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M Taglialatela

Publications and source records attributed to M Taglialatela.

At least 19 recordsLinked to original sources
PubMed1992

The Na(+)-Ca2+ exchanger activity in cerebrocortical nerve endings is reduced in old compared to young and mature rats when it operates as a Ca2+ influx or efflux pathway.

The activity of the Na(+)-Ca2+ exchanger, which regulates the entry and the extrusion of Ca2+ ions from nerve endings was investigated in Percoll-purified cerebrocortical synaptosomes of aged rats. 45Ca2+ uptake in a Na(+)-free medium and 45Ca2+ efflux in a 145 mM Na+ medium were significantly reduced in cerebrocortical synaptosomes from aged rats (24 months) as compared to those occurring in young (4 months) and mature (14 months) rats. 45Ca2+ influx induced by 55 mM K+, a concentration of K+ ions which selectively promotes Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), was significantly reduced in mature and aged rats as compared to that occurring in young rats. The impairment of these mechanisms in aged rats is not accompanied by any variation of fura-2 monitored Ca2+ levels under resting and depolarizing conditions.

Animals↗
PubMed1992

The Na(+)-Ca++ exchanger in central nerve endings: the relationship between its pharmacological blockade and dopamine release from tuberoinfundibular hypothalamic neurons.

2', 4'-Dimethylbenzamiloride (DMB), an inhibitor of Na(+)-Ca++ antiporter dose-dependently (10-100 microM) inhibited Na(+)-dependent 45Ca++ efflux from brain synaptosomes. This compound was also able to stimulate basal release of [3H]DA from superfused TIDA neurons. Another amiloride analogue, 5-N-methyl-N-guanidinocarbonylmethylamiloride (MGCMA, 100-300 microM), which lacks of inhibitory properties on the Na(+)-Ca++ antiporter, failed to modify basal [3H]DA release from TIDA neurons. In addition, when the antiporter operates as a Ca(++)-influx pathway, DMB dose-dependently inhibited Na(+)-dependent 45Ca++ uptake in brain synaptosomes, whereas it did not prevent K(+)-induced 45Ca++ uptake, which reflets the activation of voltage-operated Ca++ channels. Finally DMB inhibited ouabain-induced [3H]DA release, which depends on the activation of the Na(+)-Ca++ exchanger due to the inhibition of the Na+/K(+)-ATPase pump.

Amiloride↗
PubMed1992

Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids.

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

Amino Acid Sequence↗
PubMed1992

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.

Amino Acid Sequence↗
PubMed1992

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Animals↗
PubMed1992

Maitotoxin, a novel activator of mediator release from human basophils, induces large increases in cytosolic calcium resulting in histamine, but not leukotriene C4, release.

Maitotoxin (MTX) is a potent marine toxin which stimulates several Ca(++)-dependent processes presumably through an increase in Ca++ permeability. We have examined the effect of MTX on the release of chemical mediators from human basophils and its mechanism of action. MTX (1-20 ng/ml) induced histamine release (37-100%) from both mixed leukocyte preparations and purified basophils. Histamine release activated by MTX was slow (t 1/2 approximately equal to 15 min), temperature and Ca++ dependent (optimal at 37 degrees C and 1-2.5 mM Ca++). Sr++ ion could substitute for Ca++ in the secretory process. Digital video microscopy analysis of purified (> 70%) basophils revealed that MTX (1-20 ng/ml) induced a slow and marked increase of cytosolic Ca++ levels that was temporally coincident with histamine release. MTX (1-20 ng/ml) stimulated the release of sulfidopeptide leukotriene C4 from mixed leukocyte preparations (approximately equal to 0.5% basophils). However, purified basophils (77 +/- 7%) showed no sulfidopeptide leukotriene C4 release even in the presence of large histamine secretion (84 +/- 14%). Two organic Ca(++)-channel entry blockers, verapamil and diltiazem (1-30 microM) inhibited the release of histamine induced by MTX, whereas the dihydropyridine nifedipine (0.1-10 microM) caused only minimal inhibition. These results suggest that MTX represents a novel stimulus useful to study the role of Ca++ in human basophil mediator release.

Basophils↗
PubMed1991

Cytoplasmic alkalinization induced by insulin through an activation of Na(+)-H+ antiporter inhibits tyrosine hydroxylase activity in striatal synaptosomes.

Insulin dose-dependently inhibited tyrosine hydroxylase (TH) activity and increased intrasynaposomal pH (pHi) in rat striatal nerve endings. Both these effects of insulin on TH and pHi were prevented by the 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA), a putative selective inhibitor of the Na(+)-H+ antiporter. Interestingly when, by changing the extracellular pH (pHo), the pHi was increased, from 7.1 up to 7.5, an equivalent inhibition of TH activity occurred. The inhibitory action exerted from insulin on TH activity disappeared when the hormone was added to synaptosomes whose pHi was lowered to 6.83. Collectively, the results of the present study showed that insulin inhibited TH activity in striatal synaptosomes. This effect seems to involve the activation of the Na(+)-H+ antiporter. This exchange system once activated, may induce an intrasynaptosomal alkalinization, a condition in which TH activity is inhibited.

Amiloride↗
PubMed1991

Voltage-dependent inhibition and facilitation of Ca channel activation by GTP-gamma-S and Ca-agonists in adult rat sensory neurons.

Intracellular application of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S, 100 microM) causes a slow down of high-threshold Ca channel activation in adult rat sensory neurons that is relieved by strong depolarizations or by the Ca agonist Bay K 8644 (5 microM). Recovery from GTP-gamma-S inhibition is usually accompanied by an increase in Ca current amplitude (facilitation) and is insensitive to holding potential (-60 to -90 mV). Inhibition and facilitation of Ca currents are also little affected by nifedipine (5 microM) or by cell incubation with omega-conotoxin (omega-CgTx, 3.2 microM). We conclude that both slowdown of Ca channel activation by GTP-gamma-S and facilitation by strong depolarizations or by Ca agonists derive from a common process in which G-protein activation, membrane voltage and Ca agonist receptors interact to modulate neuronal Ca channel gatings.

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

Exchange of conduction pathways between two related K+ channels.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Amino Acid Sequence↗
PubMed1991

Internal and external TEA block in single cloned K+ channels.

Tetraethylammonium (TEA) has been used recently to probe natural and mutational variants of voltage-dependent K+ channels encoded by cDNA clones. Its usefulness as a probe of channel structure prompted us to examine the molecular mechanism by which TEA blocks single-channel currents in Xenopus oocytes expressing the rat brain K+ channel, RCK2. TEA at the intracellular surface of membrane patches decreased channel open time and increased the duration of closed intervals. Tetrapentylammonium had similar but more potent effects. Extracellular application of TEA caused an apparent reduction of single-channel amplitude. Block was slower at the high-affinity internal site than at the low-affinity external site. Internal TEA selectively blocks open K+ channels, and the voltage dependence of the block indicates that the binding site lies within the membrane electric field at a point 25% of the distance from the cytoplasmic margin. External TEA also interacts with the open channel but is less sensitive to membrane potential. The results indicate that the internal and external TEA binding sites define the inner and outer margins of the aqueous pore.

Animals↗
PubMed1991

Patterns of internal and external tetraethylammonium block in four homologous K+ channels.

Tetraethylammonium (TEA) is a small ion that is thought to block open K+ channels by binding either to an internal or to an external site. For this reason, it has been used to probe the ion conduction pathway or pore of K+ channel mutants and a K+ channel chimera. The results suggested that the region between transmembrane segments 5 and 6 (S5-S6 linker) was involved in the formation of both the internal and the external TEA binding sites and the K+ conduction pathway. Therefore, we compared internal and external TEA block of the currents expressed in Xenopus oocytes injected with RNAs from four related K+ channel clones, DRK1, RCK1, RCK2, and r-NGK2, which have only subtle structural differences in the S5-S6 linker. r-NGK2 was the most sensitive to external TEA and the least sensitive to internal TEA application. For DRK1 the profile was reversed. RCK1 was blocked equally well from either side, whereas RCK2 was more strongly blocked by internal TEA. The internal block was voltage dependent, whereas the external block was virtually voltage independent. As predicted from block of whole-oocyte currents, internal TEA produced a slow block of DRK1 and RCK2 single-channel currents but had almost no effect on r-NGK2 single-channel currents. Tetrapentylammonium produced a stronger block than TEA at the internal site, and the block was relieved by inward K+ currents, therefore suggesting that the internal TEA binding site is located within the K+ conduction pathway. These results, together with the TEA block of single-channel currents, establish what has until now been inferred by extrapolation from other studies, i.e., that TEA is an open-channel blocker in K+ channel clones. DRK1 mutants with extensive amino- and carboxyl-terminal deletions showed the same blocking profile as the parent DRK1. We conclude that TEA blocks these K+ channels at two sites, which define the inner and outer mouths of the channel pores. Comparison of the primary amino acid sequences in the S5-S6 linker suggests which residues may be responsible for the different patterns of TEA block.

Animals↗
PubMed1990

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes.

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.

Animals↗
PubMed1990

Ibopamine-induced reduction of serum prolactin level and milk secretion in puerperal women.

Ibopamine, a peripheral dopamine agonist, was administered to 80 postpartum women to assess its effect on prolactin (PRL) and milk production. The acute administration of 400 mg significantly decreased serum PRL for more than 240 min. Women given ibopamine 400 mg t.d.s. for 5 to 10 days showed suppression of PRL and milk letdown was prevented in the latter group. No side effects were observed on repeated administration. Ibopamine may be a useful alternative to other dopaminergic compounds for the inhibition of puerperal lactation.

Adult↗
PubMed1990

Possible involvement of Ca++ ions, protein kinase C and Na(+)-H+ antiporter in insulin-induced endogenous dopamine release from tuberoinfundibular neurons.

Insulin (63 microM) stimulated endogenous dopamine (DA) release from tuberoinfundibular neurons. This effect was independent on the presence of extracellular glucose and did not involve the outward transport of DA, mediated by its membrane carrier. By contrast this effect was completely prevented by the removal of extracellular Ca++ ions in presence of the Ca(++)-chelator ethyleneglycol-2-(2-aminoethyl)-tetracetic acid (EGTA). Furthermore 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine (H7), a compound which behaves as a putative inhibitor of protein kinase C (PK-C) (10 microM), completely counteracted the stimulation of endogenous DA release induced by insulin. Amiloride (300 microM) and its 5-amino nitrogen atom-substituted derivative, 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA) (10 microM), a highly selective inhibitor of the Na(+)-H+ membrane antiporter, were both able to prevent the stimulatory action exerted by insulin on endogenous DA release. Collectively, these results suggest that the transductional events by which insulin stimulated endogenous DA release from TIDA neurons may involve the activation of PK-C, the enhancement of Ca++ influx and the stimulation of the Na(+)-H+ exchange system.

Amiloride↗
PubMed1990

Na(+)-Ca2+ exchange activity in central nerve endings. I. Ionic conditions that discriminate 45Ca2+ uptake through the exchanger from that occurring through voltage-operated Ca2+ channels.

Ca2+ entrance in central nerve endings can occur through voltage-operated Ca2+ channels and/or through the Na(+)-Ca2+ antiporter. The aim of the present study was to evaluate, in brain synaptosomes, the possible contribution of these two Ca2+ entrance pathways in the process of 45Ca2+ uptake elicited by different extracellular ionic conditions. The decrease in extracellular Na+ concentration from 145 mM to 95 mM and its concomitant substitution with complemental concentration of K+ (5-55 mM) caused an increase in 45Ca2+ uptake, whereas an equimolar concentration of choline (50 mM), although in the presence of the same Na+ concentration (95 mM), failed to stimulate 45Ca2+ uptake. Only when the extracellular Na+ concentration was further lowered from 95 mM to 0 mM and substituted with equivalent amounts of choline (50-145 mM) did a dose-dependent stimulation of 45Ca2+ uptake occur. In addition, when the lowering of the extracellular Na+ concentration from 95 mM to 0 mM was compensated for by K+ concentrations higher than 55 mM (55-150 mM), 45Ca2+ uptake was higher than that elicited by Na+ ion substitution with equimolar amounts (50-145 mM) of choline. The amount of 45Ca2+ uptake induced by 55 mM K+ did not differ either in Na(+)-preincubated or in Na(+)-depleted synaptosomes. Synaptosomal membrane potential, monitored with the potential-sensitive fluorescent dye bis-(1,3-diethyltiobarbiturate)trimethineoxonol, showed a progressive depolarization when extracellular K+ concentrations were raised from 5 to 150 mM, reaching a plateau at 55 mM extracellular K+ concentration, whereas when choline (145 mM) completely substituted for extracellular Na+ ions, synaptosomal membrane potential did not show any depolarization. Collectively, these results demonstrate that 45Ca2+ uptake induced by 55 mM K+ ions occurs selectively through voltage-operated Ca2+ channels, whereas, in choline-substituted media, starting from 70 mM choline, Ca2+ ions seemed to utilize the Na(+)-Ca2+ antiporter to penetrate into synaptosomes. In contrast, when extracellular K+ concentrations are raised above 55 mM, 45Ca2+ entrance may occur through two cumulative mechanisms, the opening of Ca2+ channels that are activated by high K(+)-induced depolarization and the activation of the Na(+)-Ca2+ antiporter, which follows the reduction of the transmembrane Na+ electrochemical gradient.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Na(+)-Ca2+ exchange activity in central nerve endings. II. Relationship between pharmacological blockade by amiloride analogues and dopamine release from tuberoinfundibular hypothalamic neurons.

The aim of the present study was to investigate the possible role played by the Na(+)-Ca2+ exchange system in the modulation of [3H]dopamine ([3H]DA) release from tuberoinfundibular hypothalamic (TIDA) neurons. 2',4'-Dimethylbenzamil (DMB) dose-dependently (10-100 microM) inhibited Na(+)-dependent 45Ca2+ efflux from brain synaptosomes. This compound (30-300 microM), as well as alpha-phenylbenzamil amiloride (30-100 microM), another inhibitor of the Na(+)-Ca2+ antiporter, was also able to stimulate basal release of [3H]DA from superfused TIDA neurons. This stimulation was completely prevented by the removal of extracellular Ca2+ ions, in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. In addition, DMB-induced [3H]DA release was unaffected by the dopamine transport inhibitor nomifensine (10 microM). On the other hand, 5-[N-methyl-N-guanidinocarbonylmethyl]amiloride (MGCMA) (100-300 microM), which lacks inhibitory properties on the Na(+)-Ca2+ exchanger but behaves as an inhibitor of the Na(+)-H+ antiporter, failed to modify basal [3H]DA release from TIDA neurons. When the Na(+)-Ca2+ antiporter operates as a Ca2+ influx pathway, as occurs upon the removal of extracellular Na+ ions, Na(+)-dependent 45Ca2+ uptake in brain synaptosomes was dose-dependently (10-300 microM) inhibited by DMB, whereas DMB itself was unable to prevent 55 mM K(+)-induced 45Ca2+ uptake, which mainly reflects the activation of voltage-operated Ca2+ channels. In keeping with these results, ouabain (500 microM)-induced [3H]DA release, which depends on the activation of the Na(+)-Ca2+ exchanger due to inhibition of the Na(+)-K(+)-ATPase pump, was prevented by superfusion of TIDA neurons with DMB (50 microM). By contrast, MGCMA (100 microM) failed to modify either Na(+)-dependent 45Ca2+ influx or ouabain-induced [3H]DA release. In conclusion, the results of the present study appear to suggest that the pharmacological inhibition of the Na(+)-Ca2+ antiporter by amiloride analogues may affect DA release from central neurons. Opposite effects are observed, depending on the direction of operation of the exchanger. In fact, when the Na(+)-Ca2+ exchanger operates as a Ca2+ efflux pathway, its pharmacological blockade can produce a stimulation of DA release. In contrast, when this antiporter operates as a Ca2+ influx pathway, as occurs as a consequence of the inhibition of the Na(+)-K(+)-ATPase pump by ouabain, its pharmacological blockade can prevent ouabain-induced DA release from TIDA neurons.

Amiloride↗