Valerie Boss

The Cyclosporin A-sensitive Nuclear Factor of Activated T Cells (NFAT) Proteins Are Expressed in Vascular Smooth Muscle Cells DIFFERENTIAL LOCALIZATION OF NFAT ISOFORMS AND INDUCTION OF NFAT-MEDIATED TRANSCRIPTION BY PHOSPHOLIPASE C-COUPLED CELL SURFACE RECEPTORS

Expression of the antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by Ca2+ signaling and positive regulation through co-activation of adenylyl cyclase and Ca2+ mobilization. The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which activate Gαq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Gαq receptor agonists augment the NFAT-mediated transcriptional response to PDGF-BB. This synergy can be explained in part by augmented intracellular Ca2+ transients elicited by multiple agonist challenges. These data indicate that agonists for phospholipase C-coupled receptors stimulate NFAT-mediated transcription in VSMC differentially, and that NFAT can function to integrate co-activating signals in the extracellular environment.

Metabotropic excitatory amino acid receptor activation stimulates phospholipase D in hippocampal slices

Abstract: Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein‐coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD) and 1S,3S‐ACPD, but not the inactive isomer, 1R,3S‐ACPD, induce a concentration‐dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R‐ACPD‐induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR‐iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L‐2‐Amino‐3‐phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis‐coupled mGluRs.

Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation

The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6-11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.

Second-Messenger Systems Coupled to Metabotropic Glutamate Receptors

Glutamate and other excitatory amino acids (EAAs) have long been known to increase the levels of various second-messenger systems in different nervous system preparations. However, until recent years, these effects were generally held to be secondary to activation of glutamate-gated cation channels, and subsequent increases in neurotransmitter release or intracellular calcium concentrations. The first direct evidence for the existence of glutamate receptors directly coupled to second-messenger systems via GTP-binding proteins (G-proteins) came in the mid1980s with the discovery of glutamate receptors coupled to activation of phosphoinositide hydrolysis. Since that time, it has become clear that members of the metabotropic glutamate receptor (mGluR) family are coupled, either directly or indirectly, to a variety of second-messenger systems, including activation of phosphoinositide hydrolysis, regulation of adenylyl cyclase, activation of phospholipase D, increased cyclic guanosine mono-phosphate (cGMP) accumulation, and arachidonic acid release.

Retroviral vectors applied to gene regulation studies.

There are instances in G-protein-coupled receptor studies when it is desirable to express a transgene in some specialized cell phenotype to understand a process better. Most such cells are more resistant to plasmid transfection than are the transformed libroblasts often used in G protein signaling studies (for example, COS, 293, and Chinese hamster ovary (CHO)). This often presents a substantial barrier against understanding context-specific mechanisms of Gα protein-coupled receptor signaling and cellular regulation. Highly efficient gene transfer methods applicable to broader ranges of cell phenotypes can surmount this problem. Retroviruses use naturally evolved mechanisms to gain entry into cells, release their transgene cargoes, and permanently tag the new host. Their most significant advantages over plasmid transfection include high gene transfer efficiencies in many cell types, with the genes integrating into host cell chromatin permanently. One limitation of the Moloney murine leukemia-based retroviruses is that cell division is necessary to take advantage of these properties. For cells that transfect poorly, retroviral methods are more reproducible, easier, and less costly over the long haul.

An l-cysteine sulfinic acid-sensitive metabotropic receptor mediates increased cAMP accumulation in hippocampal slices

The excitatory amino acid (EAA), L-cysteine sulfinic acid (L-CSA), elicited a dose-dependent increase in cAMP accumulation in adult rat hippocampus that was not blocked by ionotropic glutamate receptor antagonists. Therefore, the possibility was examined that L-CSA activates the (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD)-sensitive metabotropic glutamate receptor (mGluR) that increases cAMP by potentiating responses elicited by adenosine or other agonists of receptors coupled to adenylate cyclase via Gs. Like 1S,3R-ACPD, L-CSA induced a cAMP response that was inhibited by the adenosine receptor antagonist, 8-para-sulfyltheophylline, and by adenosine deaminase. In contrast to the 1S,3R-ACPD-induced cAMP response, the L-CSA-induced response was not potentiated by the adenosine uptake inhibitor, dipyridamole. Taken together with the previous finding that L-CSA does not potentiate cAMP responses elicited by agonists of receptors that activate Gs, these data suggest that L-CSA increases cAMP accumulation by activating a metabotropic EAA receptor that is different from the 1S,3R-ACPD-sensitive mGluR associated with potentiation of cAMP responses.

Mild intermittent hypoxia does not induce stress responses in the neonatal rat brain.

We previously demonstrated that intermittent hypoxia evokes persistent changes in extracellular striatal dopamine, locomotor activity and executive function, using a rodent model emulating apnea of prematurity in which rat pups are exposed to 20-second bursts of hypoxic gas mix containing 10% oxygen (60 events/h; 6 h/day) from postnatal days 7 to 11. To determine whether subtle repetitive hypoxic insults also induce expression of stress-related genes, we employed real-time RT-PCR to assay gene transcription in neonatal rats subjected to the same paradigm. In addition, we also measured expression of stress-induced transcripts in an age-matched cohort following a more severe oxidative stressor: permanent focal ischemia. Four transcripts were elevated following the ischemic insult: heat shock protein 70 (Hsp70), CL100, nurr77, and heme oxygenase-1. In contrast, these transcripts were not regulated in the majority of neonatal rats exposed to an intermittent hypoxia protocol. Hsp70 was strongly induced, and CL100 and nurr77 were slightly induced in only 2 of 11 post-hypoxic rats compared to controls. These data demonstrate that a single ischemic event elicits expression of specific stress-related genes, whereas 5 days of brief intermittent hypoxic insults typically do not. Thus, it is unlikely that the neurochemical and behavioral morbidity observed in juvenile and adult rodents exposed to intermittent hypoxia during a critical period of brain development are related to stress-induced changes in gene expression.