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Is all Ca2+-dependent release vesicular and is all Ca2+-independent release cytoplasmic?
9th Meeting oftheESN
D-[3Ii] ASPARTATE A IS L-GLUTAMATE SUBSTITUTE FOR RELEASE
VALID IN
STUDIES?
R. Griffith3 School of Biological and University of St. Andrews,
Medical Sciences, Scotland.
Although D-[3H]aspartate (D-[3H]asp) has been used extensively as a substitute for L(Glu) in uptake and release glutamate and for identifying CNS pathways, studies, its validity, particularly for use in release There is questioned. has been studies evidence to support at least two routes of calcium-dependent release; Glu (i) a (vesicular) component reflecting exocytotic and (ii) a calcium-independent release, component of probab'le cytosolic origin. A study of the depolarisation-induced, calciumdependent release of exogenously-supplied Dresults in anomalous shows r3Hlasp comparative investigations using synaptosomes and primary neuronal cultures as model in caution in vitro systems, thus suggesting data interpretation. In contrast, D-f3Hlasp can be considered a valid probe for studying calcium independent Glu release which appears to arise from the cytoplasm by operation of the plasma membrane transporter in the efflux direction following depolarisation.
SYNTHESIS OF AXONAL PROTEINS AND fWAs IN THE ISOlAlED SQUID GlANTAXON. A. Giuditta Department of General and Environmental
w3
RELEASE Ca*+-DEPENDENT Ca*+ALL AND IS INDEPENDENT RELEASE CYTOPLASMIC?
IS ALL VESICULAR David Dept.
G. Nicholls of Biochemistry,
Dundee,
Scotland.
The exocytosis of fast-acting amino acid neurotsansmitters is tightly coupled to the entry of Ca*+ through a specific class of presynaptic Ca*' channels, before Ca2+ has time to equilibrate with the cytoplasm. vesicular release occurs from L compartment which exchanges only slowly with the cytoplasm and is characterized by its sensitivity to clostridial and a high its energy-dependency neurotoxins, selectivity of glutamate over aspartate. Cytoplasmic, Ca2'-independent, release occurs in vitro in response to agents which chronically lower the transmembrane Nat-electrochemical gradient allowing thermodynamic reversal of the Na'-coupled acidic amino acid carrier. This release mode may be of importance during stroke. It has been reported that a-lactrotoxin induces a massive Ca'+-independent release of transmitters from a number of preparations. However in the case of toxin-evoked glutamate release from DNS terminals virtually all release is cytoplasmic. There is no that glutamate exocytosis can be convincing evidence evoked other than by Ca2+ entry through voltageactivated channels under physiologically relevant conditions.
CALPAIN LIVBLS DIIITRElOTXATION Gordon Growth
DDRING OF PC12
CELLS.
Guroff and Mari Oshima, Section on Factors, NICHD, NIH, Bethesda, MD
20892.
Physiology,
Univemlty of Naples. Naplea. Italy Auoplasmic pmteins are synthesized in the isolated squid giant axon by a cytoribosomal system. According to some authors, the polysomes responsible for thii synthesis are localized in the pwiaxonsl gllal cells. Our work, however, has shown that the axoplasm of the giant axon contains all the componentsofthe~ system of protein synthesis, induding soluble protein factors and enzymes, all species of tRNAs, sizable amounts of rRNA, and several hundred speck of mRN& Some of the axoplasmic mRN& have bsen identitied as actin, tubulln and other axoplasmic proteins. In additbn, incubatim of isolated axons with 7% methionine has slbwad the IdentJfkatbn of active axoplaomic polysomes, i.e. polysomes aaaooiated with nascent peptide chains. me isdated squid giant axon also sustains the synthesis of the maln axopbrmk RNAs, moat of which enter the axoplasm as ribonucleopmtein partkles. Similar data have been obtained using the perfused giant axon. In view of the extramitochonddal nature of these RNAs, and of the presence of glial nuclei in the belated axon, the data suggest a glial origin of the RNA3 involved in axonal protein synthesis.
Since its discovery in brain in 1964 calpain has been implicated in a number of neurobiological processes. Some of the most persuasive evidence involves its role in the turnover of cytoskeletal proteins. Since the morphological differentiation of neurons is accompanied by such profound changes in the cytoskeletal structure, it seemed reasonable to ask if there were changes in the activity of calpain during such differentiation. The PC12 model undergoes robust morphological differentiation in response to nerve growth factor. Treatment of the cells with nerve growth factor causes a 50% decrease in the activity of calpain over several days of differentiation. Other ligands, such as fibroblast growth factor and dibutyryl cyclic AMP, that caused a similar differentiation, also lowered calpain activity, but when morphological differentiatio? was prevented, for example, by growing the cells in suspension, nerve growth factor had no effect on calpain levels. The decrease in calpain activity was, however, not due to a decrease in calpain itself, but to an increase in the activity of the specific inhibitor of the enzyme, calpastatin. The mechanism of calpastatin activation is not clear, since neither calpastatin mRNA nor protein are increased, but the recent observation that nerve growth factor treatment causes an increase in calpastatin phosphorylation may provide a clue.
D-[3Ii] ASPARTATE A IS L-GLUTAMATE SUBSTITUTE FOR RELEASE
VALID IN
STUDIES?
R. Griffith3 School of Biological and University of St. Andrews,
Medical Sciences, Scotland.
Although D-[3H]aspartate (D-[3H]asp) has been used extensively as a substitute for L(Glu) in uptake and release glutamate and for identifying CNS pathways, studies, its validity, particularly for use in release There is questioned. has been studies evidence to support at least two routes of calcium-dependent release; Glu (i) a (vesicular) component reflecting exocytotic and (ii) a calcium-independent release, component of probab'le cytosolic origin. A study of the depolarisation-induced, calciumdependent release of exogenously-supplied Dresults in anomalous shows r3Hlasp comparative investigations using synaptosomes and primary neuronal cultures as model in caution in vitro systems, thus suggesting data interpretation. In contrast, D-f3Hlasp can be considered a valid probe for studying calcium independent Glu release which appears to arise from the cytoplasm by operation of the plasma membrane transporter in the efflux direction following depolarisation.
SYNTHESIS OF AXONAL PROTEINS AND fWAs IN THE ISOlAlED SQUID GlANTAXON. A. Giuditta Department of General and Environmental
w3
RELEASE Ca*+-DEPENDENT Ca*+ALL AND IS INDEPENDENT RELEASE CYTOPLASMIC?
IS ALL VESICULAR David Dept.
G. Nicholls of Biochemistry,
Dundee,
Scotland.
The exocytosis of fast-acting amino acid neurotsansmitters is tightly coupled to the entry of Ca*+ through a specific class of presynaptic Ca*' channels, before Ca2+ has time to equilibrate with the cytoplasm. vesicular release occurs from L compartment which exchanges only slowly with the cytoplasm and is characterized by its sensitivity to clostridial and a high its energy-dependency neurotoxins, selectivity of glutamate over aspartate. Cytoplasmic, Ca2'-independent, release occurs in vitro in response to agents which chronically lower the transmembrane Nat-electrochemical gradient allowing thermodynamic reversal of the Na'-coupled acidic amino acid carrier. This release mode may be of importance during stroke. It has been reported that a-lactrotoxin induces a massive Ca'+-independent release of transmitters from a number of preparations. However in the case of toxin-evoked glutamate release from DNS terminals virtually all release is cytoplasmic. There is no that glutamate exocytosis can be convincing evidence evoked other than by Ca2+ entry through voltageactivated channels under physiologically relevant conditions.
CALPAIN LIVBLS DIIITRElOTXATION Gordon Growth
DDRING OF PC12
CELLS.
Guroff and Mari Oshima, Section on Factors, NICHD, NIH, Bethesda, MD
20892.
Physiology,
Univemlty of Naples. Naplea. Italy Auoplasmic pmteins are synthesized in the isolated squid giant axon by a cytoribosomal system. According to some authors, the polysomes responsible for thii synthesis are localized in the pwiaxonsl gllal cells. Our work, however, has shown that the axoplasm of the giant axon contains all the componentsofthe~ system of protein synthesis, induding soluble protein factors and enzymes, all species of tRNAs, sizable amounts of rRNA, and several hundred speck of mRN& Some of the axoplasmic mRN& have bsen identitied as actin, tubulln and other axoplasmic proteins. In additbn, incubatim of isolated axons with 7% methionine has slbwad the IdentJfkatbn of active axoplaomic polysomes, i.e. polysomes aaaooiated with nascent peptide chains. me isdated squid giant axon also sustains the synthesis of the maln axopbrmk RNAs, moat of which enter the axoplasm as ribonucleopmtein partkles. Similar data have been obtained using the perfused giant axon. In view of the extramitochonddal nature of these RNAs, and of the presence of glial nuclei in the belated axon, the data suggest a glial origin of the RNA3 involved in axonal protein synthesis.
Since its discovery in brain in 1964 calpain has been implicated in a number of neurobiological processes. Some of the most persuasive evidence involves its role in the turnover of cytoskeletal proteins. Since the morphological differentiation of neurons is accompanied by such profound changes in the cytoskeletal structure, it seemed reasonable to ask if there were changes in the activity of calpain during such differentiation. The PC12 model undergoes robust morphological differentiation in response to nerve growth factor. Treatment of the cells with nerve growth factor causes a 50% decrease in the activity of calpain over several days of differentiation. Other ligands, such as fibroblast growth factor and dibutyryl cyclic AMP, that caused a similar differentiation, also lowered calpain activity, but when morphological differentiatio? was prevented, for example, by growing the cells in suspension, nerve growth factor had no effect on calpain levels. The decrease in calpain activity was, however, not due to a decrease in calpain itself, but to an increase in the activity of the specific inhibitor of the enzyme, calpastatin. The mechanism of calpastatin activation is not clear, since neither calpastatin mRNA nor protein are increased, but the recent observation that nerve growth factor treatment causes an increase in calpastatin phosphorylation may provide a clue.