Patch-clamp capacitance analysis of the effects of alpha-SNAP on exocytosis in adrenal chromaffin cells

1996 ◽  
Vol 109 (9) ◽  
pp. 2417-2422
Author(s):  
A.V. Kibble ◽  
R.J. Barnard ◽  
R.D. Burgoyne
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Deletion Mutant ◽  
Initial Rate ◽  
Membrane Capacitance ◽  
Adrenal Chromaffin Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Adrenal Chromaffin

We have examined the effect of alpha-SNAP on exocytosis in adrenal chromaffin cells by direct assay of exocytosis using patch-clamp capacitance analysis. Cells were recorded using the whole cell patch-clamp configuration and the cells dialysed with control pipette solution or with a pipette solution containing alpha-SNAP or the deletion mutant alpha-SNAP(41–295). The deletion mutant was found to be unable to bind to syntaxin allowing a test of the requirement for syntaxin-binding for any effect of alpha-SNAP on exocytosis. Following cell dialysis for 10 minutes, cells were depolarised five times at 2 minute intervals. At each depolarisation step cells dialysed with alpha-SNAP showed a significant increase in both the initial rate and extent of exocytosis which was seen as a rise in membrane capacitance. This increase in exocytosis was not observed with alpha-SNAP(41–295) which instead produced some inhibition of the extent but had no effect on the initial rate of exocytosis. These results show directly that alpha-SNAP has a specific and marked stimulatory effect on exocytosis in chromaffin cells.

scholarly journals Patch clamp study on dopaminergic inhibition of adrenal chromaffin cells

1989 ◽  
Vol 49 ◽  
pp. 178
Author(s):  
Yasutake Mine ◽  
Akinori Akaike ◽  
Masashi Sasa ◽  
Shuji Takaori
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin ◽  
Clamp Study

Effects of ginsenosides on Ca2 channels and membrane capacitance in rat adrenal chromaffin cells

1998 ◽  
Vol 46 (3) ◽  
pp. 245-251 ◽  
Author(s):  
Hack-Seang Kim ◽  
Jung-Hwa Lee ◽  
Yong-Sook Goo ◽  
Seung-Yeol Nah
Keyword(s):  
Chromaffin Cells ◽  
Membrane Capacitance ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin ◽  
Ca2 Channels

Cation channel activated by muscarinic agonists on porcine adrenal chromaffin cells

1995 ◽  
Vol 269 (1) ◽  
pp. E43-E52 ◽  
Author(s):  
E. J. Forsberg ◽  
Q. Li ◽  
Y. Xu
Keyword(s):  
Chromaffin Cells ◽  
Secretory Response ◽  
Muscarinic Agonist ◽  
Adrenal Chromaffin Cells ◽  
Muscarinic Agonists ◽  
Whole Cell ◽  
Perforated Patch ◽  
Inward Currents ◽  
Adrenal Chromaffin ◽  
Single Channel Currents

A large portion (70%) of the secretory response to muscarinic agonists in porcine adrenal chromaffin cells has previously been shown to be dependent on extracellular Ca2+ (Xu et al., J. Neurochem. 56: 1899-1896, 1991). Results presented here show that muscarinic agonists activate a cation-selective channel which is permeable to divalent cations. The muscarinic agonist, methacholine, was found to activate the uptake of Mn2+, which paralleled the ability of methacholine to activate 45Ca2+ uptake as shown previously. Secretion induced by methacholine was not affected by nifedipine, a compound that inhibits dihydropyridine-sensitive voltage-gated Ca2+ channels. In voltage-clamped cells, methacholine activated whole cell currents, which reversed at approximately -20 mV in standard salt solutions. However, with the standard whole cell configuration, the currents were slow to activate and were often erratic. In contrast, when the perforated-patch (nystatin) technique was used to measure whole cell currents, methacholine rapidly activated sustained inward currents. Ion-substitution experiments indicated that the inward currents were carried by Na+, Ba2+, or Ca2+ but not by Cl-. Single-channel currents activated by methacholine were observed in outside-out vesicles, which were electrically accessed using the perforated-patch technique. These channels reversed at -15 mV, had a slope conductance of 20 pS, and were 14-fold more likely to be open in the presence of methacholine. These channels are probably responsible for the extracellular Ca(2+)-dependent secretory response to muscarinic receptor stimulation in porcine adrenal chromaffin cells.

GABA-Mimetic Actions of Withania somnifera on Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

2013 ◽  
Vol 41 (05) ◽  
pp. 1043-1051 ◽  
Author(s):  
Hua Yin ◽  
Dong Hyu Cho ◽  
Soo Joung Park ◽  
Seong Kyu Han
Keyword(s):  
Patch Clamp ◽  
Receptor Antagonist ◽  
Withania Somnifera ◽  
Substantia Gelatinosa ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Inward Currents

The plant Withania somnifera (WS), also known as Ashwagandha, has been used widely in traditional medicine systems in India and Nepal (Ayurveda), and has been accepted to cure various ailments. In this study, the whole-cell patch clamp technique was performed to examine the mechanism of action of WS on the SG neurons of the Vc from mouse brainstem slices. In whole-cell patch clamp mode, methanol extract of Withania somnifera (mWS) induced short-lived and repeatable inward currents in all SG neurons tested (31.3±8.51 pA, n = 7) using a high chloride pipette solution. The mWS-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na + channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, AP5, an NMDA receptor antagonist and strychnine, a glycine receptor antagonist. The mWS induced currents were blocked by picrotoxin, a GABAA receptor antagonist. These results show that mWS has an inhibitory effects on SG neurons of the Vc through GABAA receptor-mediated activation of chloride ion channels, indicating that mWS contains compounds with sedative effects on the central nervous system. These results also suggest that mWS may be a potential target for modulating orofacial pain processing.

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

2011 ◽  
Vol 301 (1) ◽  
pp. C86-C98 ◽  
Author(s):  
Juliana M. Rosa ◽  
Cristina J. Torregrosa-Hetland ◽  
Inés Colmena ◽  
Luis M. Gutiérrez ◽  
Antonio G. García ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Channel Blocker ◽  
Primary Cultures ◽  
Adrenal Chromaffin Cells ◽  
Cell Functions ◽  
Fluorescence Techniques ◽  
Voltage Dependent ◽  
Bovine Chromaffin Cells ◽  
Adrenal Chromaffin

Calcium (Ca2+)-dependent endocytosis has been linked to preferential Ca2+ entry through the L-type (α1D, CaV1.3) of voltage-dependent Ca2+ channels (VDCCs). Considering that the Ca2+-dependent exocytotic release of neurotransmitters is mostly triggered by Ca2+ entry through N-(α1B, CaV2.2) or PQ-VDCCs (α1A, CaV2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca2+ current ( ICa), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca2+ ([Ca2+]e). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca2+ caused substantially smaller endocytotic responses compared with those produced by K+ depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20–30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca2+ entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

scholarly journals Multiple Forms of Endocytosis In Bovine Adrenal Chromaffin Cells

1997 ◽  
Vol 139 (4) ◽  
pp. 885-894 ◽  
Author(s):  
Corey Smith ◽  
Erwin Neher
Keyword(s):  
Chromaffin Cells ◽  
Initial Rate ◽  
Secretory Activity ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Adrenal Chromaffin ◽  
Cell Capacitance

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.

Voltage-Dependent, Pertussis Toxin Insensitive Inhibition of Calcium Currents by Histamine in Bovine Adrenal Chromaffin Cells

2000 ◽  
Vol 83 (3) ◽  
pp. 1435-1442 ◽  
Author(s):  
Kevin P. M. Currie ◽  
Aaron P. Fox
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Chromaffin Cells ◽  
Calcium Influx ◽  
Adrenal Chromaffin Cells ◽  
Pipette Solution ◽  
Patch Pipette ◽  
Calcium Buffering ◽  
Voltage Dependent ◽  
Adrenal Chromaffin

Histamine is a known secretagogue in adrenal chromaffin cells. Activation of G-protein linked H1 receptors stimulates phospholipase C, which generates inositol trisphosphate leading to release of intracellular calcium stores and stimulation of calcium influx through store operated and other channels. This calcium leads to the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels ( I Ca). Therefore, these channels are important targets for the regulation of secretion. In particular N- and P/Q-type I Ca are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on I Ca in chromaffin cells is unknown. This paper reports that histamine inhibited I Cain cultured bovine adrenal chromaffin cells and this response was blocked by the H1 antagonist mepyramine. With high levels of calcium buffering in the patch pipette solution (10 mM EGTA), histamine slowed the activation kinetics and inhibited the amplitude of I Ca. A conditioning prepulse to +100 mV reversed the kinetic slowing and partially relieved the inhibition. These features are characteristic of a membrane delimited, voltage-dependent pathway which is thought to involve direct binding of G-protein βγ subunits to the Ca channels. However, unlike virtually every other example of this type of inhibition, the response to histamine was not blocked by pretreating the cells with pertussis toxin (PTX). The voltage-dependent, PTX insensitive inhibition produced by histamine was modest compared with the PTX sensitive inhibition produced by ATP (28% vs. 53%). When histamine and ATP were applied concomitantly there was no additivity of the inhibition beyond that produced by ATP alone (even though the agonists appear to activate distinct G-proteins) suggesting that the inhibition produced by ATP is maximal. When experiments were carried out under conditions of low levels of calcium buffering in the patch pipette solution (0.1 mM EGTA), histamine inhibited I Ca in some cells using an entirely voltage insensitive pathway. We demonstrate that activation of PTX insensitive G-proteins (most likely Gq) by H1 receptors inhibits I Ca. This may represent a mechanism by which histamine exerts inhibitory (in addition to previously identified stimulatory) effects on catecholamine release.

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