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D Siemen

Publications and source records attributed to D Siemen.

At least 19 recordsLinked to original sources
PubMed2000

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat.

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.

Adenosine Triphosphate↗
PubMed2000

The involvement of caspases in the CD95(Fas/Apo-1)- but not swelling-induced cellular taurine release from Jurkat T-lymphocytes.

Following a delay of 45 min, stimulation of the CD95 (Fas/Apo-1)-receptor in Jurkat T-lymphocytes leads to the release of the osmolyte taurine, an event coinciding with apoptotic cell shrinkage. The present study has been performed to elucidate the cellular mechanisms involved in CD95-induced taurine release as compared to swelling-induced taurine release, and to explore whether taurine modifies apoptotic DNA fragmentation and cell shrinkage. Taurine release stimulated by osmotic cell swelling is insensitive to the tyrosine kinase inhibitor herbimycin A and the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) but is blunted in the absence of extracellular Ca2+. Conversely, the Ca2+ ionophore ionomycin stimulates taurine release. However, the taurine release following CD95 stimulation is not paralleled by an increase of cytosolic Ca2+ and not inhibited by complexation of extracellular Ca2+. None of herbimycin A, the phosphatase inhibitor vanadate, spingomyelinase or Lck56 deficiency prevent CD95-induced taurine release. In contrast, the caspase inhibitor zVAD, but not the caspase inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD), almost abolishes CD95-induced taurine release. Both caspase inhibitors blunt CD95-induced cell shrinkage and DNA fragmentation, zVAD being more effective than YVAD. Preloading of the cells with 40 mM taurine but not with 40 mM mannitol significantly inhibits CD95-induced DNA fragmentation (by 28%) and apoptotic cell shrinkage (by 25%). In conclusion, CD95-receptor triggering leads to caspase-dependent stimulation of cellular taurine release, which facilitates, but is not sufficient for, the triggering of apoptotic DNA fragmentation and cell shrinkage.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Norepinephrine-induced sustained inward current in brown fat cells: alpha(1)-mediated by nonselective cation channels.

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the alpha(1)-agonist cirazoline but not by the beta(3)-agonist CGP-12177A. Reduction of extracellular Cl(-) concentration had no effect, but omission of extracellular Ca(2+) or Na(+) totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of approximately 30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from approximately 0.002 to approximately 0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the alpha(1)-agonist phenylephrine than with the beta(3)-agonist CGP-12177A, and with the Ca(2+) ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of approximately 30 pS nonselective cation channels via alpha(1)-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca(2+) concentration.

Adenylyl Cyclases↗
PubMed1999

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line.

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.

Calcium↗
PubMed1999

Cell volume regulatory mechanisms in apoptotic cell death.

One of the hallmarks of apoptosis is cell shrinkage, which--at constant extracellular osmolarity--requires a decrease of cellular osmolarity. Moreover, apoptosis can be elicited by increase of extracellular osmolarity and the resistance of cells towards apoptosis correlates with their ability to regulate their volume in hypertonic environment. On the other hand, CD95-receptor-mediated apoptosis is blunted at moderate increases of extracellular osmolarity. Given the role of cell volume alterations it is not surprising that apoptosis is paralleled by marked alterations of cell volume regulatory mechanisms. Stimulation of the CD95-receptor, which confers apoptosis to a variety of cells, leads to activation of cell volume regulatory anion channel ORCC. However, activation of ORCC is paralleled by inhibition of cell volume regulatory K+ channel Kv1.3. It is only 40 to 60 minutes after triggering of the CD95-receptor when the cells release the organic osmolyte taurine and shrink.

Animals↗
PubMed1998

Large conductance channel in plasma membranes of astrocytic cells is functionally related to mitochondrial VDAC-channels.

Large conductance anion channels with similar electrophysiological characteristics were found in plasma membranes and in outer mitochondrial membranes of various cell types. Although their large conductance and their peculiar voltage dependence point to a close relation, it was questioned whether they belong to the same family. We therefore compared some biochemical features of a plasmalemmal channel with those known from the mitochondrial channel. Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN). The underlying channels exhibited a conductance of 401 +/- 50 pS. Open probability was highest between +/- 10 mV and gradually approached zero beyond +/- 25 mV. Activity as induced by voltage ramps between +/- 40 mV appeared after a delay of up to several min. The delay could be reduced by bathing either side of the patch in an acidic Ringer solution (pH 6.2). 1 mM Al3+ increased the open time at potentials more positive than 20 mV. 10 mM dextran sulfate (MW 8000) caused reversible flickering, increasing the closed probability. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) also caused a reversible flickering into the closed state, reducing the apparent single channel amplitude by up to 70% at 0.5 mM DIDS. Application of 5 mM ATP resulted in reversible blockade; ATP was more effective from the outside than from the inside (blocking activity 65% vs. 16% of the patches). We conclude that the large conductance anion channel from astrocytic cells displays electrophysiological and pharmacological characteristics that resemble those of VDAC (Voltage Dependent Anion Channel) from the outer mitochondrial membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Regulation of the activity of 27 pS nonselective cation channels in excised membrane patches from rat brown-fat cells.

The regulation of the activity of the approximately 30 pS nonselective cation channel (NSC channel) was studied by the patch-clamp technique in inside-out patches obtained from rat brown-fat cells. NSC channel activity was induced by excision; reduced redox state induced by dithiothreitol accelerated the kinetics in the excised state. The NSC channels were inhibited by the fenamates flufenamic acid and mefenamic acid but not by NS-1619 or SKF-96365. The channels were inhibited by purine nucleotides but not by polyamines. No evidence for protein kinase C, CaM kinase or protein kinase A activation of the NSC channel was obtained. NSC-channel activity was stimulated in a concentration-dependent manner by Ca2+ but the EC50 was very high (0.81 mM), in comparison to expected cytosolic Ca2+ levels. In the presence of ATP, even higher Ca2+ levels were necessary for comparable NSC-channel activation. The increase in Po was not associated with an increase in open-time constants. We conclude that although high Ca2+ levels can experimentally activate the NSC channel, a further mediatory step must probably be postulated in order to link alpha1-adrenergic stimulation to NSC-channel activation.

Adipose Tissue, Brown↗
PubMed1997

108-pS channel in brown fat mitochondria might Be identical to the inner membrane anion channel.

Single-channel and whole-mitoplast patch-clamp recordings were employed to characterize the 108-pS (Cl-) channel in brown fat mitochondrial mitoplasts. We demonstrated the ability of this channel to conduct di- and trivalent anions, such as sulfate, phosphate, and benzenetricarboxylates, and its blockage by propranolol, 1,4-dihydropyridine-type Ca2+ antagonists, and Cibacron blue. Moreover, we have revealed its pH dependence for the first time. As a basis for the characteristic potential dependence of the whole-mitoplast current, we identified an open probability, increasing with depolarizing (positive) potentials, Eh, and being almost zero in the hyperpolarizing range. Events at negative Eh exhibit a short flickering behavior, whereas at positive Eh, they become much longer. This voltage dependence is influenced by pH in such a way that, at acidic pH, the 108-pS channel possesses a low open probability throughout the observed potential range, whereas at alkaline pH, the channel switches to long openings, even at a negative potential. All these properties lead us to conclude that the inner membrane anion channel, which has been characterized only by light scattering studies, and the 108-pS inner membrane channel, which has been characterized electrophysiologically, are one and the same process.

Adipose Tissue, Brown↗
PubMed1996

Kinetic parameters of the ionic currents in myelinated axons: characterization of temperature effects in a hibernator and a nonhibernator.

Na+ and K+ currents were measured by the patch-clamp method in the paranodal region of single sciatic nerve fibres of rats and of warm-adapted and cold-adapted golden hamsters. Kinetic parameters and temperature dependence of the Na+ currents were determined. The time constant for activation (about 0.2 ms for rats and hamsters) as well as the time constant for inactivation (about 1.6 ms for rats and hamsters) at 15 degrees C and at -35 mV compared well with single fibre voltage-clamp data from the rat. Differences amongst the three groups of animals were not significant. The temperature coefficient, Q10, for the activation and the inactivation time constant as well as for the time-to-peak of the Na+ current ranged between 2.3 and 3.1. No data have previously been published on the temperature dependence of the delayed-rectifier K channels of mammalian nerve fibres. Most of the K+ current was carried by intermediate (KI) and fast (KF) K channels. Dendrotoxin block indicated that "approximate"55% of the K+ current was due to KI channels, with no significant difference amongst the three groups of animals tested. The Arrhenius plot of the time constant of K+ current activation, "tau"n, yielded a mean Q10 of 3.3 at -40 mV (4. 0 at + 60 mV). No significant differences of the channel kinetics between rats, warm-adapted hamsters and cold-adapted hamsters were detected. We observed, however, a significant decrease of the Na channel density in the paranodal region of cold-adapted hamsters.

Animals↗
PubMed1993

A voltage-dependent and a voltage-independent potassium channel in brown adipocytes of the rat.

Single-channel recordings of a voltage-dependent potassium channel in brown adipocytes of the rat confirm recordings of macroscopic currents. Single-channel conductance (gamma) is 8 pS at 20 degrees C in KF solution inside vs. a modified Ringer's solution outside. With KCl solution outside, gamma is 17 pS for outward currents and 21 pS for inward currents. The majority of the channels inactivate with a time constant around 200 ms; deactivation occurs within milliseconds. The channel is blocked by tetraethylammonium (TEA) with an inhibiting constant of 1.8 mM. The type of block is fast. Selectivity sequence for monovalent cations is K+ > Rb+ >> NH4+ >> Li+ > or = Na+ approximately Cs+. Cs+ at the outside causes a voltage-dependent block of inward currents. This channel is remarkably similar to the delayed rectifier of the F-type in the node of Ranvier. Occasionally, an additional K+ channel was found. This channel is voltage-insensitive, not blocked by 10 mM TEA, and has not been recorded in brown adipocytes before. Physiological relevance of this channel could be the steady-state membrane potential.

Adipocytes↗
PubMed1993

Reversible blockade of the calcium-activated nonselective cation channel in brown fat cells by the sulfhydryl reagents mercury and thimerosal.

We have used patch-clamp techniques to study the effect of the sulfhydryl group oxidizing agents mercury and thimerosal on calcium-activated nonselective cation channels from brown adipose tissue. 100 nmol/l mercury and 50 mumol/l thimerosal induced a complete block. Blockade could be reversed by reduction of the mercaptide by dithiotreitol (DTT). Mercury was found to be the most potent blocker (IC50-value 21 x 10(-9) mol/l), whereas thimerosal (IC50-value 1.5 x 10(-6) mol/l) was as effective as 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC). The DCDPC effect, however, could not be reversed by DTT, indicating different blocking mechanisms. It is concluded that SH-groups are involved in gating of the calcium-activated nonselective channel.

Adipose Tissue, Brown↗
PubMed1991

Inner mitochondrial membrane anion channel is present in brown adipocytes but is not identical with the uncoupling protein.

Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8 +/- 8.7 pS (n = 16, 21 degrees C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 microM) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP.

Adipose Tissue, Brown↗
PubMed1989

Permeability of the non-selective channel in brown adipocytes to small cations.

The non-selective channel for monovalent cations of cultured brown adipocytes was studied concerning its permeability to alkali metal ions, NH4+, Tris+, Ca2+, and Ba2+. Experiments were done by means of the patch clamp technique using inside-out patches. With symmetrically increasing sodium concentrations the ion fluxes saturated. They are described by a dissociation constant (KNa) of 155 mmol/l and a maximum single channel conductance of 50 pS. Permeabilities were determined in relation to those for sodium yielding values of 0.80 for potassium and 1.55 for ammonium. The complete permeability sequence for ammonium and the alkali metals is: NH4+ greater than Na+ greater than Li+ greater than K+ greater than or equal to Rb+ congruent to Cs+ . Ca2+ and Ba2+ as well as the buffer ion Tris+ are not able to pass the channel measurably. It is shown that the conductance behaviour of the non-selective channel is not sufficiently described by the Goldman-Hodgkin-Katz theory. Deviations from independence are saturation with increased activity of the permeant ion and non-linear current voltage relations in symmetrical solutions. A simple two barrier model with one binding site in the center of the electric field is shown to be more appropriate.

Adipose Tissue, Brown↗
PubMed1987

Non-selective cationic channel in primary cultured cells of brown adipose tissue.

Single channel current events were recorded from membranes of cultured brown adipose tissue cells with the patch-clamp technique. In excised inside-out patches the predominant type of events showed a slope conductance of about 30 pS (25 degrees C). The current was carried by both, Na+ ions and K+ ions. Discrimination between them was poor. The frequency of events increased with increasing temperature. Their amplitude was temperature dependent as well (Q10 approximately equal to 1.4). A single exponential was not sufficient for fitting the histograms of "on-time" or "off-time". We conclude that these events belong to a type of non-selective cation channel described previously for other tissues.

Adipose Tissue, Brown↗