The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

The role of erbB2 receptor in human breast cancer metastasis

Overexpression of c-erbB-2 gene-encoded p185 has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of c-erbB-2 can enhance metastatic potential of human breast cancer cells, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the c-erbB-2 gene transfected 435.eB cells. In vivo experimental metastasis assays demonstrated that mice injected erbB2-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in metastatic potential in vivo were accompanied by increased invasiveness in vitro . The transfectants and the parental cells all had similar growth rates and transformation potential. These findings suggest that c- erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities. ^ Homophilic adhesion may affect invasive and metastatic potential of tumor cells. We found that Heregulin-β1 (HRG-β1), a growth factor that activates receptor kinases erbB3 and erbB4, can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-β1-increased cell aggregation, we observed that HRG-β1 increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using different kinase inhibitors, we found that the HRG-β1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-β1-activated PI3K is required for enhancing breast cancer cell aggregation. These results have provided one mechanism by which HRG-β1-activated signaling of erbB receptors may affect invasive/metastatic properties of breast cancer cells. ^ To identify the structural motifs within the erbB2 receptor that are required for erbB2 increased metastatic potential in breast cancer cells, we injected different forms of mutated erbB2 expressing MDA-MB-435 cell line transfectants with or without the EGF-like domain of heregulin-β1 protein (HRG/egf) into ICR-SCID mice to test the metastatic survival rate. The results show that an intact kinase domain of erbB2 receptor is required for erbB2 enhanced metastatic potential in these cells. The C-terminal tyrosine 1248 residue of erbB2 may also play a role in enhancing metastatic potential. Moreover, the results suggest that HRG/egf promote the metastatic potential of human breast cancer cells in vivo. ^

Intra-hippocampal administration of the VEGF receptor blocker PTK787/ZK222584 impairs long-term memory.

A number of studies have established a role for vascular endothelial growth factor (VEGF) in angiogenesis. Recent reports have shown that VEGF overexpression in the hippocampus improves learning and memory and is associated with enhanced neurogenesis. PTK787/ZK222584 (PTK/ZK) is a reported inhibitor of VEGFR signaling that is currently being tested for its effects on lung and colon cancer. However, the influence of this drug on cognition has not been examined. In the present study, we questioned if post-training administration of PTK/ZK influences hippocampus-dependent memory. When administered to rats immediately following massed training in the Morris water maze, PTK/ZK impaired spatial memory retention tested 48 h later. This impairment was evidenced by increased latency to the hidden platform and fewer platform crossings. However, this impairment was not associated with a change in neurogenesis during this time frame. PTK/ZK infusion did not reduce VEGFR or AKT phosphorylation, but increased the phosphorylation of ERK. These studies suggest that VEGFR inhibitors such as PTK/ZK may negatively influence cognition.

Role of protein kinase C in short-term sensitization of {\it Aplysia}

Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning, such as sensitization. Sensitization is induced, at least in part, by the transmitter serotonin (5-HT) and expressed in several forms, including facilitation of sensorimotor connections. Spike broadening has been believed to be a key mechanism underlying facilitation of nondepressed synapses. Previously, this broadening was believed to be dependent primarily on cAMP/protein kinase A (PKA)-mediated reduction of a noninactivating, relatively voltage-independent K$\sp{+}$ current termed the S-K$\sp+$ current (I$\sb{\rm K{,}S}$). Recent evidence, however, suggests that 5-HT-induced somatic spike broadening is composed of at least two components: a cAMP-dependent, rapidly developing component and a cAMP-independent, slowly developing component.^ Phorbol esters, activators of protein kinase C (PKC), mimicked the cAMP-independent component of 5-HT-induced broadening. Staurosporine, which inhibits PKC, had little effect on the rapidly developing component of 5-HT-induced broadening, but inhibited significantly the slowly developing component. These results suggest that PKC is involved in the cAMP-independent component of 5-HT-induced broadening. The membrane currents responsible for the slowly developing component of broadening were examined. Activation of PKC mimicked, and partially occluded, 5-HT-induced modulation of membrane currents above 0 mV, where a voltage-dependent K$\sp+$ current (I$\sb{\rm K{,}V}$) is significantly activated. This modulation was complex because it was associated with a reduction in the magnitude of I$\sb{\rm K{,}V}$, as well as a slowing of both activation and inactivation kinetics of I$\sb{\rm K{,}V}$. These results support the hypothesis that PKC modulates I$\sb{\rm K{,}V}$ and that this modulation contributes to the slowly developing component of 5-HT-induced broadening. Based on these results and others, a new scheme for 5-HT-induced spike broadening is proposed in which the modulatory effects are mediated via two second messenger/protein kinase systems converging and diverging on multiple ionic conductances.^ The relationship between spike broadening and synaptic facilitation was also examined. Pharmacological reduction of I$\sb{\rm K{,}V}$ by low concentrations of 4-aminopyridine (4-AP) led to spike broadening and facilitation of the nondepressed sensorimotor connections, indicating that spike broadening via the reduction of I$\sc{K,V}$ can facilitate the synaptic connection. Further analyses, however, revealed that 4-AP-induced facilitation has qualitative differences from 5-HT- and PKC-induced facilitation. These results suggest that 5-HT- and PKC-induced facilitation of nondepressed synapses is mediated, at least in part, by spike-duration independent (SDI) processes. Under certain conditions, the PKC inhibitor, staurosporine, significantly inhibited the 5-HT-induced facilitation of sensorimotor connections.^ Finally, it was found that activation of PKC increased a basal level of cAMP and that PKC caused desensitization of the 5-HT receptor, which may be a possible negative feedback mechanism through which an extracellular ligand, 5-HT, is regulated. These results suggest that these two second messenger/protein kinase pathways can interact in the sensory neuron. Thus, neuronal plasticity that may contribute to learning and memory appears to involve several complex and interactive processes. ^

Mechanism of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in head and neck squamous cell carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer and neuroblastoma in clinical trials. 4HPR induces growth inhibition and apoptosis in various cancer cells including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR induces apoptosis by several mechanisms including increasing reactive oxygen species (ROS), or inducing mitochondrial permeability transition (MPT). 4HPR has also been shown to modulate the level of different proteins by transcriptional activation or posttranslational modification in various cellular contexts. However, the mechanism of its action is not fully elucidated. In this study, we explored the mechanism of 4HPR-induced apoptosis in HNSCC cells. ^ First, we identified proteins modulated by 4HPR by using proteomics approaches including: Powerblot western array and 2-dimensional polyacrylamide gel electrophoresis. We found that 4HPR modulated the levels of several proteins including c-Jun. Further analysis has shown that 4HPR induced activation of Activator Protein 1 (AP-1) components, c-Jun and ATF-2. We also found that 4HPR increased the level of Heat shock protein (Hsp) 70 and phosphorylation of Hsp27. ^ Second, we found that 4HPR induced prolonged activation of JNK, p38/MAPK and extracellular signal-regulated kinase (ERK). We also demonstrated that the activation of these kinases is required for 4HPR-induced apoptosis. JNK inhibitor SP600125 and siRNA against JNK1 and JNK2 suppressed, while overexpression of JNK1 enhanced 4HPR-induced apoptosis. p38/MAPK inhibitor PD169316 and MEK1/2 inhibitor PD98059 also suppressed 4HPR-induced apoptosis. We also demonstrated that activation of JNK, p38/MAPK and ERK is triggered by ROS generation induced by 4HPR. We also found that translation inhibitor, cycloheximide, suppressed 4HPR-induced apoptosis through inhibition of 4HPR-induced events (e.g. ROS generation, cytochrome c release, JNK activation and suppression of Akt). We also demonstrated that MPT is involved in 4HPR-induced apoptosis. ^ Third, we demonstrated the presence of NADPH oxidase in HNSCC 2B cells. We also found that 4HPR increased the level of the p67phox, a subunit of NADPH oxidase which participates in ROS production and apoptosis induced by 4HPR. ^ The novel insight into the mechanism by which 4HPR induces apoptosis can be used to improve design of future clinical studies with this synthetic retinoid in combination with specific MAPK modulators. ^

Role of entorhinal cortical plasticity in spatial and contextual memory

It is well accepted that the hippocampus (HIP) is important for spatial and contextual memories, however, it is not clear if the entorhinal cortex (EC), the main input/output structure for the hippocampus, is also necessary for memory storage. Damage to the EC in humans results in memory deficits. However, animal studies report conflicting results on whether the EC is necessary for spatial and contextual memory. Memory consolidation requires gene expression and protein synthesis, mediated by signaling cascades and transcription factors. Extracellular-signal regulated kinase (ERK) cascade activity is necessary for long-term memory in several tasks, including those that test spatial and contextual memory. In this work, we explore the role of ERK-mediated plasticity in the EC on spatial and contextual memory. ^ To evaluate this role, post-training infusions of reversible pharmacological inhibitors specific for the ERK cascade that do not affect normal neuronal activity were targeted directly to the EC of awake, behaving animals. This technique provides spatial and temporal control over the inhibition of the ERK cascade without affecting performance during training or testing. Using the Morris water maze to study spatial memory, we found that ERK inhibition in the EC resulted in long-term memory deficits consistent with a loss of spatial strategy information. When animals were allowed to learn and consolidate a spatial strategy for solving the task prior to training and ERK inhibition, the deficit was alleviated. To study contextual memory, we trained animals in a cued fear-conditioning task and saw an increase in the activation of ERK in the EC 90 minutes following training. ERK inhibition in the EC over this time point, but not at an earlier time point, resulted in increased freezing to the context, but not to the tone, during a 48-hour retention test. In addition, animals froze maximally at the time the shock was given during training; similar to naïve animals receiving additional training, suggesting that ERK-mediated plasticity in the EC normally suppresses the temporal nature of the freezing response. These findings demonstrate that plasticity in the EC is necessary for both spatial and contextual memory, specifically in the retention of behavioral strategies. ^

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis.

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Ca++/calmodulin-dependent protein kinase II: Messenger RNA in developing rat brain

Ca$\sp{++}$/calmodulin-dependent protein kinase II (CaM-KII) is highly concentrated in mammalian brain, comprising as much as 2% of the total protein in some regions. In forebrain, CaM-KII has been shown to be enriched in postsynaptic structures where it has been implicated in maintaining cytoskeletal structure, and more recently in signal transduction mechanisms and processes underlying learning and memory. CaM-KII appears to exist as a holoenzyme composed of two related yet distinct subunits, alpha and beta. The ratio of the subunits in the holoenzyme varies with different brain regions and to some degree with subcellular fractions. The two subunits also display distinct developmental profiles. Levels of alpha subunit are not evident at birth but increase dramatically during postnatal development, while levels of beta subunit are readily detected at birth and only gradual increase postnatally. The distinct regional, subcellular and developmental distribution of the two subunits of CaM-KII have prompted us to examine factors involved in regulating the synthesis of the subunit proteins.^ This dissertation addresses the regional and developmental expression of the mRNAs for the individual subunits using in situ hybridization histochemistry and northern slot-blot analysis. By comparing the developmental profile of each mRNA with that of its respective protein, we have determined that initiation of gene transcription is likely the primary site for regulating CaM-KII protein levels. Furthermore, the distinct cytoarchitecture of the hippocampus has allowed us to demonstrate that the alpha, but not beta subunit mRNA is localized in dendrites of certain forebrain neurons. The localization of alpha subunit mRNA at postsynaptic structures, in concert with the accumulation of subunit protein, suggests that dendritic synthesis of CaM-KII alpha subunit may be important for maintaining postsynaptic structure and/or function. ^

Multisite phosphorylation of ornithine decarboxylase in transformed macrophages

Ornithine decarboxylase (ODC), the initial inducible enzyme in the polyamine biosynthetic pathway, exists in the transformed macrophage RAW264 cell line as a phosphoprotein following cell stimulation. The hypothesis that ODC is phosphorylated at multiple sites in stimulated RAW264 cells was investigated. ODC isolated from tetradecanoyl-phorbol-13-acetate (TPA)-stimulated cells metabolically radiolabeled in the presence of $\sp{32}$P$\sb{\rm i}$ was subjected to cyanogen bromide (CNBr) cleavage followed by phosphopeptide mapping and two dimensional phosphoamino acid analysis. These phosphorylation studies demonstrated six in situ phosphorylated CNBr-generated fragments having apparent molecular weights of 17, 14.3, 8, 6.5, 4, and 2.7 kDa and also revealed that ODC is phosphorylated in RAW264 cells on at least 5 serine and 2 threonine residues.^ In addition, the in vivo specific activity and phosphorylation pattern of ODC in response to various kinase cascade stimulants was studied. A differential response in ODC specific activity and a variation in the relative distribution of $\sp{32}$P-labeling of serine and threonine residues on the ODC molecule was noted in response to fetal bovine serum, cAMP and isobutylmethylxanthine, lipopolysaccharide, or TPA.^ Based on information derived from consensus sequence motifs, three protein kinases responsible for the phosphorylation of ODC in vitro were identified. Purified ODC was phosphorylated in vitro by casein kinase II (CK II), extracellular signal-regulated kinase 1 (ERK1), and its activator, extracellular signal-regulated kinase kinase (MEK). CK II phosphorylated ODC on serine residues contained on three CNBr-generated peptides with apparent molecular weights of 14.3, 6.5, and 2.7 kDa. Both ERK1 and MEK phosphorylated ODC on serine and threonine residues on a CNBr-generated peptide fragment with an apparent molecular weight of 6.5 kDa. The in vitro radiolabeled peptides corresponded in molecular mass with some of the CNBr fragments of ODC phosphorylated in situ in stimulated RAW264 cells.^ This study concludes that ODC is phosphorylated in the transformed macrophage RAW264 cell line at multiple sites in response to various kinase cascade stimulants. These stimulants also led to a differential response in specific activity and phosphorylation pattern of ODC in RAW264 cells. Three protein kinases have been identified which phosphorylate ODC in vitro on peptides and amino acid residues which correspond with those phosphorylated in situ. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^