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E Stefani

Publications and source records attributed to E Stefani.

At least 109 records · Page 6Linked to original sources
PubMed1992

A novel N18TG2 x mesencephalon cell hybrid expresses properties that suggest a dopaminergic cell line of substantia nigra origin.

A dopaminergic neuroblastoma was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell line N18TG2. The resulting interspecies hybrid, named MES23.5, has retained a stable phenotype and karyotype for a continuous culture period of 1 year. The hybrid exhibits several properties that suggest that the parent primary neurons originated in the substantia nigra. The cell line contains tyrosine hydroxylase, which is identifiable both by biochemical and immunological methods and synthesizes dopamine, but no other catecholamine. Additionally, the cell line expresses apparent voltage-gated CA2+ channels as measured by high-affinity omega-conotoxin binding. The MES23.5 omega-conotoxin receptors are of similar affinity class to those found in adult rat mesencephalon. No dihydropyridine receptors, as measured by PN200-100 ligand binding, are present. None of these properties are found in the N18TG2 parent. At least three neuronal features, namely, tyrosine hydroxylase, dopamine synthesis, and omega-conotoxin receptor expression, are quantitatively elevated after sustained treatment with cAMP analogs. The cell line expresses a complex range of neural properties found in the dopaminergic neurons of the substantia nigra, and may therefore be useful elucidating further details of their cell biology.

Animals↗
PubMed1991

Molecular basis of gating charge immobilization in Shaker potassium channels.

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.

Amino Acid Sequence↗
PubMed1991

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.

Blotting, Western↗
PubMed1991

Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.

Animals↗
PubMed1991

Antibodies as probes for ligand gating of single sarcoplasmic reticulum Ca2(+)-release channels.

A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.

Adenosine Triphosphate↗
PubMed1991

Immunoglobulins from animal models of motor neuron disease and from human amyotrophic lateral sclerosis patients passively transfer physiological abnormalities to the neuromuscular junction.

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons of unknown etiology. In support of the potential role of autoimmunity in ALS, two immune-mediated animal models of motoneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, the presence of IgG within motoneurons and at the neuromuscular junction, and with respect to altered physiology of the motor nerve terminal. To provide direct evidence for the primary role of humoral immunity, passive transfer with immunoglobulins from the two animal models and human ALS was carried out. Mice injected with serum or immunoglobulins from the animal disease models and human ALS but not controls demonstrated IgG in motoneurons and at the neuromuscular junction. The mice also demonstrated an increase in miniature end-plate potential (mepp) frequency, with normal amplitude and time course and normal resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal. The ability to transfer motoneuron dysfunction with serum immunoglobulins provides evidence for autoimmune mechanisms in the pathogenesis of both the animal models and human ALS.

Adult↗
PubMed1991

Calcium-activated K+ channels: metabolic regulation.

Calcium-activated potassium (KCa) channels are highly modulated by a large spectrum of metabolites. Neurotransmitters, hormones, lipids, and nucleotides are capable of activating and/or inhibiting KCa channels. Studies from the last few years have shown that metabolites modulate the activity of KCa channels via: (1) a change in the affinity of the channel for Ca2+ (K 1/2 is modified), (2) a parallel shift in the voltage axis of the activation curves, or (3) a change in the slope (effective valence) of the voltage dependence curve. The shift of the voltage dependence curve can be a direct consequence of the change in the affinity for Ca2+. Recently, the mechanistic steps involved in the modulation of KCa channels are being uncovered. Some interactions may be direct on KCa channels and others may be mediated via G-proteins, second messengers, or phosphorylation. The information given in this review highlights the possibility that KCa channels can be activated or inhibited by metabolites without a change in the intracellular Ca2+ concentration.

Animals↗
PubMed1991

Cyclophosphamide alters the clinical and pathological expression of experimental autoimmune gray matter disease.

Guinea pigs inoculated with bovine spinal cord ventral horn homogenate develop a syndrome termed experimental autoimmune gray matter disease (EAGMD) characterized by extremity weakness, bulbar signs, and a loss of lower and upper motoneurons. To provide evidence for the role of autoimmune mechanisms, we have administered the immunosuppressant cyclophosphamide prior to and after gray matter immunization. Pretreatment with cyclophosphamide prevented the appearance of clinical signs of disease and decreased the loss of spinal cord motoneurons, the appearance of damaged motoneurons, and the antibody titer to motoneurons. Treatment 7 days after immunization attenuated the expression of disease. Treatment immediately after signs also improved the clinical and pathological findings. In all cyclophosphamide-treated animals there was less IgG within motoneurons and less inflammation. These results support the role for autoimmune mechanisms in motoneuron loss and degeneration in EAGMD.

Animals↗
PubMed1991

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle.

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.

Animals↗
PubMed1991

Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.

Animals↗
PubMed1991

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.

Animals↗
PubMed1991

Differential effects of ryanodine and tetracaine on charge movement and calcium transients in frog skeletal muscle.

1. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique. Calcium transients were monitored with the fluorescent indicator Rhod-2. 2. Ryanodine modified the kinetics and the total amount of charge moved during depolarizing pulses (Q(on)), while it did not significantly modify the charge after repolarization (Q(off)). The extracellular application of 100 microM-ryanodine elicited a temporary initial increase of the delayed component of charge movement (Q gamma) and the calcium transient. Both phenomena were later blocked with the same temporal course and to the same extent. 3. The blockade of Q gamma and the calcium transient was also observed with ryanodine concentrations of 1-10 microM. For membrane potentials positive to -10mV, the Qon measured was larger in the presence of ryanodine; Qoff was not modified. 4. Tetracaine (400-500 microM) blocked a similar delayed component of Qon, identified as Q gamma, as well as the calcium transient monitored simultaneously. This effect was observed in the first minutes after the addition of tetracaine to the extracellular solution. 5. Tetracaine blocked a faster initial component of Qon for voltages positive to -10 mV, corresponding to the voltage range of activation of the calcium current. At these same membrane potentials, Qoff was also reduced to a similar extent to Qon. 6. Ryanodine and tetracaine showed different effects on calcium current. Whereas the slow calcium current was not modified upon the addition of ryanodine, it was completely blocked in the presence of tetracaine. The blockade of the slow calcium current made evident the fast calcium current. The effects of tetracaine on the charge movement, the calcium transient and the slow calcium current were reversible. 7. These results suggest that ryanodine and tetracaine may act at different sites. Ryanodine exerts its effect on the sarcoplasmic reticulum ryanodine receptor, blocking calcium release and Q gamma, while tetracaine at these concentrations may affect the release channel and the dihydropyridine receptor, causing a blockade of the charge movement, calcium transient and calcium current.

Animals↗
PubMed1991

Calcium current and charge movement of mammalian muscle: action of amyotrophic lateral sclerosis immunoglobulins.

1. The Vaseline-gap voltage clamp technique was used to record dihydropyridine (DHP)-sensitive Ca2+ currents (ICa) and charge movement in single cut fibres from the rat extensor digitorum longus (EDL) muscle. Amyotrophic lateral sclerosis (ALS) immunoglobulin G (IgG) action on ICa and charge movement has been characterized. 2. ALS IgG reduced ICa amplitude. The peak ICa of EDL fibres (mean +/- S.E.M.) at 0 mV, expressed as amperes per membrane capacitance, was -4.79 +/- 0.029 A F-1, while after 30 min incubation in ALS IgG it was -2.52 +/- 0.04 A F-1. IgG from healthy patients, and from patients with other diseases (familial ALS, myasthenia gravis, chronic relapsing inflammatory polyneuritis, multiple sclerosis and one sample from Lambert-Eaton syndrome, LES) did not affect ICa, while IgG from patients with Guillain-Barré syndrome and one other sample from a patient with LES affected the ICa in a similar way as ALS IgG. 3. The time constant of ICa activation (alpha m) at 0 mV was 44.8 +/- 1.4 ms in control, and 36.6 +/- 1.5 ms after an incubation of 30 min in ALS IgG. The steady-state activation curve (m infinity) was shifted to more positive potentials by ALS IgG. 4. The rate constants of activation (range -20 to 30 mV) were altered by ALS IgG: alpha m decreased while beta m increased. These data suggest that ALS IgG favours the permanence of the Ca2+ channels in the closed state. 5. The time constant of Ca2+ channels deactivation at -90 mV with a pre-pulse to 0 mV was 4.4 +/- 0.5 ms in control and 4.1 +/- 0.6 ms in ALS IgG. The relationship between the deactivation time constant and membrane potential was not significantly modified by ALS IgG. 6. ICa inactivation was not affected by ALS IgG. The potentials of half-inactivation were -32.1 and -36.6 mV in control and ALS IgG, respectively. Similarly, the rate constants of inactivation (alpha h and beta h) remained unaltered by ALS IgG. 7. We successfully blocked ICa with 100 microM-TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride), without major effects on charge movement. We adopted this procedure to study charge movement. ALS IgG reduced charge movement without significant effects on the effective valence and voltage dependence. Qon and Qoff, the charges during and after the pulse, were similarly affected by ALS IgG. 8. The actions of ALS IgG on DHP-sensitive Ca2+ current and charge movement suggest an interaction between ALS IgG and some component of the DHP-receptor complex.

Adult↗
PubMed1991

IgG from amyotrophic lateral sclerosis affects tubular calcium channels of skeletal muscle.

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons. We studied the action of the immunoglobulin G (IgG) from ALS and disease control patients on dihydropyridine (DHP)-sensitive Ca2+ channels in single mammalian skeletal muscle fibers with the double Vaseline gap technique. The peak of the Ca2+ current (ICa) and the charge movement were reduced when the fibers were incubated in ALS IgG. These effects were lost when the IgG was boiled or adsorbed with skeletal tubular membranes. ALS IgG reduced skeletal muscle ICa in a similar fashion as nifedipine; the ICa blockade was voltage dependent, and the associated charge movement was reduced. These observations suggest that IgG from ALS patients reacts with the skeletal muscle DHP-sensitive Ca2+ channels or some associated regulatory moiety.

Adult↗
PubMed1991

Potassium currents in freshly dispersed myometrial cells.

K+ currents in freshly dispersed cells from rat myometrium at estrus were studied with the patch-clamp technique (whole cell). Three types of K+ currents were identified: 1) a fast-activating current (IKf), 2) a slowly activating current (IKs), and 3) a transient current (IKt). IKf had a half-activation voltage of 12 mV and a time constant of activation (tau on) of approximately 3 ms at +50 mV. IKs had a tau on of approximately 9 ms at +50 mV and a half-activation potential of 33 mV. Both IKf and IKs were sustained and became potentiated by the entrance of Ca2+ from the patch pipette. These two Ca(2+)-activated K+ currents were inhibited by 100 nM external charybdotoxin and were blocked by external tetraethylammonium (TEA, 2-20 mM). The third current (IKt) was transient, had a faster tau on (approximately 1 ms), and a decay phase with a time constant of approximately 8 ms at +50 mV. This current had a half-activation potential of 22 mV. IKt was not potentiated by intracellular Ca2+, was sensitive to 4-aminopyridine (1 mM), was insensitive to external charybdotoxin (100 nM) and TEA (2 mM), and spontaneously decreased with time.

Animals↗
PubMed1991

Calcium-activated potassium channels from coronary smooth muscle reconstituted in lipid bilayers.

This work is the initial characterization of Ca(2+)-activated K+ (KCa) channels from coronary smooth muscle reconstituted into lipid bilayers. The channels were obtained from a surface membrane preparation of porcine coronary smooth muscle. KCa channels were the predominant K+ channels in this preparation. The conductance histogram (n = 137 channels) revealed two main populations of "maxi" KCa channels with conductances of 245 and 295 pS. Each population could be subdivided in two "isoforms" or "isochannels" with different functional properties (voltage and Ca2+ sensitivities and kinetics). The analysis of "burst" probability of opening showed that at pCa 4 the two isochannels of 245 pS (KCa-1 and KCa-1') had half-activation potentials (V1/2) of -80 and 6 mV, respectively. The isochannels of 295 pS (KCa-2 and KCa-2') had V1/2 of -28 and -66 mV, respectively. KCa-1 had the highest Ca2+ sensitivity; at -60 mV, the concentration of half-activation value for Ca2+ was 1.2 +/- 0.3 microM (n = 5). External tetraethylammonium reduced channel amplitude in a voltage-dependent manner; dissociation constant was 180 +/- 6 and 466 +/- 41 microM at -40 and +80 mV, respectively (n = 5). Charybdotoxin (5-50 nM) produced typical long closings. These effects were similar in all the channels. We conclude that coronary smooth muscle possesses isoforms of maxi KCa channels with Ca2+ and voltage sensors with different properties, which may confer to each channel a specific functional role.

Action Potentials↗