Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors.

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

Distinctive cellular roles for novel protein kinase C isoenzymes.

A number of in vitro studies have implicated protein kinase Cdelta (PKCdelta) and PKCepsilon in the regulation of the immune system. In recent years, this has been convincingly demonstrated in mice deficient for PKCdelta and PKCepsilon. The reported phenotypes for these transgenic mice indicate that PKCdelta suppresses immunoresponsiveness and inhibits the proliferation of B-lymphocytes, while PKCepsilon is required for macrophages to mount an effective immune response to bacterial pathogens. In either case, these isoenzymes appear to cooperate in fine-tuning certain immunoreactions by either suppressing (PKCdelta) or stimulating (PKCepsilon) the transcription of various cytokines. This review will compare and contrast the structures of these two nPKC isoenzymes and their respective roles in the modulation of cytokine production and various other cellular processes, such as growth, differentiation, apoptosis, and tumor suppression.

Biochemical and morphogenic effects of the interaction between protein kinase C-epsilon and actin in vitro and in cultured NIH3T3 cells.

Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.

Presynaptic kappa-opioid and muscarinic receptors inhibit the calcium-dependent component of evoked glutamate release from striatal synaptosomes.

In addition to cytosolic efflux, reversal of excitatory amino acid (EAA) transporters evokes glutamate exocytosis from the striatum in vivo. Both kappa-opioid and muscarinic receptor agonists suppress this calcium-dependent response. These data led to the hypothesis that the calcium-independent efflux of striatal glutamate evoked by transporter reversal may activate a transsynaptic feedback loop that promotes glutamate exocytosis from thalamo- and/or corticostriatal terminals in vivo and that this activation is inhibited by presynaptic kappa and muscarinic receptors. Corollaries to this hypothesis are the predictions that agonists for these putative presynaptic receptors will selectively inhibit the calcium-dependent component of glutamate released from striatal synaptosomes, whereas the calcium-independent efflux evoked by an EAA transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), will be insensitive to such receptor ligands. Here we report that a muscarinic agonist, oxotremorine (0.01-10 microM), and a kappa-opioid agonist, U-69593 (0.1-100 microM), suppressed the calcium-dependent release of glutamate that was evoked by exposing striatal synaptosomes to the potassium channel blocker 4-aminopyridine. The presynaptic inhibition produced by these ligands was concentration dependent, blocked by appropriate receptor antagonists, and not mimicked by the delta-opioid agonist [D-Pen2,5]-enkephalin. The finding that glutamate efflux evoked by L-trans-PDC from isolated striatal nerve endings was entirely calcium independent supports the notion that intact basal ganglia circuitry mediates the calcium-dependent effects of this agent on glutamate efflux in vivo. Furthermore, because muscarinic or kappa-opioid receptor activation inhibits calcium-dependent striatal glutamate release in vitro as it does in vivo, it is likely that both muscarinic and kappa receptors are inhibitory presynaptic heteroceptors expressed by striatal glutamatergic terminals.

Translocation assays of protein kinase C activation.

The protein kinase C (PKC) family members include at least 11 different isoforms that, based on their different requirements for activation, have been divided into three subfamilies, the Ca(2+)-dependent (cPKCα, (ß(I), ß(II), and γ), the Ca(2+)-independent (nPKCδ,ɛ,η,θ, and µ), and phorbol ester-insensitive (aPKCζ and ι, the human counterpart of mouse PKCλ) subfamilies. Much research on this growing family of protein kinases has concentrated on the possibility that these enzymes may have assumed distinct responsibilities for the control of complex and diverse cellular functions. The current working hypothesis is that the differential sensitivity of PKC isoforms to endogenous agonists and their differential targeting to discrete subcellular domains may dictate what substrates are phosphorylated by a given isoform. For this reason, an important initial goal in the analysis of the connection between PKC activation and a cellular response, such as neurodegeneration, is to identify the endogenous PKC isoforms that become "membrane-associated" in response to an appropriate stimulus. Translocation assays have classically been used to screen the individual isoforms of PKC for the production of active PKC-membrane complexes. The principal criteria used to distinguish between inactive and active forms of PKC are that, in the later case, the solubility of the enzyme is reduced when intact cells are treated with an appropriate agonist (e.g., phorbol esters) and that the association of a given PKC isoform with the particulate fraction resists extraction with Ca(2+)-chelators, but not nonionic detergents.

Molecular analysis of the interactions between protein kinase C-epsilon and filamentous actin.

Protein kinase C-epsilon (PKC-epsilon) contains a putative actin binding motif that is unique to this individual member of the PKC gene family. We have used deletion mutagenesis to determine whether this hexapeptide motif is required for the physical association of PKC-epsilon and actin. Full-length recombinant PKC-epsilon, but not PKC-betaII, -delta, -eta, or -zeta, bound to filamentous actin in a phorbol ester-dependent manner. Deletion of PKC-epsilon amino acids 222-230, encompassing a putative actin binding motif, completely abrogated this binding activity. When NIH 3T3 cells overexpressing either PKC-epsilon or the deletion mutant of this isozyme were treated with phorbol ester only wild-type PKC-epsilon colocalized with actin in zones of cell adhesion. In binary reactions, it was possible to demonstrate that purified filamentous actin is capable of directly stimulating PKC-epsilon phosphotransferase activity. These and other findings support the hypothesis that a conformationally hidden actin binding motif in the PKC-epsilon sequence becomes exposed upon activation of this isozyme and functions as a dominant localization signal in NIH 3T3 fibroblasts. This protein-protein interaction is sufficient to maintain PKC-epsilon in a catalytically active conformation.

Phylogenetic analysis of membrane trafficking proteins: a family reunion and secondary structure predictions.

The realization that a highly conserved family of membrane proteins are localized to transport vesicles and selectively interact with proteins anchored at appropriate target sites of membrane fusion inspired a simple and compelling explanation of how proteins might be transferred and segregated within the cell, the "SNARE hypothesis". This model holds that vesicle and target membrane proteins (designated as v-SNARE and t-SNARE proteins, respectively) wind around one another to form a three-stranded coiled coil structure, termed the prefusion complex. While the molecular topology of the prefusion complex has not been established, the concept that phylogenetically diverse SNARE proteins may become interlocked in a stable coiled coil is particularly attractive, because such a tertiary fold would only be permitted between strictly matched binding partners. For this reason, we have performed a phenetic analysis of all known SNARE sequences to assess the evolutionary and structural relatedness of these ancient protein families. Our phylogenetic analysis and consensus structure predictions revealed that syntaxin and SNAP-25 homologs are significantly related and constitute a superfamily of t-SNARE proteins that fall naturally into four major classes with distinct architectural motifs. The synaptobrevins sorted into three different classes of v-SNARE proteins. Comparison of the consensus structure predictions within each lineage or class of SNARE proteins strongly implied that coiled coil domains may not be required for fusion complex assembly in simple eukaryotic cells. It is our hypothesis that SNARE proteins in the late secretory pathway of mammalian cells may have elaborated more complex secondary structures (coiled coils), at about the time metazoan organisms diverged from yeast, that provide a sterically rigid foundation for positioning a conserved binding domain, the amphipathic alpha-helix.

Brain myosin V is a synaptic vesicle-associated motor protein: evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex.

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca2+ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg2+. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.

Action of New World scorpion venom and its neurotoxins in secretion.

New World scorpion venom contains protein toxins specific for ion channels in the plasmalemma of excitable cells. The effects were examined of whole venoms from Tityus serrulatus, T. bahiensis and T. stigmurus, and some purified toxins in isolated nerve endings (synaptosomes) and pancreatic acinar cells. Both systems initiated exocytosis in a dose-dependent response to the venom or its bioactive protein toxins. Actions differed, however, such that pancreatic acinar cells required Ca2+ while cerebrocortical synaptosomes responded by a Ca(2+)-dependent mechanism, except in the case of one toxin, IV-5, that elicited a Ca(2+)-independent response. Membrane depolarization caused by scorpion venom toxins was measured via radioisotopic discharge of tetra[3H]phenylphosphonium bromide. The role of protein kinase C in second-messenger coupling in pancreatic acinar cells is favored over ion-exclusive routes characteristic of synaptosomes.

Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function.

Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKC epsilon and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKC epsilon within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC-binding protein. The binding of PKC epsilon to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKC epsilon. Once established, this protein-protein interaction securely anchored PKC epsilon to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKC epsilon isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.

Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 over-expression.

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) or 13-hydroperoxydodecadienoic acid (13-HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15-HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl-2 proto-oncogene were 6- to 8-fold more resistant to apoptotic killing by tumor necrosis factor-alpha, but only 0.4-fold more resistant to 15-HPETE. Thus, Bcl-2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

Kappa opioid agonists produce anxiolytic-like behavior on the elevated plus-maze.

The selective kappa agonist U-50,488H was evaluated on the elevated plus-maze test of anxiety. U-50,488H was administered intraperitoneally to male Sprague-Dawley rats 20 min before testing, first in an open field apparatus, then followed immediately on the elevated plus-maze. No significant change in spontaneous locomotor activity was measured in the open field apparatus, suggesting that U-50,488H was devoid of sedative effects in the dose range tested (0.1-1000 micrograms/kg, IP). Doses between 10 and 1000 micrograms/kg produced significant increases in elevated plus-maze behavior that were consistent with anxiolytic actions for U-50,488H. These anxiolytic-like effects were antagonized by naloxone (2.0 mg/kg, IP), suggesting an opioid receptor site of action. In addition, we tested the kappa 1-selective U-50,488H-derivative, U-69,593 (100 micrograms/kg, IP), which was also shown to mimic the anxiolytic-like effects produced by U-50,488H. These results suggest that low doses of the selective kappa 1 agonists U-50,488H and U-69,593 are endowed with anxiolytic properties in rodents and that the kappa opioid system may be involved in the behavioral response to anxiety.

Persistent enhancement of sustained calcium-dependent glutamate release by phorbol esters: requirement for localized calcium entry.

Sustained activation of protein kinase C significantly enhanced a secondary (slow) phase in the depolarization-induced release of glutamate from isolated hippocampal nerve endings. The phorbol ester, 4 beta-phorbol 12,13-dibutyrate, was used to sustain the activation of presynaptic protein kinase C for a prolonged (10-min) period, and then this relatively water-soluble phorbol ester was removed by superfusion before a 2-min stimulus of continuous membrane depolarization. These conditions were used to investigate the persistent effects of sustained protein kinase C activation on the magnitude of the slow phase of evoked glutamate release, in which the efficiency of synaptic vesicle mobilization and recycling may be primary determinants of response magnitude. It is reported here that sustained protein kinase C activation selectively increased the Ca(2+)-dependent component of glutamate release during a prolonged phase of K(+)-induced depolarization. The magnitude of this persistent effect on Ca(2+)-dependent glutamate release was directly related to the dose of 4 beta-phorbol 12,13-dibutyrate and the duration of exposure that was used to prime the release apparatus, was observed using two alternative synaptosomal preparations, and was evident regardless of the depolarizing stimulus used (elevated [KCl] or 4-aminopyridine). However, 4 beta-phorbol 12,13-dibutyrate did not alter the release induced by the Ca2+ ionophore ionomycin. Thus, the persistent effects of protein kinase C activation on a prolonged phase of glutamate release were dependent on the route of Ca2+ influx. The finding that voltage-regulated Ca2+ channel blockers were able to neutralize completely the 4 beta-phorbol 12,13-dibutyrate-dependent facilitation of K(+)-evoked glutamate release provided further support for this conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent enhancement of sustained calcium-dependent glutamate release by phorbol esters: role of calmodulin-independent serine/threonine phosphorylation and actin disassembly.

The phorbol ester 4 beta-phorbol 12,13-dibutyrate increases the final extent of Ca(2+)-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca2+. Previous work from our laboratory demonstrated that this 4 beta-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4 beta-phorbol 12,13-dibutyrate, requires localized Ca2+ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca2+/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4 beta-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca2+/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca(2+)-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the eb1ect of 4 beta-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.

Dentate granule cells as a central cardioregulatory site in the rat.

Dentate granule cells can be selectively destroyed by intrahippocampal injections of colchicine. This study evaluates the consequences of granule cell destruction on blood pressure regulation in the normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR). Bilateral destruction of dentate granule cells at 6 weeks of age produced a significant increase in blood pressure in the WKY that lasted for approximately 3 weeks, and a biphasic effect (increase then decrease) in the SHR that resulted in a significant hypotensive period that persisted for 6 weeks. Granule cell destruction at 11 weeks produced a maximal hypertension in the SHR that preceded age-matched controls by 4 weeks, but produced only a small transient increase in WKY blood pressure. Dentate granule cells are the exclusive source of prodynorphin-derived peptides in the hippocampal formation and their synthesis is regulated by glucocorticoids. Evidence suggests glucocorticoids may be involved in the regulation of blood pressure and hypertension. We determined that chronic high levels of corticosterone significantly reduced hippocampal dynorphin B levels in normotensive Sprague-Dawley rats. In addition, we confirmed that naive SHRs also contain significantly lower levels of hippocampal dynorphin B. These results suggest (i) that dentate granule cells represent a discrete neural site that may exert a tonic inhibitory influence on blood pressure, (ii) that dentate granule cells are not required for the full expression of hypertension in the SHR, and (iii) that chronic high levels of corticosterone can reduce dynorphin B levels in the dentate granule cells of normotensive rats.

mRNA at the synapse: analysis of a synaptosomal preparation enriched in hippocampal dendritic spines.

Previous studies have demonstrated that the branched spines of the mossy fiber-CA3 hippocampal synapse contain a particularly large number of polyribosomes (Chicurel and Harris, 1989, 1992). We analyzed a preparation of synaptosomes isolated from this region and have found it to contain a restricted RNA population: certain mRNAs, presumably derived from the dendritic spines and the fine astrocytic processes surrounding the pre- and postsynaptic elements of the synapse, are enriched in the synaptosome preparation as compared to the total hippocampus; other mRNAs are less prevalent or altogether absent. In addition, neural BC1, a small noncoding RNA thought to be involved in pre- or posttranslational regulatory processes in dendrites, is a major RNA component of the dendritic spine. These results support the hypothesis that local translational regulation of gene expression may be important in establishing and modulating synaptic function.

Evidence for the corelease of dynorphin and glutamate from rat hippocampal mossy fiber terminals.

Hippocampal mossy fiber (MF) nerve endings may be isolated in a subcellular fraction (P3) that releases both prodynorphin-derived peptides and glutamate (Glu) in a calcium-dependent manner when depolarized. However, this isolation procedure does not yield a pure preparation of MF synaptosomes. The present study evaluates the proportion of dynorphin (Dyn) and Glu that is released from synaptosomes in the P3 fraction that are of MF origin. We have addressed this issue by determining the degree to which a selective lesion of the dentate granule cell/MF system in vivo concomitantly reduces the exocytosis of Dyn and Glu from the P3 subcellular fraction. Unilateral injections of colchicine into the dentate gyrus resulted in a substantial and selective degeneration of the granule cell/MF pathway in the rat hippocampal formation. The overall integrated density of the Timm-stained band, which corresponds to the position of the MF terminal field, was estimated to be reduced by 75%. After this extensive loss of MF boutons, the K(+)-evoked release of Dyn and Glu from the P3 fraction was reduced by 95 and 51%, respectively. The loss of Timm staining and evoked Dyn release indicate that colchicine effectively eliminated MF synaptosomes from the P3 fraction. Those subcellular entities that were not destroyed by colchicine comprised approximately 50% of the protein and evoked Glu release measured by using the P3 fraction. In addition, the present results demonstrate that the inhibitory potency of the kappa opioid agonist U-50,488H was not altered by the elimination of MF boutons from this synaptosomal preparation. This finding indicates that U-50, 488H is capable of suppressing Glu exocytosis from both MF and non-MF synaptosomes. These results are consistent with the hypothesis that Dyn peptides and Glu are co-released from hippocampal MF terminals.

Transduction of a protein kinase C-generated signal into the long-lasting facilitation of glutamate release.

The present study investigated the delayed and persistent effects of 4 beta-phorbol 12,13-dibutyrate (PDBu) on the K(+)-evoked release of endogenous glutamate and dynorphin B-like immunoreactivity from a subcellular fraction (P3) that is enriched in hippocampal mossy fiber synaptosomes. It is demonstrated that the alpha, beta, gamma, epsilon, and zeta isoforms of protein kinase C (PKC) are present in the P3 fraction obtained using the guinea pig hippocampus as starting tissue. The K(+)-evoked release of glutamate was found to be selectively enhanced when mossy fiber-enriched synaptosomes were preincubated with PDBu for 15 minutes and extensively washed with a PDBu-free medium. The persistent enhancement of glutamate release observed under this condition was not reversed by the protein kinase inhibitor staurosporine and was desensitized to the potentiating effects of an acute reexposure to PDBu. The overall content and activity of PKC was not substantially altered during the initial 15 minutes of treatment with PDBu (10 microM). More prolonged pretreatments with PDBu altered the substrate specificity of PKC and decreased the content of all PKC isoforms, but did not reverse the facilitation of glutamate release that followed preincubation in the presence of PDBu. It is concluded that the persistent activation of PKC enhances K(+)-evoked glutamate release from hippocampal mossy fiber-enriched synaptosomes and that, once established, this presynaptic facilitation is sustained by a process that is no longer directly dependent on continued PKC phosphotransferase activity.

Inhibition of glutamate release: a potential mechanism of action for the anticonvulsant U-54494A in the guinea pig hippocampus.

U-54494A, a 1,2-diamine, is a potent inhibitor of glutamate release in a synaptosomal preparation that is highly enriched with hippocampal mossy fiber (MF) nerve endings. At a concentration of 100 microM, U-54494A significantly reduced the availability of cytosolic free calcium (Ca2+) in depolarized MF-enriched synaptosomes by 30% and inhibited the K(+)-evoked release of endogenous glutamate by 85%. The extent to which glutamate release was inhibited allows us to conclude that U-54494A acts directly on the MF subpopulation of glutamatergic nerve endings in the guinea pig hippocampus. In addition, this anticonvulsant effectively countered the presynaptic facilitation of K(+)-evoked glutamate release that is induced by kainic acid (KA). Thus, while KA (1 mM) by itself nearly doubled the rate of K(+)-evoked glutamate release, there was no net increase in the presence of both KA and U-54494A (100 microM). However, the opposed effects of these two compounds on glutamate release do not appear to be due to a direct interaction. In the presence of U-54494A (100 microM), KA (1 mM) significantly enhanced the K(+)-evoked release of glutamate. Finally, it is demonstrated that the KA-induced enhancement of glutamate release does not require the depolarization-induced entry of extracellular Ca2+.

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes.

Opioid agonists specific for the mu, delta, and kappa opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The kappa opioid agonists U-62,066E and (-) ethylketocyclazocine, but not the mu agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAGO) nor the delta agonist [D-Pen2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca2+ levels, indicating a possible mechanism for kappa agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca2+ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca2+ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the delta-opioid specific antagonist ICI 174,864 and the mu opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that kappa opioid agonists inhibit transmitter release from mossy fiber terminals through both kappa opioid and non-kappa opioid receptor mediated mechanisms.