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J R Lemos

Publications and source records attributed to J R Lemos.

50 records · Page 3Linked to original sources
PubMed1989

Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals.

The properties of the Ca2+ channels mediating transmitter release in vertebrate neurons have not yet been described with voltage-clamp techniques. Several types of voltage-dependent Ca2+ channels are known to exist on neuronal somata, but the small size and inaccessibility of most vertebrate nerve endings have precluded direct characterization of the presynaptic channels. However, large nerve endings, which release the peptides oxytocin and vasopressin in a Ca2(+)-dependent manner, can be dissociated from the rat neurohypophysis. Using both single-channel and whole-cell patch-clamp techniques, we have characterized two types of Ca2+ channels that coexist in these terminals. One is a large-conductance, high-threshold, dihydropyridine-sensitive channel that contributes a slowly inactivating current. The second is a smaller conductance channel, which is also activated at high thresholds, but underlies a rapidly inactivating, dihydropyridine-insensitive current. Both types of Ca2+ channels may participate in the peptide release process.

Action Potentials↗
PubMed1987

Isolated neurosecretory nerve endings as a tool for studying the mechanism of stimulus-secretion coupling.

In the present paper we discuss the properties of a recently developed preparation of isolated neurosecretory nerve endings obtained from the rate neurohypophysis. These nerve terminals release two neurohormones, oxytocin and vasopressin, which are easily assayed by radioimmunoassay. Depolarization-induced secretion is dependent on the same parameters as those regulating release from the whole neural lobe. The isolated nerve endings can be permeabilized by means of digitonin; a treatment which gives direct access to the cytoplasm allowing the study of the minimal requirements for inducing neuropeptide release. Furthermore, some nerve endings are large enough to allow the use of the patch-clamp technique. In the present paper we present evidences which show that the isolated neurohypophysial nerve terminals represent a protent tool for studying the mechanism of stimulus-secretion.

Animals↗
PubMed1986

Ionic channels and hormone release from peptidergic nerve terminals.

Although there is considerable evidence that depolarization of nerve cell terminals leads to the entry of Ca2+ and to the secretion of neurohormones and neurotransmitters, the details of how ionic currents control the release of neuroactive substances from nerve terminals are unknown. The small size of most nerve terminals has precluded direct analysis of membrane ionic currents and their influence on secretion. We now report that it is possible, using patch-clamp techniques, to study stimulus--secretion coupling in isolated peptidergic nerve terminals. Sinus gland terminals from Cardisoma are easily isolated following collagenase treatment and appear morphologically and electrically very similar to non-dissociated nerve endings. We have observed two types of single-channel currents not previously described. The first ('f') channel is activated by intracellular Na+ and the second ('s') by intracellular Ca2+. Both show little selectivity between Na+ and K+. In symmetrical K+, these cation channels have mean conductances of 69 and 213 pS, respectively. Furthermore, at least three types of Ca2+ channels can be reconstituted from nerve terminal membranes prepared from sinus glands. Nerve terminals can also be isolated from the rat neural lobe. These neurosecretosomes release oxytocin and vasopressin, in response to membrane depolarization, only in the presence of external Ca2+. The depolarization of the nerve endings is associated with an increase in intracellular free Ca2+ concentration and this increase, measured using a fluorescent indicator, is abolished by Ca2+ channel blockers. Channels similar in their properties to the f and s channels also exist in rat neural lobe endings. Since these channels have not been found in other neurones or neuronal structures they may be unique to peptidergic nerve terminals.

Animals↗
PubMed1985

Phosphoproteins associated with the regulation of a specific potassium channel in the identified Aplysia neuron R15.

The neurotransmitter serotonin (5HT) activates a specific K+ conductance in the identified Aplysia neuron R15. This response to 5HT has been shown previously to be mediated by cAMP and cAMP-dependent protein phosphorylation. We have measured protein phosphorylation within neuron R15 in vivo, following the intracellular injection of [gamma-32P]ATP, and have demonstrated that 5HT modulates the phosphorylation of a number of proteins in R15. The present study was undertaken to determine which of these phosphoproteins are closely associated with, and may be responsible for, the K+ conductance increase. Treatment of neuron R15 with a cAMP analog produces some but not all of the 5HT-induced phosphoprotein changes, indicating that some are not cAMP-dependent and thus can be dissociated from the cAMP-dependent K+ conductance increase. Similar results are obtained by intracellular injection of the adenylate cyclase inhibitor guanosine 5'-O-(2-thiodiphosphate), which completely blocks the 5HT-evoked K+ conductance increase but fails to block some of the 5HT-induced phosphorylation changes. Examination of the phosphoprotein pattern at short times after 5HT application has demonstrated that some of the phosphoprotein changes, but not others, are closely associated in time with the appearance of the physiological response. These and other pharmacological and kinetic experiments have allowed the identification of two phosphoproteins, of Mr = 29,000 and 70,000, which cannot be dissociated from the 5HT-induced K+ conductance increase whatever the experimental manipulation. Thus, one or both of these phosphoproteins may be involved in the regulation of the 5HT-sensitive K+ channel in neuron R15.

Animals↗
PubMed1985

Calcium- and cyclic nucleotide-dependent protein kinases and their substrates in the Aplysia nervous system.

Homogenates of the Aplysia nervous system contain protein kinase activities sensitive to cAMP, cGMP, and Ca2+/calmodulin. The cAMP- and cGMP-dependent activities are either soluble enzymes or are only loosely bound to membranes, since they can be detected only in crude but not in washed membrane fractions, and are present in 20,000 or 100,000 X g supernatants prepared from homogenates. In contrast there are both soluble and tightly membrane-bound Ca2+/calmodulin-dependent protein kinase activities. The three activities present in supernatant fractions can be separated by chromatography on DE-cellulose, indicating that they are due to distinct enzyme species. Substrates for these enzymes were analyzed by two-dimensional gel electrophoresis. Protein phosphorylation within the identified Aplysia neuron R15 in vivo was measured by the intracellular injection of [gamma-32P]ATP. cAMP stimulates the phosphorylation of nine proteins and decreases phosphorylation of two proteins in this cell. This in vivo pattern was compared with in vitro phosphorylation measured in homogenates of whole ganglion. Most of the phosphoproteins affected by cAMP in neuron R15 in vivo are also substrates for cAMP-dependent protein kinase in vitro. Thus, the in vitro system will be a useful tool for detailed biochemical analysis of phosphoproteins which have been identified as being physiologically relevant in vivo.

Animals↗
PubMed1984

Topographical distributions of 32K and 48K cAMP-regulated phosphoproteins: relationships to dopamine and serotonin innervations in striatum, substantia nigra and cerebral cortex.

The topographical distribution of a cAMP-regulated phosphoprotein of an apparent molecular weight (Mr) of 32,000 (32K) was examined in homogenates prepared from microdiscs punched out from serial frozen slices of the striatum. The amount of this phosphoprotein progressively diminished from the rostral to the caudal part of the striatum as did both the dopamine-innervation and the dopamine (D1)-sensitive adenylate cyclase. After kainic acid lesion of the rostral part of the striatum, the 32K phosphoprotein disappeared in this area and we observed a 48% decrease in the amount of 32K phosphoprotein found in the substantia nigra. 6-Hydroxydopamine lesions of the nigro-striatal dopaminergic pathway did not affect the 32K phosphoprotein either in striatum or substantia nigra. These results suggest that in the nigro-striatal pathway, the 32K phosphoprotein is closely associated with dopaminoceptive neurons containing D1 receptors. In the cerebral cortex the association of 32K phosphoprotein with dopaminoceptive neurons is more questionable since we did not find a higher density of this phosphoprotein in areas containing a high amount of D1 receptor (frontal cerebral cortex) than in areas containing a low amount of D1 receptor (parietal cerebral cortex). In the course of this study we found another cAMP-regulated phosphoprotein of an Mr of 48,000 (48K). The amount of this phosphoprotein increased progressively from the rostral to the caudal part of the striatum, a pattern of distribution close to that of serotonin terminals. This protein was also present in the substantia nigra. Kainic acid lesioning of the rostral part of the striatum did not affect the amount of the 48K phosphoprotein within the substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Synaptic stimulation alters protein phosphorylation in vivo in a single Aplysia neuron.

Protein phosphorylation was examined in the identified Aplysia neuron R15, in vivo, after the intracellular injection of [gamma-(32)P]ATP. Two-dimensional gel electrophoretic analysis indicates that at least 70 proteins are phosphorylated within R15 during a 50-min labeling period. Application of serotonin (5HT) results in an increase in K(+) conductance in R15 and a concomitant change in the phosphorylation pattern: there are increases or decreases in the phosphorylation of some proteins, and at least five phosphoproteins appear that are not observed in control cells. Dopamine causes a decrease in voltage-dependent inward conductance in R15 and also alters the phosphorylation pattern: several of the phosphorylation changes are similar to those produced by 5HT, while others are unique to dopamine. Stimulation of the branchial nerve leading to the abdominal ganglion results in a long-lasting synaptic hyperpolarization of R15. The conductance changes underlying this response include an increase in K(+) conductance (identical to that produced by 5HT) together with a decrease in voltage-dependent inward conductance (identical to that produced by dopamine). The phosphorylation changes induced in R15 by branchial nerve stimulation resemble a combination of the changes induced by 5HT and dopamine. The results demonstrate that synaptic stimulation can modulate the phosphorylation of specific proteins in a single identified postsynaptic neuron and are consistent with the hypothesis that protein phosphorylation can regulate the regulate the activity of neuronal ion channels.

Journal Article↗
PubMed1984

Intracellular injection of guanyl nucleotides alters the serotonin-induced increase in potassium conductance in Aplysia neuron R15.

The effects of the adenylate cyclase inhibitor GDP beta S on the response of Aplysia neuron R15 to serotonin (5HT) were investigated. Previous studies have demonstrated that 5HT causes an increase in K+ conductance in R15 and that the response is mediated by cAMP. At concentrations in the micromolar range, GDP beta S inhibits the stimulation of adenylate cyclase by 5HT in particulate fractions from Aplysia ganglia. When micromolar concentrations of GDP beta S are injected into neuron R15, there is no effect on the resting membrane conductance, but the increase in K+ conductance normally elicited by 5HT is completely inhibited. Furthermore, the decrease in inward current normally elicited by dopamine (DA), which does not appear to involve cAMP, is not affected by micromolar concentrations of GDP beta S. In addition, application of 8-benzylthio cAMP to R15 can evoke an increase in K+ conductance even after the injection of GDP beta S, which indicates that events subsequent to the activation of adenylate cyclase are not inhibited by the GDP analogue. In contrast, when millimolar concentrations of GDP beta S are injected into R15, direct effects on membrane conductance are observed and the response of R15 to 5HT is enhanced. Although these effects of high concentrations of GDP beta S are only poorly understood, the results with micromolar concentrations are consistent with the hypothesis that stimulation of adenylate cyclase is necessary for the 5HT-induced increase in K+ conductance in neuron R15.

Adenylyl Cyclases↗
PubMedDate not supplied

Single channels and ionic currents in peptidergic nerve terminals.

Control of secretion, by a mechanism in which membrane depolarization leads to Ca2+ entry, has been extensively studied. The small size and inaccessibility of most nerve terminals, however, have precluded direct analysis of membrane ionic currents and their influence on secretion (with some notable exceptions). Recently, patch-clamp methods have been applied to several secretory systems for both voltage-clamp and single-channel recordings. We now report the extension of this analysis to isolated peptidergic nerve terminals. We used terminals obtained from a crustacean neurohaemal organ, the sinus gland. Analyses of currents under whole-terminal voltage clamp showed inward currents carried by Na+ and by Ca2+, and outward currents carried predominantly by K+. Furthermore, we have observed two types of single-channel currents that may be unique to nerve terminals. Both show little selectivity between Na+ and K+. The first channel is activated by intracellular Na+ and the second by intracellular Ca2+. These channels have conductances of 69 and 213 pS, respectively, in symmetrical 310 mM KCl. It should now be possible to compare electrical activity recorded intracellularly from intact nerve endings, with whole-cell and single-channel currents and with the release of peptide neurohormones from isolated neuronal terminals.

Animals↗