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Biomedical subjects

G Prestipino

Publications and source records attributed to G Prestipino.

32 records · Page 2Linked to original sources
PubMed1992

Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake.

A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.

Amino Acid Sequence↗
PubMed1991

AMPA and kainate-operated channels reconstituted in artificial bilayers.

Cationic channels which can be activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate play a key role in the generation of excitatory postsynaptic potentials in the brain of all vertebrates. On a protein carrying binding sites for both these ligands, affinity-purified from the Xenopus nervous system, electrical measurements have been performed to investigate its functional properties after the pure complex had been incorporated into planar lipid bilayers. Domoate, AMPA or kainate added to the reconstituted protein activated cationic channels, which were blocked by typical antagonists for this neurotransmitter system. These data suggest that the reconstituted protein is an ionotropic receptor of the unitary non-NMDA subtype.

Animals↗
PubMed1989

Purification and reconstitution of potassium channel proteins from squid axon membranes.

Voltage-dependent K+ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K+-channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NTX-affinity purified proteins had Mr values of 60,000 +/- 6,000, 160,000 +/- 15,000 and 220,000 +/- 20,000. Their incorporation into bilayers resulted in single-channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K+ channels involved in the squid axon action potential.

Animals↗
PubMed1989

Localization of putative nicotinic cholinoreceptors in the early development of Paracentrotus lividus.

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.

Animals↗
PubMed1989

Effects of alpha-latrotoxin on the early developmental events of the sea urchin Paracentrotus lividus: a histochemical study.

The effects of the spider toxin alpha-latrotoxin (alpha-LTX) on gametes, zygotes, and early embryos of the sea urchin Paracentrotus lividus have been investigated by in vivo experiments and by histochemical studies of acetylcholinesterase (AChE) activity. Treatment of unfertilized eggs with nanomolar amounts (1 to 0.3 nmol/l) of alpha-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization layer by sperms. Instead, fertilized eggs exposed to alpha-LTX showed noticeable alterations in cell surface topography, including the appearance of prominent membrane-limited blebs. Moreover, the zygotes treated with 1 nmol/l alpha-LTX failed to cleave. The histochemical staining of treated zygotes revealed a very strong AChE activity in the cortical region, including blebs. An enzyme reaction was also found in the perivitellin space. Our results suggest the hypothesis that some alpha-LTX receptors may appear after fertilization, supporting the awareness that fertilized eggs display excitable cell features.

Acetylcholinesterase↗
PubMed1987

Blocking of the squid axon K+ channel by noxiustoxin: a toxin from the venom of the scorpion Centruroides noxius.

We have studied the selective effects of noxiustoxin (NTX), a fraction of the venom of the scorpion Centruroides noxius, on the K currents of perfused squid giant axons using the voltage-clamp technique. At concentrations below 1.5 microM, NTX blocked K currents in a voltage-independent manner, with little effect on their turning-on and turning-off kinetics. Above 1.5 microM, the block by NTX became voltage-dependent and could be partially removed by repetitive pulsing and strong depolarizations. Long repolarizations and more negative holding potentials favoured the slow restoration of channel block. Reduction of K currents by internally perfusing the fibers with solutions of low K+ concentration (200 mM), affected very little the removal of NTX-block during repetitive pulsing, suggesting that block removal depended on membrane potential and not on outward movements of K+ ions through open channels. In high extracellular K+ (300 mM) the blocking action of NTX was reduced and the instantaneous I-V characteristics showed a marked outward rectification. At 20 microM NTX, inward tail currents measured on step repolarizations to -70 mV were fully blocked, suggesting a direct interaction of the toxin with the open channel. The effects of the total venom Centruroides noxius Hoffmann was also studied. External application of 0.25 mg/ml of the venom caused a marked reduction of both Na and K currents, an effect similar to that of other scorpion venoms.

Animals↗
PubMed1987

Effects of low-intensity pulsed electromagnetic fields on the early development of sea urchins.

The effects of weak electromagnetic signals on the early development of the sea urchin Paracentrotus lividus have been studied. The duration and repetition of the pulses were similar to those used for bone healing in clinical practice. A sequence of pulses, applied for a time ranging from 2 to 4 h, accelerates the cleavages of sea urchin embryo cells. This effect can be quantitatively assessed by determining the time shifts induced by the applied electromagnetic field on the completion of the first and second cleavages in a population of fertilized eggs. The exposed embryos were allowed to develop up to the pluteus stage, showing no abnormalities.

Animals↗
PubMed1986

Reconstitution of the purified alpha-latrotoxin receptor in liposomes and planar lipid membranes. Clues to the mechanism of toxin action.

The receptor of alpha-latrotoxin (the major toxin of the black widow spider venom), purified from bovine synaptosomal membranes, was reconstituted into small unilamellar liposomes. These (but not control) liposomes exhibited high-affinity, specific binding of [125I]alpha-latrotoxin. In the receptor-bearing liposomes alpha-latrotoxin induced depolarization and stimulated 45Ca efflux. These responses to alpha-latrotoxin, that were observed only in the presence of external divalent cations, resembled those previously demonstrated in mammalian brain synaptosomes. The alpha-latrotoxin-activated ion fluxes are therefore, at least in part, the result of the direct interaction of the toxin with its receptor. When control and receptor-bearing liposomes were pre-incubated with alpha-latrotoxin and then added to a solution bathing a planar lipid bilayer membrane, single channel cationic conductances were observed. In the presence of the receptor, the conductances induced by alpha-latrotoxin were markedly different from those observed without the receptor, but not identical to those observed without the receptor, but not identical to those recently characterized by patch clamping in the cells of a line (PC12) sensitive to alpha-latrotoxin. These results demonstrate that the reconstituted receptor is functional, and suggest that the cationic channel activated by the toxin-receptor interaction is modulated by additional component(s) in the membrane of synapses and cells.

Animals↗
PubMed1984

Selective modification of the squid axon Na currents by Centruroides noxius toxin II-10.

We have studied the effects of the purified toxin II-10, from the venom of the scorpion Centruroides noxius Hoffmann, on the Na and K currents of voltage clamped squid giant axons. Extracellular applications of 10 microM of toxin II-10 produced a selective depression of peak Na currents, with no significant effects on the time course of K currents. On pharmacologically separated Na currents, low concentrations of toxin II-10 (0.28-1 microM) caused a reversible decrease in inward and outward peak INa, with little effect on either the maintained level of the currents or their turning-off. At high concentrations (greater than 3 microM), toxin II-10 drastically reduced the peak conductance and increased both the level of the maintained conductance, and the time course of its turning-off. It is suggested that, when applied extracellularly on squid axons, toxin II-10 primarily reduces the peak Na conductance by modifying the activation of fast-inactivating Na channels. At high concentrations (10 microM), the toxin also modifies the rate constants of the transition from the inactivated to the second open state of the channel (Chandler and Meves, 1970) thus producing an increased level of the maintained Na conductance. It is also very likely, however, that peak conductance and maintained conductance reflect two separate populations of Na channels on which toxin II-10 has a differentiated action. Under these conditions, toxin II-10 would be the first reported toxin which can pharmacologically separate the two types of channels.

Amino Acid Sequence↗
PubMed1983

High intracellular pH reversibly prevents gating-charge immobilization in squid axons.

Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332).

Animals↗
PubMed1978

Action of extracellular pH on Na+ and K+ membrane currents in the giant axon of Loligo vulgaris.

Voltage-clamp currents and resting membrane potential of squid giant axons have been studied at extracellular pH varying between 4 and 10. The membrane currents, analyzed according to the Hodgkin-Huxley equations, showed that sodium permeability, PNa(E), and potassium conductance gK(E), curves were shifted toward positive voltages by different amounts and slightly depressed as the external pH was lowered. Under the same conditions, taum(E) and taun(E) were found to be enhanced and shifted to a larger extent in the same direction. The rate constants alpham and alphan were shifted substantially toward positive voltages, but betam and betan changed hardly at all. The shift of the alpham(E) curve was analyzed in terms of a fixed surface charge model; it indicates that unspecific negative groups with an approximate pKa of 4.5 are located in the vicinity of sodium active sites with an average charge separation of 8 A. A similar figure is obtained for the potassium system from the shift of the alphan(E) curve.

Animals↗