Myelin sheath survival after guanethidine-induced axonal degeneration.

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.

The light-harvesting chlorophyll a/b-protein complex from barley thylakoid membranes. Polypeptide composition and characterization of an oligomer.

Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll a/b-protein complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide. The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll a/b-protein is closer to the in vivo form rather than the monomer.

Mn2+ can substitute for Ca2+ in causing catecholamine secretion but not for increasing tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells.

The ability of the divalent cation manganese (Mn2+) to substitute for calcium (Ca2+) both in triggering catecholamine release and in stimulating catecholamine synthesis, as indicated by an increase in tyrosine hydroxylase (TOH) phosphorylation, has been determined in bovine adrenal medullary chromaffin cells maintained in tissue culture. Mn2+ was found to enter chromaffin cells through pathways activated by nicotinic receptor stimulation and potassium depolarisation, and via the Na1:Ca0 exchange mechanism in Na(+)-loaded cells. Like Ca2+, entry of Mn2+ through these pathways triggered immediate catecholamine release and, like Ca2+, maintained quantitatively comparable release at least up to 40 min. Unlike Ca2+, Mn2+ did not stimulate an increase in TOH phosphorylation in intact chromaffin cells, even over a prolonged time course, but Mn2+ did stimulate increased TOH phosphorylation in lysed cell preparations showing that its lack of effect in the intact cells was not due to inhibition of the specific phosphorylation pathway. In lysed cell preparations, Mn2+ stimulated also phosphorylation of a different spectrum of proteins to Ca2+, and of the same proteins to different extents. In particular, P80 (MARCKS protein) was more intensely phosphorylated in the presence of Mn2+ than in the presence of Ca2+. Since TOH phosphorylation always occurs when intracellular Ca2+ is increased, the absence of an increase with Mn2+ indicates that none of its intracellular effects could have occurred as a consequence of Mn2+ mobilisation of intracellular Ca2+. In summary, the data show that Mn2+ is a surrogate for Ca2+ in triggering and maintaining catecholamine release, but does not substitute for Ca2+ in stimulating TOH phosphorylation.

Autophosphorylation of neuronal calcium/calmodulin-stimulated protein kinase II.

A unique feature of neuronal calcium/calmodulin-stimulated protein kinase II (CaM-PK II) is its autophosphorylation. A number of sites are involved and, depending on the in vitro conditions used, three serine and six threonine residues have been tentatively identified as autophosphorylation sites in the alpha subunit. These sites fall into three categories. Primary sites are phosphorylated in the presence of calcium and calmodulin, but under limiting conditions of temperature, ATP, Mg2+, or time. Secondary sites are phosphorylated in the presence of calcium and calmodulin under nonlimiting conditions. Autonomous sites are phosphorylated in the absence of calcium and calmodulin after initial phosphorylation of Thr-286. Mechanisms that lead to a decrease in CaM-PK II autophosphorylation include the thermolability of the enzyme and the activity of protein phosphatases. A range of in vitro inhibitors of CaM-PK II autophosphorylation have recently been identified. Autophosphorylation of CaM-PK II leads to a number of consequences in vitro, including generation of autonomous activity and subcellular redistribution, as well as alterations in conformation, activity, calmodulin binding, substrate specificity, and susceptibility to proteolysis. It is established that CaM-PK II is autophos-phorylated in neuronal cells under basal conditions. Depolarization and/or activation of receptors that lead to an increase in intracellular calcium induces a marked rise in the autophosphorylation of CaM-PK II in situ. The incorporation of phosphate is mainly found on Thr-286, but other sites are also phosphorylated at a slower rate. One consequence of the increase in CaM-PK II autophosphorylation in situ is an increase in the level of autonomous kinase activity. It is proposed that the formation of an autonomous enzyme is only one of the consequences of CaM-PK II autophosphorylation in situ and that some of the other consequences observed in vitro will also be seen. CaM-PK II is involved in the control of neuronal plasticity, including neurotransmitter release and long-term modulation of postreceptor events. In order to understand the function of CaM-PK II, it will be essential to ascertain more fully the mechanisms of its autophosphorylation in situ, including especially the sites involved, the consequences of this autophosphorylation for the kinase activity, and the relationships between the state of CaM-PK II autophosphorylation and the physiological events within neurons.

Oxidative stress and the fetotoxicity of alcohol consumption during pregnancy.

Pregnant Quackenbush Special mice were exposed to ethanol under semiacute (3.0 g/kg body weight intragastrically, days 7 to 12 of pregnancy), and chronic conditions (15% ethanol in drinking water for 5 weeks before and during pregnancy) to assess whether embryo-fetotoxic actions of the drug involve oxidative stress effects. Effects were monitored both in the maternal system and embryo. Alcohol compromised the maternal system by increasing the generation of lipid peroxides in the liver. It also decreased glutathione and vitamin E levels, and glutathione peroxidase and superoxide dismutase activities in this organ. Glutathione peroxidase activity in the maternal blood decreased. Only minor alcohol-induced changes occurred in the uterine endometrium, including decreased xanthine oxidase and increased gamma-glutamyl transpeptidase. Similarly, only few changes were induced in day-12 embryos by alcohol. In this case, glutathione content and xanthine oxidase activity decreased while glutathione reductase activity increased following exposure to the chronic regime. With the possible exception of the maternal liver where evidence of oxidative damage was detected, these results do not reflect substantial changes in the antioxidant defences of either the pregnant mouse or embryo. However, the changes may contribute to the growth retarding and other fetotoxic effects of alcohol when they are totalled into the multifactorial actions of the drug.

Protein phosphorylation in guinea-pig myenteric ganglia and brain: presence of calmodulin kinase II. protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins.

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The alpha subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the beta subunit by autophosphorylation. The ratio of beta to alpha subunit was considerably higher in ganglia than in brain and ganglia beta subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinase and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Opioid inhibition of nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells.

The ability of a number of opioid agonists and antagonists to affect nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells has been investigated. High (10 microM) concentrations of the opioid agonist bremazocine produced a significant inhibition of nicotine-induced 45Ca2(+)-uptake throughout the 15 min time course examined. The opioid subtype-selectivity of this inhibition was investigated; mu and delta selective agonists produced only minor effects whereas the kappa selective agonist U50-488H and the endogenous opioid peptides dynorphin(1-13) and metorphamide almost abolished nicotine-induced 45Ca2(+)-uptake. The U50-488H inhibition was significant at 10 nM concentrations with an IC50 of approximately 1 microM. U50-488H inhibition could not be reversed or reduced by the opioid antagonists naxolone, diprenophine or Mr2266. Furthermore, Mr2266 and its optical isomer Mr2267 also produced marked inhibition of 45Ca2(+)-uptake. The inhibition was specific to nicotine-induced 45Ca2(+)-uptake in that a similar level of uptake evoked by potassium depolarization was unaffected by high concentrations of U50-488H. These data indicate that opioid inhibition of nicotine-induced 45Ca2(+)-uptake does not involve classical, stereospecific opioid receptors and suggests the involvement of a pharmacologically distinct opioid recognition site. It is speculated that this may be associated with the nicotine receptor-ionophore complex.

Altered protein phosphorylation in intact rat cortical synaptosomes after in vivo administration of fluphenazine.

Phenothiazines such as fluphenazine are able to inhibit calcium-stimulated protein kinases in vitro in both lysed and intact synaptosomes. In this study protein phosphorylation was assayed in intact synaptosomes isolated from the cerebral cortex of rats treated chronically (21 days, 10 mg/kg, i.p.) or acutely (1 hr, 10 mg/kg, i.p.) with fluphenazine. When intact synaptosomes from chronically treated animals were prelabeled with 32Pi, there were two effects on protein phosphorylation: an increase in the basal labeling of many phosphoproteins and a decrease in depolarization-evoked protein phosphorylation. Acute injections had even more pronounced effects, but the direction and nature of the effects were the same. No effects on K+-stimulated calcium entry or on protein phosphatase activity were detected. When lysed synaptosomes from chronically treated animals were labeled in the presence of [gamma-32P]ATP, a small decrease in calmodulin-dependent and cAMP-dependent protein phosphorylation was observed. The results suggest that two different in vivo mechanisms may underlie these effects, and these are discussed. We proposed that intact synaptosomes may be a good model in which to study the in vivo mechanisms of the action of fluphenazine since they appear to retain at least some effects of the drug after subcellular fractionation.

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells. Clonidine stimulates basal but inhibits nicotinic receptor evoked phosphorylation.

Clonidine inhibited the uptake of calcium and the overall phosphorylation of tyrosine hydroxylase induced by nicotinic receptor activation in bovine adrenal medullary chromaffin cells in culture. However, clonidine did not inhibit the increase in these parameters that accompanied K+ depolarisation of the cells. There was also no effect of clonidine on the overall phosphorylation of tyrosine hydroxylase when cells were stimulated by muscarine. Nicotinic receptor activation increased the phosphorylation of Ser-19, Ser-31, and Ser-40 on tyrosine hydroxylase, and this was inhibited by clonidine in a concentration-dependent manner. On the other hand, clonidine had no effect on calcium uptake, yet increased the phosphorylation of Ser-19 under basal conditions. Using calcium and calmodulin-stimulated protein kinase II obtained from rat brain clonidine increased the autophosphorylation of the alpha-subunit of the kinase by 37%, and also its activity against an exogenous peptide substrate by 29%. These data are consistent with the hypothesis that clonidine inhibits nicotinic receptor-induced tyrosine hydroxylase phosphorylation by decreasing calcium influx into chromaffin cells, perhaps by an action at the nicotinic receptor. Clonidine also increases the basal phosphorylation of tyrosine hydroxylase at Ser-19, perhaps by directly activating calcium and calmodulin-stimulated protein kinase II.

Autophosphorylation of calmodulin-stimulated protein kinase II in intact synaptosomes.

The major phosphoproteins observed after lysis of synaptosomes and incubation in the presence of [gamma-32P]ATP and calmodulin are the autophosphorylated 50-kDa and 60-kDa subunits of calmodulin-stimulated protein kinase II (CMK II). However, when intact synaptosomes are preincubated with 32Pi, these subunits are hardly labeled even after depolarization. The aim of this study was to determine the extent to which methodological factors contribute to this discrepancy. The distribution of CMK II between the outside and the inside of synaptosomes was determined by incubating intact and lysed synaptosomes with [gamma-32P]ATP. Some 38% of the 50-kDa subunit was found on the inside of synaptosomes, and at this location it would be accessible to ATP generated within synaptosomes during the preincubation with 32Pi and could be autophosphorylated. The rest (62%) was on the outside of the synaptosomes, presumably associated with postsynaptic densities, where it could not be autophosphorylated. The effect of preincubation at 37 degrees C on CMK II autophosphorylation was determined by incubating intact synaptosomes for 45 min. This reduced calmodulin-stimulated autophosphorylation of the 50-kDa subunit in lysed synaptosomes by 38% and in intact synaptosomes by 29%. Thus, 9% of the 50-kDa autophosphorylation activity within synaptosomes was lost by thermal inactivation during preincubation. The extent of this loss of activity depended on the synaptosomal protein concentration during preincubation. CMK II activity against its major endogenous substrate synapsin I and an exogenous peptide substrate was also decreased by preincubation. The effect of the ionic environment on CMK II autophosphorylation was determined by incubating lysed synaptosomes with [gamma-32P]ATP in the absence or presence of ions at concentrations that mimic the extra or intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of calmodulin-stimulated protein kinase II from two-day and adult chicken forebrain.

Soluble calmodulin-stimulated protein kinase II has been purified from 2-day and adult chicken forebrain. At both ages the holoenzyme eluted from a Superose-6B column with an apparent molecular weight of approximately 700,000 daltons and contained three subunits. The subunits were found to be the counterparts of the alpha, beta, and beta' subunits of the enzyme purified from adult rat brain in that they had one-dimensional phosphopeptide maps that were indistinguishable from those of the corresponding subunit in the rat enzyme and they migrated in SDS-polyacrylamide gels with the same apparent molecular weights. However, the doublet formed by the beta subunit was much more clearly resolved in the chicken enzyme and the beta' subunit, which was much more abundant in the adult chicken than in the adult rat, was also found to be a doublet. The ratio of the concentrations of the alpha and beta subunits changed during development. By autoradiography following autophosphorylation, the alpha:beta ratios of the 2-day and adult enzymes were 0.89 +/- 0.07 and 1.92 +/- 0.26, respectively; by silver staining the alpha:beta ratios were 0.95 +/- 0.11 and 1.85 +/- 0.17, respectively. The concentration of the beta' subunit was equal to that of the beta subunit at both ages. Autophosphorylation produced a decrease in the electrophoretic mobility of the alpha and beta subunits in SDS-polyacrylamide gels and a marked decrease in the calcium dependence of the substrate phosphorylation activity of the enzyme at both ages. The purified enzyme from chicken brain appeared to be more stable under standard in vitro assay conditions than the rat enzyme, and this was particularly so for the enzyme from 2-day forebrain.

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells.

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions.

The metabolic and functional viability of synaptosomes was examined in 5 subcellular fractions obtained after centrifugation of an S1 fraction from rat cerebral cortex on a discontinuous Percoll gradient (Brain Research, this volume, 1987). Fraction 4 was the most enriched for viable synaptosomes since, although it accounted for only 11.8% of the total protein recovered from the gradient, this fraction contained 23.7% of the basal synapsin I phosphorylation activity, the greatest degree of depolarisation-stimulated increase in synapsin I phosphorylation, 36.1% of the total [3H]noradrenaline uptake capacity and 46.9% of the total [3H]noradrenaline release capacity. Noradrenaline release from fraction 4 was consistent with a neuronal mechanism as it was increased with increasing K+ concentrations and was dependent on calcium. Fractions 1 and 2 contained few viable synaptosomes as judged by their capacity for noradrenaline uptake and release, yet these fractions accounted for some 62.6% of the endogenous content of noradrenaline. In part their lack of viability was due to a low content of intrasynaptosomal mitochondria, while their high content of endogenous noradrenaline was due to the presence of synaptic vesicles released from damaged nerve terminals. The synaptosomes in fraction 3 were metabolically and functionally viable, but their capacity for uptake and release of noradrenaline was lower than for fraction 4. The synaptosomes in fraction 5 showed only a small depolarisation-stimulated release of noradrenaline, suggesting a lack of viability. Part of the capacity for uptake of [3H]noradrenaline into fraction 5 was attributed to the presence of extrasynaptosomal mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid method for isolation of synaptosomes on Percoll gradients.

A new rapid method for fractionation of crude synaptosomes (postmitochondrial pellet, P2) on a discontinuous 4-step Percoll gradient is described. The homogeneity and integrity of the 5 major subcellular fractions were determined by analysis of the distribution of protein, lactate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, synapsin I (a synaptic vesicle marker) and the myelin basic proteins. The biochemical results were substantiated by quantitative electron microscopy. Fractions 3, 4 and 5 were enriched in synaptosomes and contained 19.7, 40.6 and 19.5% of the intact, identifiable synaptosomes in P2, respectively. Fraction 1 was enriched in membranous material, fraction 2 in myelin and fraction 5 in extrasynaptosomal mitochondria. The synaptosomes in fractions 3, 4 and 5 differed in their size, and their content of mitochondria, synapsin I and neurotransmitters. These results suggest that partial separation of different pools of synaptosomes has been achieved. The synaptosomes in fractions 3, 4 and 5 are viable, as they take up calcium, phosphate and noradrenaline; they are metabolically normal as judged by their ability to perform protein phosphorylation and they respond normally to depolarization by increasing calcium uptake, protein phosphorylation and neurotransmitter release. The synaptosomes in fraction 4 are relatively homogeneous and appear to be free of contamination from lysed synaptosomes and synaptic plasma membranes. This constitutes a major advantage of the Percoll method over traditional procedures which involve centrifugation to equilibrium. We have therefore confirmed (J. Neurochem., 43 (1984) 1114-1123) the advantages of Percoll use over traditional procedures, while further reducing the time taken, and extended our analysis to show that the present procedure provides a fractionation of synaptosomes into different pools of viable synaptosomes.

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions.

A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.

Ca(2+) influx stimulated phospholipase C activity in bovine adrenal chromaffin cells: responses to K(+) depolarization and histamine.

The role of Ca(2+) influx in activating phospholipase C in bovine adrenal chromaffin cells has been investigated. Phospholipase C activity in response to K(+) depolarization (56 mM) was blocked by the L-type Ca(2+) channel antagonist nifedipine and partially inhibited by the omega-conotoxins GVIA and MVIIC. In contrast, phospholipase C activity in response to histamine receptor activation was unaffected by omega-conotoxin GVIA and partially inhibited by omega-conotoxin MVIIC or nifedipine. This response was however markedly inhibited by the non-selective Ca(2+) channel antagonists La(3+) or 1-[beta-[3-(4-Methoxyphenyl)propoxy]-4-methoyphenethyl]-H-imidazol e (SKF-96365). Despite this Ca(2+) dependence phospholipase C activity was not increased during periods of "capacitative" Ca(2+) inflow generated by histamine-, caffeine- or thapsigargin-mediated depletion of internal Ca(2+) stores. Thus, while Ca(2+) influx in response to K(+) depolarization or G-protein receptor activation can increase phospholipase C activity in these cells, in the latter case it appears to be ineffective unless there is concurrent agonist occupation of the receptor.

Depolarization-dependent protein phosphorylation in rat cortical synaptosomes: the effects of calcium, strontium and barium.

Depolarization of synaptosomes increases the phosphorylation of a number of proteins in a calcium-dependent manner. The concentration of calcium required for optimum stimulation was 0.1 mM, with higher concentrations up to 2.5 mM being progressively less effective. Calcium was significantly better than strontium at increasing depolarization-dependent protein phosphorylation, while barium had no stimulating effect at concentrations above 0.1 mM. The order of potency of these ions is consistent with a calmodulin-stimulated protein kinase being activated on entry of calcium into synaptosomes, but is not consistent with the known efficacy of these ions in stimulating neurotransmitter release. The data show for the first time that phosphorylation of proteins may not be a prerequisite for neurotransmitter release.

Intrasynaptosomal free calcium levels in rat forebrain synaptosomes: modulation by sigma (sigma) receptor ligands.

The sigma receptor ligands (+) and (-)pentazocine and BD1008 (1-100 microM) were added to rat forebrain synaptosomes. Their effects on intrasynaptosomal free calcium ([Ca2+(+)]i) levels under basal conditions and after depolarisation with high potassium buffer (45 mM KCl), veratridine (25 microM) and 4-aminopyridine (4-AP, 1 mM) were determined. The sigma ligands elicited significant, concentration-dependent decreases in basal [Ca2+]i levels with an order of potency (-)pentazocine > (+)pentazocine = BD1008. The sigma ligands (at the maximum effective concentrations) also significantly inhibited the rise in [Ca2+]i levels produced by depolarisation with KCl, veratridine and 4-AP. The effect of (+) and (-)pentazocine (100 microM) to inhibit the depolarisation-dependent increase in [Ca2+]i levels was greater when veratridine and 4-aminopyridine were used to depolarise the synaptosomes than with KCl, whereas the effect of BD1008 (100 microM) was approximately equipotent using all three depolarising agents. However, BD1008 was more potent to inhibit the KCl-induced rise in [Ca2+]i compared to (+) and (-)pentazocine. The data demonstrate for the first time that sigma ligands decrease [Ca2+]i levels in rat forebrain synaptosomes and this suggests a possible mechanism for the changes to neuronal protein phosphorylation and neurotransmitter release previously observed with sigma ligands.

The major calmodulin-stimulated phosphoprotein of synaptic junctions and the major post-synaptic density protein are distinct.

The major post-synaptic density protein (mPSDp) present in isolated synaptic junction fractions is distinct from the major phosphoprotein (50Kpp) that is labelled by an endogenous calmodulin-stimulated protein kinase. mPSDp and the 50Kpp have different apparent molecular weights on sodium dodecyl sulphate polyacrylamide gels and the presence of 50Kpp in brain soluble fractions indicates that the two proteins have different subcellular distributions.