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H Schindler

Publications and source records attributed to H Schindler.

At least 19 recordsLinked to original sources
PubMed1992

Potent block of Cl- channels by antiallergic drugs.

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p < 0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.

Animals↗
PubMed1992

Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in planar lipid bilayers.

We have previously described an inositol polyphosphate receptor (IPxRec), purified from detergent-solubilized bovine cerebellum microsomes, that displays potassium ion channel activity in planar lipid bilayers. We now find that the IPxRec is closely related to clathrin assembly protein AP-2. The IPxRec and AP-2 purified from bovine brain clathrin-coated vesicles share several structural and functional features: (i) similar subunit composition; each has four major polypeptides that have similar mobility (Mr values of 111,000, 100,000, 50,000, and 17,000) and relative intensity by SDS/PAGE analysis; (ii) similar size as studied by molecular sieve chromatography (Mr 400,000); (iii) identical N-terminal amino acid sequences for the Mr 50,000 subunits and Mr 111,000/100,000 doublets; (iv) immunoreactivity of the AP-2 Mr 111,000/100,000 doublet to polyclonal antibodies affinity purified against the doublet proteins of the IPxRec; (v) display of the in vitro diagnostic feature of assembly proteins--i.e., they induce the assembly of clathrin cages; and (vi) ion channel activity selective for potassium ions with the same unitary conductance when incorporated into planar lipid bilayers. One difference was found. AP-2 channels were not blocked by inositol 1,3,4,5-tetraphosphate as reported for IPx receptor channels. These studies suggest a possible connection between the IPx signaling pathways and receptor-mediated endocytosis.

Adaptor Proteins, Vesicular Transport↗
PubMed1992

Structural and functional characterization of an inositol polyphosphate receptor from cerebellum.

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.

Animals↗
PubMed1992

Activity of cardiac L-type Ca2+ channels is sensitive to cytoplasmic calcium.

Ca(2+)-induced inactivation of L-type Ca2+ channels is proposed as an important negative feedback mechanism regulating Ca2+ entry. Here, for the first time, evidence for modification of heart L-type Ca2+ channel activity by cytoplasmic calcium is provided from excised inside-out membrane patches. Ba2+ currents through cardiac L-type Ca2+ channels exhibited only modest inactivation in the absence of cytoplasmic Ca2+. Elevation of cytoplasmic Ca2+ to micromolar concentrations strikingly affected L-type Ca2+ channel activity as evaluated from ensemble average Ba2+ currents. Inactivation was markedly increased concomitant with a reduction of peak inward current, which was almost completely eliminated at about 15 microM cytoplasmic Ca2+ concentration. Half maximal suppression of Ba2+ currents was observed at 2.3 microM Ca2+. The observed modifications of L-type Ca2+ channel activity show that cytoplasmic Ca2+ induces channel closure. Below 4 microM Ca2+, channels can be reversibly reactivated during repetitive depolarizations, while at high Ca2+ concentrations (approximately 15 microM) most Ca2+ channels reside in a closed state. This may allow for a delicate regulation of Ca2+ entry, and consequently of heart contraction.

Animals↗
PubMed1992

The dihydropyridine niguldipine inhibits T-type Ca2+ currents in atrial myocytes.

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca2+ currents in guinea pig atrial cells. Ca2+ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 microM (+/-)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20 +/- 10% of control, n = 5) and L-type Ca2+ currents (to 28 +/- 12% of control, n = 5). Current to voltage relationships clearly showed that both Ca2+ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca2+ currents was found to be voltage-dependent, i.e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC50 for T-type Ca2+ current inhibition by (+/-)-NIG was determined as 0.18 microM. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca2+ currents. This study demonstrates the Ca2+ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca2+ channels.

Animals↗
PubMed1992

Scanning force microscopy studies of the S-layers from Bacillus coagulans E38-66, Bacillus sphaericus CCM2177 and of an antibody binding process.

In many prokaryotic cells (eubacteria and archaebacteria) the outermost cell envelope component is composed of a regularly structured protein surface layer (S-layer). The two-dimensional S-layer from Bacillus coagulans E38-66 and Bacillus sphaericus CCM2177 has been investigated by SFM at molecular resolution under physiological conditions (i.e., in buffer solution). We find the E38-66 S-layer lattice to be oblique with lattice parameters of a = 9-10 nm, b = 7-8 nm and gamma = 80 degrees -90 degrees (E38-66). The CCM2177 lattice is square with a = 12-14 nm, in good agreement with TEM data. We have used the unique possibility of the SFM to study the kinematics of biological processes and have performed experiments on the adhesion of polyclonal antibodies to the recrystallized E38-66 protein layer on a time scale of about two to ten seconds per image frame. This represents a first step in directly visualizing molecular recognition reactions.

Antibodies, Bacterial↗
PubMed1991

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.

Animals↗
PubMed1991

Lipid-protein surface films generated from membrane vesicles: selfassembly, composition, and film structure.

Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 microliters vesicle suspensions containing 25 micrograms protein yield at least 50 cm2 film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure (greater than or equal to 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (+/- 0.2) nm2.

Animals↗
PubMed1991

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches.

The activity of single L-type Ca2+ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca2+ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent run-down as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca2+ channel regulation with free access to both sides of the membrane.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Purified IP3 receptor from smooth muscle forms an IP3 gated and heparin sensitive Ca2+ channel in planar bilayers.

The IP3 receptor of aortic smooth muscle, purified to near homogeneity, was incorporated into vesicle derived planar bilayers. The receptor forms channels which are gated by Ins(1,4,5)P3 (0.5 microM) and are permeable to Ca2+ (Ca2+ greater than K+ much greater than Cl-). Channel activation is specific for Ins(1,4,5)P3. Essentially no activation of channel currents was found for Ins(1,3,4)P3 or Ins(1,3,4,5)P4 at 10 microM. Heparin (25 micrograms/ml) blocked induced currents completely at all levels of activity while ATP (50 microM) increased mean current levels 2 to 4 fold. Ins(1,4,5)P3 activated mean currents increased non-linearly with voltage above about -40 mV applied voltage. Mean current levels could be reversibly adjusted by voltage to the single channel level (0 to -50 mV) or to macroscopic levels (-50 to -100 mV) over periods exceeding 1 h. Single channel events are characterized by fast transitions between predominantly non-resolved sublevels. Estimates of maximal single event currents yield a slope conductance of 32 +/- 4 pS (0 to -60 mV, 50 mM CaCl2). Thus, the purified IP3 receptor forms a channel with functional properties characteristic of IP3 triggered Ca2+ release.

Animals↗
PubMed1989

The cardiac sodium channel shows a regular substate pattern indicating synchronized activity of several ion pathways instead of one.

Cardiac sodium channel substates were induced by using different gating modifiers, namely S-DPI 201-106 (s), toxin II from Anemonia sulcata (a), veratridine (v) and mixtures of these agents (s + v, a + v). Current ratios (normalized substate currents), slope conductances, reversal potentials and saturation characteristics were evaluated for the individual channel substates. The results can be summarized as follows: (i) Current ratios fell into a pattern of six equidistant values (I to VI) irrespective of the modification applied (0.20, 0.34, 0.51, 0.69, 0.85, 1.00). Slope conductances, determinable for substates II, V and VI (4.8, 11.7 and 14.0, respectively), are also consistent with six conductance substates which are integer multiples of a smallest conductance (state I). (ii) The permeability ratio PNa+/PK+ (i.e., reversal potential of substate currents) of the sodium channel was conserved both for different modifications, i.e., by s, a, s + v and a + v, and for the different substates (at least for II, IV and VI) observed for each modification. (iii) Sodium binding to the channel is substate independent. Analysis of slope conductances of states II and VI for three sodium chloride concentrations (71.5, 140 and 303 mM) revealed different maximal conductances (geVImax = 2.9.geIImax) but similar apparent affinities for sodium (KNa + VI = 286 mM; KNa + II = 303 mM). These findings are shown to seriously challenge the commonly unquestioned conception that 'single-current events' reflect ion passage through only one single pathway. The alternative view, that not one pore, but either six or three pores with synchronized gating ('oligochannel') underlie 'single-channel events', is shown to readily account for the observed substate properties and appears not to contradict known properties of 'the sodium channel'. This fundamentally new view of the sodium channel aims to invoke further efforts to distinguish between conceptually distinct models of structure-function relationships for a variety of channels which show multiple substates and conserved ion selectivity.

Animals↗
PubMed1989

[The current risk of gallbladder and bile duct surgery].

The lethal operative risk in gallbladder and bile duct operations decreased from 2.16% in the period 1964-1976 to 1.32% in the period 1979-1987.--Main operative risk factors are gallbladder and bile duct complications and the age beyond the 6th decade of life.--The lethality of choledocholithiasis actually amounts to the 37-fold of uncomplicated cholecystolithiasis and the lethality of all primary and secondary gallstone operations after the 6th decade of life amounts to the 38-fold in comparison with younger patients.--A further reduction of operative risk appears possible by reason: 1. Consequent early operation. 2. immediate operative or endoscopic removal of extrahepatic cholestasis, 3. complex intraoperative diagnostics of bile duct and papilla Vateri, 4. preoperative single dose AB prophylaxis or short-term therapy, 5. general low dose heparin prophylaxis, 6. primary endoscopic therapy in residual stones or irreversible stenosis of papilla Vateri and 7. interdisciplinary diagnosis and treatment of senile complications.

Aged↗
PubMed1988

Reconstitution of purified cardiac muscle calcium release channel (ryanodine receptor) in planar bilayers.

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).

Adenosine Triphosphate↗
PubMed1988

Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers.

The purified 1,4-dihydropyridine receptor from skeletal muscle has been incorporated into planar bilayers, and its channel characteristics have been investigated. Conductances showed the characteristics of an L-type Ca2+ channel: divalent cation selectivity (PBa/PNa approximately equal to 30), blockage of Na+ conductance by micromolar Ca2+, and blockage of the Ca2+ channel by D890 and by Cd2+. The alpha 1 subunit of the receptor must be phosphorylated by the cAMP-dependent protein kinase to give channel activity. BAY K 8644 did not activate nonphosphorylated channels, and (+)-PN200-110 caused dramatic prolongation of mean open times when applied after phosphorylation. Channel properties were found to be dependent on association of receptor molecules in the bilayer. Single receptor molecules form channels of 0.9 pS (100 mM Ba2+) and show no voltage-dependent gating. Upon association, both voltage-dependent gating and higher conductance events are recovered; stabilized conductance levels assume values of even multiples of 0.9 pS, predominately 7.5 and 15 pS and multiples of these values up to 60 pS. Thus, individual channels become functionally coupled (synchronous opening and closing) with association, reinstating the characteristics of one larger unitary channel. It is concluded that the L-type Ca2+ channel represents an oligomer of 1,4-dihydropyridine-receptor protein complexes, each of which constitutes a channel, where the array of channels (oligochannel) opens and closes in concerted action.

Animals↗
PubMed1988

Purified ryanodine receptor of skeletal muscle sarcoplasmic reticulum forms Ca2+-activated oligomeric Ca2+ channels in planar bilayers.

The ryanodine receptor of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle has been purified and found by electron microscopy to be equivalent to the feet structures that are involved in situ in the junctional association of transverse tubules with terminal cisternae of SR. We now find that when the purified receptor is incorporated into vesicle-derived planar bilayers, it forms Ca2+-specific channels, which are dependent on submicromolar Ca2+ for activity. In the presence of 1 mM ATP, the channel shows essentially no activity at 10 nM Ca2+ but becomes highly activated at 50 nM Ca2+. At suboptimal Ca2+ levels (100 nM), the channel is strongly activated by 1 mM ATP and can be blocked by ruthenium red, both effects being prevented by higher Ca2+ levels (1 microM). Mg2+, added from the cis side at millimolar concentrations, blocks Ca2+ flux through the channel from trans to cis (equivalent to flux from luminal to myoplasmic compartment). Ryanodine stabilizes the open state of the channel and blocks the action of ruthenium red to close the channel. Thus, the purified ryanodine receptor incorporated into a bilayer has the Ca2+-channel characteristics consistent with the calcium release observed in isolated terminal cisternae vesicles. Furthermore, ryanodine induced the appearance of a sublevel gating mode characterized by long open conductance states, which were integral multiples of the smallest observed conductance, 3.8 pS in 50 mM Ca2+. The purified receptor consists essentially of a single-sized high molecular weight polypeptide (Mr. approximately equal to 360,000), which on reconstitution forms the square rectangles diagnostic of the feet structures. We conclude that the identity of the Ca2+-release channel of SR is the foot structure, which consists of an oligomer of the high molecular weight polypeptide.

Adenosine Triphosphate↗