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.

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations

1994 ◽  
Vol 267 (4) ◽  
pp. C1152-C1159 ◽  
Author(s):  
S. Grinstein ◽  
R. Romanek ◽  
O. D. Rotstein
Keyword(s):  
Single Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Patch Configuration ◽  
In Cells

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

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.

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.

Neuropeptide galanin inhibits omega-conotoxin GVIA-sensitive calcium channels in parasympathetic neurons

1995 ◽  
Vol 73 (4) ◽  
pp. 1374-1382 ◽  
Author(s):  
L. A. Merriam ◽  
R. L. Parsons
Keyword(s):  
Calcium Channels ◽  
Patch Clamp Technique ◽  
Maximal Inhibition ◽  
Whole Cell ◽  
Recording Technique ◽  
Perforated Patch ◽  
Cell Recording ◽  
Parasympathetic Neurons ◽  
Current Activation ◽  
Patch Technique

1. We determined the effect of the neuropeptide galanin on barium currents (IBa) flowing through voltage-gated calcium channels. We voltage clamped parasympathetic neurons dissociated from mudpuppy cardiac ganglia using both the standard whole cell and the perforated-patch variations of the patch-clamp technique. 2. Galanin produced a concentration-dependent inhibition of IBa. The maximal inhibition was 50-60% and the concentration that produced half-maximal inhibition (IC50) was 0.42 nM. In mud-puppy parasympathetic neurons, omega-conotoxin-GVIA (CTX)-sensitive channels are the predominant type of calcium channels, and only a small portion of IBa is contributed by dihydropyridine-sensitive channels. Galanin preferentially inhibited a portion of the CTX-sensitive current. 3. In currents recorded with the standard whole cell technique, activation of IBa was slowed in the presence of galanin. In contrast, in the majority of neurons studied with the perforated-patch technique, galanin decreased IBa without altering the kinetics of current activation. With both recording methods, the decrease in IBa was greatest with voltage steps to 0 mV and persisted with steps to +50 mV. For control currents, large depolarizing voltage steps (+70 to +120 mV) did not markedly facilitate IBa when either recording technique was used. However, the degree of facilitation in galanin was significantly greater with the standard whole cell recording technique. 4. IBa exhibited inactivation under the conditions of these experiments. Inactivation of IBa recorded during a 900-ms depolarizing voltage step was fitted to a double exponential. Galanin decreased the amplitude of IBa but did not alter the time constants of inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterologous Expression of a P2 x-Purinoceptor in Rat Chromaffin Cells Detects Vesicular ATP Release

1997 ◽  
Vol 78 (6) ◽  
pp. 3069-3076 ◽  
Author(s):  
Bettye Hollins ◽  
Stephen R. Ikeda
Keyword(s):  
Carbon Fiber ◽  
Heterologous Expression ◽  
Chromaffin Cells ◽  
Time Course ◽  
Atp Release ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Vesicular Release ◽  
Inward Currents ◽  
Adrenal Chromaffin

Hollins, Bettye and Stephen R. Ikeda. Heterologous expression of a P2 x -purinoceptor in rat chromaffin cells detects vesicular ATP release. J. Neurophysiol. 78: 3069–3076, 1997. A cloned P2 x -purinoceptor was transiently expressed in single isolated rat adrenal chromaffin cells and evaluated for the detection of released ATP. After cytoplasmic injection of the P2 x complementary RNA (cRNA; 4–24 h), application of ATP produced an inwardly rectifying current over the voltage range −130 to −10 mV as measured by the whole cell patch-clamp technique. The dose-response curve for ATP was sigmoidal with a 50% effective concentration of 18.2 μM. Suramin, a P2 x -antagonist, attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or application of histamine, stimuli that trigger exocytosis, resulted in the appearance of suramin-sensitive spontaneous transient inward currents (at −60 mV) that resembled excitatory postsynaptic currents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed coincidence of the amperometric current with the synaptic currentlike events suggesting that ATP and catecholamines were released from the same vessicle. These data demonstrate that expression of a P2 x -purinoceptor in chromaffin cells produces a functional autoreceptor capable of detecting vesicular release of ATP. In combination with carbon fiber amperometry, simultaneous vesicular release of two neurotransmitters from a single chromaffin cell could be monitored. The P2 x -purinoceptor, however, produced a regenerative effect on release apparently resulting from the high Ca2+ permeability of the receptor. Thus modification of the P2 x -purinoceptor would be required before the system could be applied to examining processes involved in stimulus-release coupling.

scholarly journals Changes in Intracellular Calcium Concentration Affect Desensitization of GABAA Receptors in Acutely Dissociated P2–P6 Rat Hippocampal Neurons

1998 ◽  
Vol 79 (3) ◽  
pp. 1321-1328 ◽  
Author(s):  
Jerzy W. Mozrzymas ◽  
Enrico Cherubini
Keyword(s):  
Intracellular Calcium ◽  
Hippocampal Neurons ◽  
Calcium Concentration ◽  
Intracellular Calcium Concentration ◽  
Potential Range ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Configuration ◽  
High Calcium ◽  
Kinetics Of

Mozrzymas, Jerzy W. and Enrico Cherubini. Changes in intracellular calcium concentration affect desensitization of GABAA receptors in acutely dissociated P2–P6 rat hippocampal neurons. J. Neurophysiol. 79: 1321–1328, 1998. The whole cell configuration of the patch-clamp technique was used to study the effects of different cytosolic calcium concentrations [Ca2+]i on desensitization kinetics of γ-aminobutyric acid (GABA)-activated receptors in acutely dissociated rat hippocampal neurons. Two different intrapipette concentrations of the calcium chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA; 11 and 0.9 mM, respectively) were used to yield a low (1.2 × 10−8 M) or a high (2.2 × 10−6 M) [Ca2+]i. In low [Ca2+]i, peak values of GABA-evoked currents (20 μM) evoked at −30 mV, were significantly larger than those recorded in high calcium [2,970 ± 280 (SE) pA vs. 1,870 ± 150 pA]. The extent of desensitization, assessed from steady-state to peak ratio was significantly higher in high calcium conditions (0.14 ± 0.007 vs. 0.11 ± 0.008). Similar effects of [Ca2+]i on desensitization were observed with GABA (100 μM). Recovery from desensitization, measured at 30 s interval with double pulse protocol was significantly slower in high [Ca2+]i than in low [Ca2+]i (54 ± 3% vs. 68 ± 2%). The current-voltage relationship of GABA-evoked currents was linear in the potential range between −50 and 50 mV. The kinetics of desensitization process including the rate of onset, extent of desensitization, and recovery were voltage independent. The run down of GABA-evoked currents was faster with the higher intracellular calcium concentration. The run down process was accompanied by changes in desensitization kinetics: in both high and low [Ca2+]i desensitization rate was progressively increasing with time as the slow component of the desensitization onset was converted into the fast one. In excised patches, the desensitization kinetics was much faster and more profound than in the whole cell configuration, indicating the involvement of intracellular factors in regulation of this process. In conclusion, [Ca2+]i affects the desensitization of GABAA receptors possibly by activating calcium-dependent enzymes that regulate their phosphorylation state. This may lead to modifications in cell excitability because of changes in GABA-mediated synaptic currents.

scholarly journals Regulation of the formation of inositol phosphates by calcium, guanine nucleotides and ATP in digitonin-permeabilized bovine adrenal chromaffin cells

1991 ◽  
Vol 279 (2) ◽  
pp. 447-453 ◽  
Author(s):  
D A Eberhard ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Inositol Phosphates ◽  
Purinergic Receptor ◽  
Receptor Activation ◽  
Maximal Response ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Adrenal Chromaffin ◽  
Phosphatidylinositol 4 ◽  
Concentration Response Curve

Both micromolar Ca2+ and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated the formation of inositol phosphates (InsPs) in digitonin-permeabilized chromaffin cells prelabelled with [3H]inositol. The production of InsPs was potentiated by ATP. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) caused a GTP-reversible shift to higher concentrations in the Ca(2+)-concentration-response curve for the release of InsPs without changing the maximal response. GTP[S] caused a shift to lower concentrations of Ca2+ and also increased the maximal response. The effects of GTP[S] and Ca2+ were synergistic. Although as much as 80% of the InsPs were derived from phosphatidylinositol 4-phosphate (PtdInsP) or 4,5-bisphosphate (PtdInsP2), the amount of InsPs produced could be several times the total amount of PtdInsP and PtdInsP2 in the cells and was largely accounted for by a decrease in PtdIns. The levels of labelled PtdInsP and PtdInsP2 increased on stimulation with Ca2+, but decreased on stimulation with GTP[S] or the combination of Ca2+ and GTP[S]. Preincubation with Ca2+ and ATP amplified the subsequent GTP[S]-induced production of InsPs. ATP and its gamma-thio and beta gamma-imido analogues stimulated the formation of InsPs in intact cells. However, only ATP potentiated the responses to Ca2+ and GTP[S] in permeable cells. Our main conclusions are: (1) a GTP-binding protein participates in the Ca(2+)-induced production of InsPs by phospholipase C, and (2) ATP markedly potentiates the stimulated formation of InsPs, an effect with arises from its role in polyphosphoinositide synthesis and does not involve purinergic receptor activation in permeabilized cells. The data also suggest that the different effects of Ca2+ and GTP[S] on polyphosphoinositide synthesis probably contribute to the synergistic action of Ca2+ and GTP[S] on the generation of InsPs.

Postsynaptic Mechanisms Are Essential for Forskolin-Induced Potentiation of Synaptic Transmission

2006 ◽  
Vol 95 (4) ◽  
pp. 2570-2579 ◽  
Author(s):  
Irina V. Sokolova ◽  
Henry A. Lester ◽  
Norman Davidson
Keyword(s):  
Synaptic Transmission ◽  
Ampa Receptors ◽  
Internal Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Nmda Receptor Blockade ◽  
Mepsc Frequency ◽  
Pka Pathway ◽  
Activity Dependent

It has been demonstrated that stimulation of protein kinase A (PKA) results in enhanced synaptic transmission in the hippocampus and other brain areas. To investigate mechanisms of the PKA-mediated potentiation of synaptic transmission, we used rat hippocampal embryonic cultures. In low-density cultures, paired recordings under the perforated patch demonstrated that 15-min forskolin treatment produced long-lasting potentiation of evoked excitatory postsynaptic currents (eEPSCs) mediated by the cAMP/PKA pathway. eEPSC amplitudes increased to 240 ± 10% of baseline after 15 min of forskolin treatment (early). After forskolin washout, eEPSCs declined to a potentiated level. Potentiation was sustained for ≥85 min after forskolin washout and, 60 min after forskolin washout, constituted 152 ± 7% of baseline (late potentiation). Disruption of presynaptic processes with the whole cell configuration and internal solution containing PKA inhibitor peptide did not affect forskolin-induced potentiation. Disruption of postsynaptic processes, in contrast, impaired early potentiation and abolished late potentiation. Study of mEPSCs confirmed the contribution of postsynaptic mechanisms. Forskolin-induced enhancement of mEPSC frequency observed under the perforated patch was attenuated by the whole cell configuration. Forskolin also induced an increase of mEPSC amplitudes in the perforated patch, but not in the whole cell, experiments. Potentiation of eEPSCs was not activity dependent, persisting in the absence of stimulation. NMDA receptor blockade did not abolish forskolin-induced potentiation. In summary, we demonstrate that forskolin-induced potentiation of eEPSCs was mediated by postsynaptic mechanisms, presumably by upregulation of AMPA receptors by phosphorylation.

scholarly journals Use Dependent Attenuation of Rat HCN1-Mediated Ih in Intact HEK293 Cells

2016 ◽  
Vol 38 (6) ◽  
pp. 2079-2093 ◽  
Author(s):  
Lennart Barthel ◽  
Olivia Reetz ◽  
Ulf Strauss
Keyword(s):  
Mammalian Cells ◽  
Hek293 Cells ◽  
Hcn Channels ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Human Embryonic Kidney Cells ◽  
Intracellular Content ◽  
Voltage Dependent ◽  
Activity Dependence

Background/Aims: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. Methods: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. Results: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. Conclusion: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore.

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