Chronic lithium treatment of B lymphoblasts from bipolar disorder patients reduces transient receptor potential channel 3 levels.

Chronic lithium treatment of B-lymphoblast cell lines (BLCLs) from bipolar-I disorder (BD-I) patients and healthy subjects ex vivo attenuates agonist- and thapsigargin-stimulated intracellular calcium (Ca(2+)) responses. As these findings suggest that chronic lithium treatment modifies receptor (ROCE) and/or store-operated Ca(2+) entry (SOCE) mechanisms, we determined whether chronic lithium treatment of BLCLs modified the expression of two members of the transient receptor potential channels (TRPC1 & 3), which participate in ROCE/SOCE. Chronic lithium treatment significantly reduced BLCL TRPC3 immunoreactivity (repeated-measures ANOVA, P=0.00005), with interaction effects of diagnosis (P=0.037) and sex (P=0.040). The lithium-induced decrease was greatest in BLCLs from female BD-I patients compared with those from healthy females (-27%) and with vehicle-treated BLCLs from female BD-I patients (-33%). However, lithium treatment did not affect TRPC1 and 3 mRNA levels, and TRPC1 immunoreactivity. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological Ca(2+) disturbances as observed in BD.

Regulation of the nitric oxide synthase-nitric oxide-cGMP pathway in rat mesenteric endothelial cells.

Most of the available data on the nitric oxide (NO) pathway in the vasculature is derived from studies performed with cells isolated from conduit arteries. We investigated the expression and regulation of components of the NO synthase (NOS)-NO-cGMP pathway in endothelial cells from the mesenteric vascular bed. Basally, or in response to bradykinin, cultured mesenteric endothelial cells (MEC) do not release NO and do not express endothelial NOS protein. MEC treated with cytokines, but not untreated cells, express inducible NOS (iNOS) mRNA and protein, increase nitrite release, and stimulate cGMP accumulation in reporter smooth muscle cells. Pretreatment of MEC with genistein abolished the cytokine-induced iNOS expression. On the other hand, exposure of MEC to the microtubule depolymerizing agent colchicine did not affect the cytokine-induced increase in nitrite formation and iNOS protein expression, whereas it inhibited the induction of iNOS in smooth muscle cells. Collectively, our findings demonstrate that MEC do not express endothelial NOS but respond to inflammatory stimuli by expressing iNOS, a process that is blocked by tyrosine kinase inhibition but not by microtubule depolymerization.

Molecular aspects of soluble guanylyl cyclase regulation.

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme (comprised of alpha and beta subunits) that generates the intracellular second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP is subsequently important for the regulation of protein kinases, ion channels, and phosphodiesterases. Since recent evidence has demonstrated that heterodimerization of the alpha/beta subunits is essential for basal and stimulated enzymatic activity, the existence of several types of isoforms for each of the two subunits, along with their varying degrees of expression in different tissues, implies that multiple regulatory mechanisms exist for sGC. Yet, progress in studying and clarifying the regulatory processes that can alter sGC expression and activity has only slowly started being elucidated. In the following paper, we elaborate on sGC structure, function, and distribution along with recently described signaling pathways that modulate sGC gene expression.

Characterization of alpha(s)-immunoreactive ADP-ribosylated proteins in postmortem human brain.

ADP-ribosylation of the stimulatory G protein alpha subunit, alpha(s), has been demonstrated in a number of different mammalian tissues. However, little is known about the occurrence and role of this process in modifying alpha(s) levels/function in human brain. In the present study, endogenous and cholera toxin (CTX)-catalyzed [32P]ADP-ribosylated products were characterized in postmortem human temporal cortex by (1) immunoprecipitation with alpha(s) antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [32P]ADP-ribosylated products, and (3) limited protease digestion. Of the three major endogenous [32P]ADP-ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48-kDa and 45-kDa bands were clearly identified as alpha(s-L) (long isoform) and alpha(s-S) (short isoform), respectively. RM/1 immunoprecipitated the 39-kDa [32P]ADP-ribosylated protein, and overlays of immunoblots and autoradiograms showed that this product corresponded to an alpha(s)-like-immunoreactive protein. Furthermore, limited protease digestion of the 39-kDa endogenous [32P]ADP-ribosylated band generated peptide fragments similar to both endogenous and CTX-catalyzed [32P]ADP-ribosylated alpha(s-S). Two major CTX-catalyzed [32P]ADP-ribosylated products were also identified as alpha(s-L) (52 kDa) and alpha(s-S) (45 kDa). These findings clearly demonstrate that alpha(s) is a substrate for endogenous and CTX-catalyzed [32P]ADP-ribosylation in postmortem human brain. Furthermore, a lower molecular weight alpha(s)-like immunoreactive protein is also expressed in human brain and is a substrate for endogenous but not CTX-catalyzed [32P]ADP-ribosylation.

Effect of thyroid deficiency on Go alpha-subunit isoforms in developing rat cerebral cortex.

Postnatal development of G alpha o isoforms in rat cerebral cortex was studied by SDS-PAGE and immunoblotting. When rat cerebral cortical membranes were resolved on separating gels containing 9% acrylamide and 8 M urea, three electrophoretically distinct G alpha o-immunoreactive proteins were evident. Comparison of their electrophoretic mobilities and partial tryptic digest pattern with recombinant G alpha o1 or G alpha o1-specific antibody revealed that the slowest and intermediate-migrating bands represent unmodified and fatty acylated forms of G alpha o1 protein, respectively. The fastest-migrating band corresponds to G alpha o2. While the fatty acylated form of G alpha o1 is the predominant species, its appearance paralleled that observed for G alpha o2 in developing rat cortex. Perinatal hypothyroidism induced by methimazole treatment did not significantly alter the appearance of cerebral cortical G alpha o1 and G alpha o2 between days 1 and 22 postpartum. Our findings support the earlier idea that heterogeneity of G alpha o proteins in mammalian brain is likely the result of different co- or post-translational processings of each splice variant of G alpha o. While the appearance of G alpha o isoforms is developmentally regulated, they likely do not play an obligatory role in neonatal brain development. Alternatively, the expression of G alpha o isoforms in developing rat cortex may be controlled by an intrinsic signal(s) that is independent of the thyroid status.

Developmental expression of G alpha o and G alpha s isoforms in PC12 cells: relationship to neurite outgrowth.

Recent evidence suggests neuronal differentiation is associated with developmentally regulated changes in the expression of certain G-protein subtypes but the relationship of such changes to neuronal differentiation and/or neurite outgrowth is poorly understood. In this study, the appearance of the G-protein alpha o and alpha s subunit isoforms was characterized in NGF-induced differentiating pheochromocytoma (PC)12 cells and correlated with neurite outgrowth, which was assessed with direct morphometric measures at 24-h intervals for a 5-day period and at 24, 48 and 72 h following NGF removal. Significant increases were observed in alpha o1 immunoreactive levels and the ratio of alpha o1/alpha o2 immunoreactivities during NGF exposure but not in the levels of either the long or short isoform of alpha s. The former changes also correlated significantly with neurite length during NGF treatment. Furthermore, no significant changes were observed in the levels of alpha o or alpha s isoforms following NGF removal. These findings suggest that alpha o isoforms are involved in neuritic extension in PC12 cells.

The effects of schedules of reinforcement on instruction-following in human subjects with verbal and nonverbal stimuli.

The experiment reported here represents a partial replication of an experiment by Newman, Buffington, and Hemmes (in press) and analyzes responding in college students as a function of three different schedules of reinforcement (FR 1, FR 2, FR 3) and either verbal discriminative stimuli (instructions) or nonverbal discriminative stimuli (different colored cards). All consequences (tokens) were based on behavior consistent either with the verbal discriminative stimulus (S(D)) or with the nonverbal S(D). The schedule of reinforcement varied across subjects, and accuracy of the verbal and nonverbal S(D)s varied across phases from. Results showed that the behavior of all continuous reinforcement (FR 1) subjects was sensitive to the accuracy of the verbal S(D)s, but the behavior of subjects in the nonverbal S(D) conditions showed more sensitivity than the behavior of subjects in verbal conditions under intermittent schedules (FR 2 and FR 3). These finding suggest that the behavior of subjects in experiments where instructions are sometimes pitted against actual contingencies of reinforcement is a function not only of the instruction, but also of the type of reinforcement schedule used.