cl− current with properties similar to the Ca2+-gated Cl channel BEST2. In addition, intracellular Ca2+ stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction.

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1989 ◽  
Vol 61 (3) ◽  
pp. 467-477 ◽  
Author(s):  
D. E. Meyers ◽  
J. L. Barker
Keyword(s):  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Calcium Currents ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Cells Cultured ◽  
In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

Neuromedin U Depolarizes Rat Hypothalamic Paraventricular Nucleus Neurons In Vitro by Enhancing IH Channel Activity

2003 ◽  
Vol 90 (2) ◽  
pp. 843-850 ◽  
Author(s):  
De-Lai Qiu ◽  
Chun-Ping Chu ◽  
Tetsuro Shirasaka ◽  
Takashi Nabekura ◽  
Takato Kunitake ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Paraventricular Nucleus ◽  
Dependent Manner ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Ih Channel ◽  
Parvocellular Neurons ◽  
Zd 7288

The effect of neuromedin U (NMU) on rat paraventricular nucleus (PVN) neurons was examined using whole cell patch-clamp recordings. Under current-clamp, 31% of PVN parvocellular neurons ( n = 243) were depolarized by 100 nM NMU, but magnocellular neurons were not affected. NMU (10 nM to 1 μM) resulted in increased basal firing rate and depolarization in a dose-dependent manner with an EC50 of 70 nM. NMU-induced depolarization was unaffected by co-perfusion with 0.5 μM TTX + 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) + 10 μM bicuculline. Extracellular application of 70 μM ZD 7288 completely inhibited NMU-induced depolarization. Under voltage-clamp, 1 μM NMU produced negligible inward current but did increase the hyperpolarization-activated current ( IH) at step potentials less than –80 mV. The effects of NMU on IH were voltage-dependent, and NMU shifted the IH conductance-voltage relationship ( V1/2) by about 10.8 mV and enhanced IH kinetics without changing the slope constant ( k). Extracellular application of 70 μM ZD 7288 or 3 mM Cs+ blocked IH and the effects of NMU in voltage-clamp. These results suggest that NMU selectively depolarizes the subpopulation of PVN parvocellular neurons via enhancement of the hyperpolarization-activated inward current.

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