Persistent activation of Galphai/o-coupled receptors results in a paradoxical enhancement of subsequent drug-stimulated adenylate cyclase activity. The exact mechanism of this up-regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP-dependent protein kinase in D2L receptor-mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD-D2L). Long-term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two-fold enhancement of forskolin-stimulated cyclic AMP accumulation. Similarly, long-term treatment with the PKA inhibitors, H89 or Rp-8Br-cAMP, also enhanced adenylate cyclase activity. In contrast, long-term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin-stimulated cyclic AMP accumulation and reduced both quinpirole- and H89-induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT-PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD-D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells.

CuCl2, ZnCl2 and NiCl2, but not CdCl2 or CoCl2, induced transmitter release from superfused rat hippocampal and striatal synaptosomes preloaded with, respectively, [3H]noradrenaline and [3H]dopamine. Cu2+ was the most potent and effective, acting in a concentration- (0.1-300 microM) and time-dependent (peak effect occurring at 2-3 min) manner. The amount of Cu2+-induced release over a 5 min period is similar to that induced by depolarization with high KCl or the K+ channel blocker 4-aminopyridine. However, the time course of the Cu2+-induced release is slower and the effect of Cu2+ is not reversed by washout. Cu2+-induced catecholamine release requires extracellular calcium (Ca2+) and is inhibited by the Ca2+ channel blocker Cd2+, and in the case of noradrenaline, by the voltage-gated Na+ channel blocker tetrodotoxin. The ability of Cu2+ to induce massive Ca2+-dependent transmitter release from brain catecholaminergic nerve terminals may contribute to the neuropathological processes associated with Cu2+ toxicity in Wilson's disease.

A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.

Abstract: The subunit composition of the N‐methyl‐d‐aspartate (NMDA) glutamate receptor affects both its channel activity and its sensitivity to modulation by a wide variety of substances. Expression studies in oocytes and physiological studies in neurons indicate that endogenous postsynaptic NMDA receptors are heterooligomeric complexes of NR1 and NR2 subunits. To deduce the subunit composition of the presynaptic NMDA receptor on noradrenergic nerve terminals, we examined the modulation of NMDA‐evoked norepinephrine (NE) release from hippocampal synaptosomes. At high glycine concentrations, the NMDA‐evoked release was not potentiated by reducing reagents, low micromolar Zn2+ or Ni2+, polyamines, or 100 µM histamine. It was also not inhibited by oxidizing agents or physiological concentrations of protons but was inhibited by high micromolar Co2+, Zn2+, and Ni2+, but not Fe3+, by high micromolar ifenprodil, and by 1 mM histamine. At low glycine concentrations, it was potentiated by spermine. These characteristics are similar to those displayed by homooligomeric complexes of NR1 subunits that contain in the N‐terminal domain the 21‐amino‐acid insert encoded by exon 5. These data provide physiological evidence that some endogenous NMDA receptor complexes may contain only the NR1 (+ exon 5) subunits.

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca2+-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of M(r) 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing there-after. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.

Generation of antibodies and direct protein sequencing were used to identify and characterize proteins associated with highly purified synaptic vesicles from rat brain. A protein doublet of low abundance of 119 and 124 kDa apparent molecular mass [synaptic vesicle-associated phosphoprotein with a molecular mass of 120 kDa (SVAPP-120)] was identified using polyclonal antibodies. SVAPP-120 was found to copurify with synaptic vesicles and to be enriched in the purified synaptic vesicle fraction to the same extent as synapsin I. Like synapsin I, SVAPP-120 is not an integral membrane protein because it was released from synaptic vesicles by high salt concentrations. This protein was demonstrated to be brain specific, and its distribution in various brain regions paralleled the distribution of synapsin I and synaptophysin. During the postnatal development of the rat cortex and cerebellum, its expression correlated with synaptogenesis. SVAPP-120 was demonstrated to be a phosphoprotein both in vivo and in vitro. It was shown to be phosphorylated on serine and to a lesser extent on threonine residues. These results provide evidence that SVAPP-120 represents a novel synaptic vesicle-associated phosphoprotein. In addition, aldolase, a glycolytic enzyme, and alpha-c-adaptin, a clathrin assembly-promoting protein, were identified on purified synaptic vesicles by direct protein sequencing.

The localization of MARCKS (myristoylated, alanine-rich C-kinase substrate), a major specific substrate for protein kinase C, has been studied in the rat brain. Light microscopic immunocytochemistry and biochemical analysis demonstrated that the protein is widespread throughout the brain and enriched in certain regions, including the piriform and entorhinal cortices, portions of the amygdaloid complex, the intralaminar thalamic nuclei, the hypothalamus, the nucleus of the solitary tract, nucleus ambiguus, and many catecholaminergic and serotonergic nuclei. Electron microscopic analysis revealed immunoreactivity in axons, axon terminals, small dendritic branches, and occasionally in dendritic spines. In neuronal processes, immunoreactivity was particularly prominent in association with microtubules, but reaction product was also seen in cytosol and adjacent to plasma membranes. No reaction product was observed in large dendrites, somata, or nuclei. A population of strongly immunoreactive glial cells was also observed. Many of these glial cells were morphologically similar to microglial cells, although some resembled astrocytes. In glial cells, both cytoplasm and plasma membranes were heavily labeled. The distribution of the MARCKS protein did not coincide precisely with the distribution of any of the subspecies of protein kinase C. The results indicate that the MARCKS protein is expressed in the majority of cell types in the CNS, and they suggest that the protein may be involved both in glial cell functions and in neuronal functions involving cytoskeletal elements in small dendritic branches and axon terminals.

Abstract: When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol‐depenpent protein kinase (protein kinase C) and an 87‐kiIodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chen|iical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87‐kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal gaijglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87‐kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87‐kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.

A protein of 87 kilodaltons (87 kDa) was previously identified as a major specific substrate for protein kinase C in neuronal and other tissues. We have now studied the effect of protein kinase C-catalyzed phosphorylation of this protein on its association with membranes in isolated nerve terminals (synaptosomes) from rat cerebral cortex. Incubation of synaptosomal membranes under conditions associated with activation of protein kinase C led to the release of the phosphorylated 87-kDa protein into the incubation medium. In intact synaptosomes, activation of protein kinase C by phorbol esters or by depolarization-induced Ca2+ influx caused an increased phosphorylation of the 87-kDa protein and its translocation from membrane to cytosol. This translocation showed time courses, calcium dependency, and reversibility similar to those observed for the protein kinase C-induced phosphorylation of the protein. These results suggest that protein kinase C-catalyzed phosphorylation of the 87-kDa protein is responsible for its subcellular translocation into the cytosol of nerve terminals.

Depolarization of isolated nerve terminals (synaptosomes) has been shown to stimulate neurotransmitter release and to increase the phosphorylation state of a number of proteins, including synapsin I, in a Ca2+-dependent manner. Synapsin I, a prominent nerve terminal phosphoprotein, interacts with the cytoplasmic surface of small synaptic vesicles and with cytoskeletal elements in a phosphorylation-dependent manner. In the present study we have found that depolarization of synaptosomes resulted in a rapid (2-5 sec) translocation of synapsin I from the particulate to the cytosolic (soluble) fraction. This translocation of synapsin I correlated with its phosphorylation state and was dependent on the presence of Ca2+ in the incubation medium. The stoichiometry of phosphorylation of soluble synapsin I was considerably higher than that of synapsin I in the particulate fraction, under both basal and depolarizing conditions. These data are consistent with the hypothesis that, in situ, the phosphorylation of synapsin I promotes its translocation from synaptic vesicles/cytoskeleton to the cytosol. This phosphorylation/translocation may be instrumental in regulating the release of neurotransmitter.

A variety of evidence indicates that activation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in nerve terminals leads to enhanced neurotransmitter release. Arachidonic acid and its 12-lipoxygenase metabolite, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), have been suggested to act as second messengers mediating presynaptic inhibition of neurotransmitter release. In the present study it was found that CaM-kinase II, purified from rat brain cortex, was inhibited both by arachidonic acid (IC50 = 24 microM) and by 12-HPETE (IC50 = 0.7 microM). Neither substance inhibited CaM-kinase I or III, protein kinase C, or the catalytic subunit of cAMP-dependent protein kinase. Specific inhibition of Ca2+/calmodulin-dependent protein phosphorylation by arachidonic acid was also demonstrated in intact synaptic terminals (synaptosomes) isolated from rat forebrain. These results suggest that arachidonate and its metabolites may modulate synaptic function through the inhibition of CaM-kinase II-dependent protein phosphorylation.

Rat cerebral cortical synaptosomes that had been prelabeled with 32P- orthophosphate were exposed to either (1) K depolarization which causes Ca2+ influx and hence would be expected to activate Ca2+-dependent enzymes, including Ca2+/calmodulin-dependent and Ca2+/diacylglycerol- dependent protein kinases (Ca/CaM kinases and protein kinase C, respectively); or (2) phorbol esters or 1-oleoyl-2-acetyl-glycerol (OAG), which selectively activate protein kinase C. Proteins whose state of phosphorylation was affected by these treatments could be divided into 3 classes. Class A includes 5 phosphoproteins that showed rapidly increased phosphorylation by synaptosomal depolarization but not by OAG or phorbol ester. Four of these proteins, synapsins Ia and Ib and proteins IIIa and IIIb, are neuron-specific, synaptic vesicle- associated proteins known to be substrates for Ca/CaM kinases I and II. These phosphoproteins were rapidly dephosphorylated upon synaptosomal repolarization. Class B is composed of 2 phosphoproteins that showed rapidly increased phosphorylation by either synaptosomal depolarization or treatment with phorbol ester or OAG. These 2 acidic proteins of Mr87 and 49 kDa are known from in vitro studies to be specific substrates for protein kinase C. Thermolytic peptide mapping indicated that the 87 kDa protein in synaptosomes was phosphorylated by protein kinase C in situ. These 2 phosphoproteins were slowly dephosphorylated upon synaptosomal repolarization. Class C comprises 4 phosphoproteins that were rapidly dephosphorylated upon synaptosomal depolarization and may be substrates for Ca2+-activated protein phosphatase(s). These data suggest that Ca2+ influx into nerve terminals activates Ca/CaM kinases I and II, protein kinase C, and unidentified protein phosphatase(s).

Synaptosomes, purified from rat cerebral cortex, were prelabeled with [3H]inositol to study phosphatidylinositol turnover in nerve terminals. Labeled synaptosomes were either depolarized with 40 mM K+ or exposed to carbamoylcholine (carbachol). K+ depolarization increased the level of inositol phosphates in a time-dependent manner. The inositol trisphosphate concentration increased rapidly and transiently, reaching maximum (250% of control) in less than 3 sec and returning to near basal levels by 30 sec. The inositol bisphosphate level also increased rapidly, but its elevated level (220% of control) was sustained during continued depolarization. The elevated level of inositol bisphosphate was reversed upon repolarization of the synaptosomes. The level of inositol monophosphate increased slowly to 120-130% of control. These effects of K+ depolarization depended on the presence of Ca2+ in the incubation medium. Carbachol stimulated the turnover of phosphatidylinositol in a dose- and time-dependent manner. The level of inositol trisphosphate increased only slightly (120-130% of control) during carbachol stimulation. The level of inositol bisphosphate increased to 210% of control, and this maximal response was seen from 15 to 60 min. Accumulation of inositol monophosphate (250% of control) was larger than that of inositol bisphosphate, but its time course was slower. Atropine and pirenzepine inhibited the carbachol effect with high affinities of 0.8 nM and 16 nM, respectively, indicating that the effect of carbachol was mediated by activation of a M1 muscarinic receptor. Incubation of synaptosomes in Ca2+-free buffer reduced the response to carbachol by 30%, and addition of EGTA abolished it. These data show that both Ca2+ influx and M1 muscarinic receptor activation stimulate phospholipase C activity in synaptosomes, suggesting that phosphatidylinositol turnover may be involved in regulating neurotransmitter release from nerve terminals.

Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosine-containing proteins distributed in the same manner as synaptophysin in all subcellular fractions. Like synaptophysin, the two high molecular weight phosphotyrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation of an exogenous substrate, poly(Glu80,-Tyr20). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.

Bacterial lipopolysaccharide (LPS), the major surface component of gram-negative bacteria, exerts a profound effect on the immune system by enhancing the release of proteins and arachidonic acid metabolites from macrophages (for review see ref. 1). The molecular mechanism(s) by which LPS induces these various secretory responses is unknown. We previously reported that LPS promotes the myristoylation of several macrophage proteins including one with a relative molecular mass (Mr) of 68K2. We have now found that by several criteria the 68K myristoylated protein is similar or identical to the 80/87K protein, a major specific substrate for protein kinase C (PKC) found in brain and fibroblasts (for review see refs 7,8). We have also found that the myristoylated PKC substrate is quantitatively associated with the membrane fraction. Myristoylation of the PKC substrate may target it to the membrane and constitute a transduction pathway for stimulus-response coupling.

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.

Abstract: Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+‐dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87‐kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87‐kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, γ‐aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+‐dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus‐secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.

An 87-kDa phosphoprotein, identified previously as a major, specific substrate for Ca2+/phospholipid/diacylglycerol-dependent protein kinase (protein kinase C) in broken cell preparations from rat brain, has been characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, mouse, and bovine brain and in Torpedo californica electric organ. The protein was also identified in a variety of nonneuronal rat and bovine tissues. The rat protein had an apparent molecular mass 4-7 kDa lower, and was slightly more acidic, than the protein in bovine tissues. The 87-kDa proteins from various bovine tissues were identical by the following criteria: each was phosphorylated by exogenous protein kinase C, was of comparable molecular mass, generated multiple spots within the pH range of 4.4-4.9 upon isoelectric focusing, yielded identical patterns upon digestion with Staphylococcus aureus V8 protease, and was recognized by a specific 87-kDa antiserum. The relative concentrations of the 87-kDa protein in bovine tissues were highest in brain, spleen, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle. In the brain, the 87-kDa protein was concentrated in the synaptosomal membrane and in the cytosol. The membrane-bound protein was extractable with nonionic detergents but not with NaCl. This species, tissue, and subcellular distribution of the 87-kDa protein is similar to that of protein kinase C.