ABSTRACT
Accumulation of beta-amyloid (Abeta) protein in brain is an important characteristic for the etiology of Alzheimer's disease. Of all the possible processes generating the neurotoxic effects by Abeta, disruption of intracellular Ca(2+) homeostasis is the primary event. In this process, various intracellular Ca(2+) regulatory mechanisms are reported to be involved. Using patch-clamp techniques, both low and high voltage activated Ca(2+) channel currents were recorded in the cultured dorsal root ganglion (DRG) neurons. Application of Abeta protein fragment, Abeta(25-35) (2 microM), for 30 s increased the amplitude in both currents. The Abeta-triggered facilitation effect of Ca(2+) channel was found in all the depolarized potentials tested, as shown in the current-voltage relationship. Furthermore, after applying single cell Ca(2+) microfluorometric method, it was found that Abeta(25-35) alone could trigger elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)) level in 90% of the cells tested. The elevation diminished completely by cumulatively adding CdCl(2), NiCl(2), thapsigargin (TG), FCCP and Zn(2+) in the normal bath solution. Combining pharmacological approaches, we found that voltage-dependent Ca(2+) channels, Ca(2+) stores and a putative Zn(2+)-sensitive extracellular Ca(2+) entry, respectively, makes 61.0, 25.1, and 13.9% contribution to the [Ca(2+)](i) increase caused by Abeta. When tested in a Ca(2+)-free buffer, mitochondria was found to contribute 41.3% of Abeta produced [Ca(2+)](i) elevation and the remaining 58.7% was attributed to endoplasmic reticulum (ER) release.
Subject(s)
Amyloid beta-Peptides/metabolism , Calcium Channels/metabolism , Calcium/metabolism , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Neurons/drug effects , Neurons/metabolism , Peptide Fragments/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Calcium Channels/drug effects , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Culture Techniques , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , Homeostasis/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Patch-Clamp Techniques , Peptide Fragments/pharmacology , Rats , Rats, Wistar , Thapsigargin/pharmacology , Uncoupling Agents/pharmacologyABSTRACT
Carbon fiber electrode (CFE) can be used in detection of exocytosis of single neuron or endocrine cell. A simplified method for preparation of CFE, as described in the present paper, greatly ensures the coordination and success of the preparation. Such CFEs have a low noise level. Exocytosis of rat adrenal chromaffin cells was investigated in the clarification of the effect of MPP+ on transmitter release. It is found that exocytosis was not stimulated by MPP+ and the high K+ induced secretion was not changed by MPP+. These observations suggest that the previously reported elevation of dopamine content in the surrounding histosolution of DA neuron is probably resulted from blockage of the re-uptake of DA by MPP+.
Subject(s)
1-Methyl-4-phenylpyridinium/pharmacology , Chromaffin Cells/metabolism , Electrodes/classification , Exocytosis/physiology , Adrenal Medulla/metabolism , Animals , Carbon , Electrochemistry , Rats , Rats, WistarABSTRACT
In rat single adrenal chromaffin cells, the effects of methacholine (MCh) on [Ca2+]i and catecholamine secretion were studied with fura-2 fluorescence and carbon fiber electrodes. In the presence of 2 mmol/L Ca2+ in the bath, locally applying 1 mmol/L MCh, either containing or not containing Ca2+, evoked both [Ca2+]i and secretion signals. In the absence of Ca2+ in the bath, MCh could still evoke [Ca2+]i and secretion. These results suggest that MCh causes release of Ca2+ from Ca2+ stores, which is sufficient to yield the evoked secretion. The Ca2+ store can be depleted by single MCh puff in the absence of Ca2+ in the bath.
Subject(s)
Adrenal Medulla/metabolism , Calcium/metabolism , Chromaffin Cells/metabolism , Receptors, Muscarinic/physiology , Adrenal Medulla/cytology , Animals , Catecholamines/metabolism , Cells, Cultured , Male , Methacholine Chloride/pharmacology , Rats , Rats, WistarABSTRACT
Using perforated and cell-attached patch clamp techniques, the characteristics of ATP sensitive K(+) channels (K(ATP)), delayed rectifier K(+) channels (K(DR)), Ca(2+) and Na+ channels on single rat pancreatic beta cell membranes were studied. The results showed that (1) the efflux and influx conductance of K(ATP) channels was about 31 and 65 pS respectively, and the reversal potential of K(ATP) was about 60 mV; (2) K(DR) was activated completely after a latency of 20 ms, and K(DR) was about 1/3 of K(ATP); (3) whole cell Ca(2+) current reached a peak (40 60 pA) at 0 mV; L-type Ca(2+) channel was the main Ca(2+) channel in beta cells, but other types of high voltage activated Ca(2+) channels existed as well; and 4) whole cell Na(+) current reached a peak (200 400 pA) at 10 mV; but the expression level of Na(+) channel in beta cells varied among the cells. About half of the beta cells virtually had no Na(+) currents.
Subject(s)
Calcium Channels/metabolism , Islets of Langerhans/metabolism , Potassium Channels/metabolism , Sodium Channels/metabolism , Animals , Cells, Cultured , Islets of Langerhans/cytology , Patch-Clamp Techniques , Rats , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
Chemical communication for neurons and endocrine cells depends on their secretion activity. Development of variety of biophysical techniques have greatly promoted the understanding of the mechanism of cellular secretion. Here we will introduce three recently developed biophysical techniques which have been frequently used to monitor secretion activity in real-time.
Subject(s)
Cells/metabolism , Electrochemistry/methods , Microscopy, Fluorescence/methods , Patch-Clamp Techniques/methods , Electrochemistry/instrumentation , Fluorescent Dyes , MicroelectrodesABSTRACT
AIM: To study the calcium buffer in SH-SY5Y cells. METHODS: By using patch clamp technique, measure voltage-gated calcium currents in undifferentiated SH-SY5Y cell line. Then, by using microfluorometric technique, detect the intracellular [Ca2+]i and the dynamic recovery course after high-K+ induced [Ca2+]i elevation. RESULTS: There are voltage-gated calcium currents in undifferentiated SH-SYSY cell line. When the time intervals between the stimulus are < 150s, the recovery course will be much more deferred because of the saturation of the intracellular calcium buffer; and when the intervals between the stimulus are > 150 s, the buffer will recruit so as to lead to the steadiness state of the recover course. CONCLUSION: Calcium buffer proteins have an important effect in the course of cellular calcium signal transduction.
Subject(s)
Calcium Channels/physiology , Calcium Signaling , Calcium/metabolism , Neuroblastoma/metabolism , Cell Line, Tumor , Electrophysiology , Humans , Patch-Clamp TechniquesABSTRACT
AIM: To study the anti-arrhythmic mechanism of 7-bromoethoxybenzene-tetrahydropalmatine (EBP). METHODS: Whole-cell current and voltage clamp on isolated guinea pig ventricular cells. RESULTS: EBP 30 mumol.L-1 prolonged APD90 from 430 +/- 47 ms to 514 +/- 61 ms (n = 5, P < 0.05) without effects on the action potential amplitude and resting potential. Delayed outward K+ current and its tail current were blocked by EBP in a concentration-dependent fashion, while EBP did not change the amplitudes of the sodium current, the L type calcium current, and the inwardly rectifying potassium current. CONCLUSION: The mechanism of anti-arrhythmic action of EBP was to prolong the APD through inhibiting the delayed rectified potassium current.