Cerebral Cortex Gsα Protein Levels and Forskolin-Stimulated Cyclic AMP Formation Are Increased in Bipolar Affective Disorder

Abstract: Experimental animal and peripheral blood cell studies point to guanine nucleotide regulatory (G) protein disturbances in bipolar affective disorder. We have previously reported elevated prefrontal cortex G_sα protein in bipolar affective disorder and have now extended these preliminary observations in a larger number of subjects, assessing the brain regional specificity of these changes in greater detail, determining the functional biochemical correlates of such changes, and evaluating their diagnostic specificity. Membrane G protein (G_sα, G_iα, G_oα, and Gβ) immunoreactivities were estimated by western blotting in postmortem brain regions obtained from 10 patients with a DSMIII-R diagnosis of bipolar affective disorder and 10 nonpsychiatric controls matched on the basis of age, postmortem delay, and brain pH. To examine whether there were functional correlates to the observed elevated G_sα levels, basal and GTPγS-and forskolin-stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, G_sα (52-kDa species) immunoreactivity was significantly (p < 0.05) elevated in prefrontal (+36%), temporal (+65%), and occipital (+96%) cortex but not in hippocampus (+28%), thalamus (-23%), or cerebellum (+21%). In contrast, no significant differences were found in the other G protein subunits (G_iα, G_oα, Gβ) measured in these regions. Forskolin-stimulated cyclic AMP production was significantly increased in temporal (+31%) and occipital (+96%) cortex but not in other regions. No significant differences were apparent in basal or GTPγS-stimulated cyclic AMP production. A significant correlation (r= 0.60, p < 0.001) was observed between forskolin-stimulated cyclic AMP formation and G_sα (52 kDa) immunoreactivity when examined across these cortical regions. The observed increase in G_sα may be specific to bipolar disorders as no significant differences were detected in G_sα levels in temporal cortex from patients with either schizophrenia (n = 7) or Alzheimer's disease (n = 7). In summary, the present study confirms and extends our earlier findings and supports the notion that increased G_sα levels and possibly G_sα-adenylyl cyclase-mediated signal transduction are relevant to the pathophysiology of bipolar affective disorder.     

Elevated Phosphatidyl-CMP Is Not the Source of Diacylglycerol Accumulation Induced by Lithium in NG108-15 Cells

Abstract: Previous studies have shown that in the neuroblastoma X glioma hybrid cell line NG108-15 lithium is able to induce an increase in diacylglycerol levels. This effect was shown to be enhanced by the presence of bradykinin. Another striking effect of lithium was a marked gain in the level of the liponucleotide phosphatidyl-CMP. Increased phosphatidyl-CMP levels were detected in the presence of lithium alone but were considerably more pronounced in the presence of both lithium and bradykinin. These results are consistent with the inhibitory action of lithium on key enzymes of the degradation pathway of inositol phosphates, resulting in a decrease in cellular inositol content and in an elevation in levels of phosphorylated inositols. Comparison of the mass of the inositol phosphates and diacylglycerol showed that the lithium-induced diacylglycerol levels were substantially greater than would be expected from phosphoinositide hydrolysis alone. One possible reason for the increase in the level of diacylglycerol through the action of lithium is the reversal of the reaction for the formation of phosphatidyl-CMP. The resulting phosphatidic acid would then need to be further dephosphorylated to diacylglycerol. The lithium-induced elevation of phosphatidyl-CMP was prevented by addition of myo -inositol (10–30 m M ), suggesting that the increase in liponucleotide level was due to depletion of cellular inositol. Under the same conditions the elevated diacylglycerol concentration remained unchanged. Consequently, phosphatidyl-CMP is not its source, and diacylglycerol may arise through an effect of lithium on the degradation of phospholipids other than phosphoinositides. The action of phospholipase C or D on phosphatidylcholine is the most likely mechanism.     

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC₅₀ = 21.5 µM, K_i = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC₅₀ = 0.800 µM, K_i = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC₅₀ values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.     

Benzotriazole stabilized lithium intermediates as a route to the elaboration of N-substituents in amides

Lithiation of N-(benzotriazol-1-ylmethyl)benzamide or N-(benzotriazol-1-ylmethyl)-2,2-dimethylbutyramide, readily prepared from the corresponding amides, formaldehyde and benzotriazole, followed by quenching with various electrophiles, such as alkyl halides, ketones or ester, gives the corresponding N-substituted derivatives. Subsequent displacement of the benzotriazole group with Grignard reagents, thiols or alcohols provides access to a wide variety of N-substituted amides in good yields. Treatment of the N-(benzotriazol-1-ylalkyl)benzamides with n- BuLi afforded the 1,1-dibenzamidoalkanes.     

Polyphosphoinositide hydrolysis by phospholipase C is accelerated by thyrotropin releasing hormone (TRH) in clonal rat pituitary cells (GH3 cells)

Thyrotropin releasing hormone (TRH) accelerates the turnover of phosphatidylinositol in GH3 cells ('phospholipid response'). From the analysis of inositol phosphates in the presence of Li+ which inhibits their dephosphorylation, it can be concluded that the hydrolysis of phosphatidylinositol 4,5-biphosphate, and possibly of phosphatidylinositol 4-phosphate by phospholipase C is markedly accelerated by TRH. It appears that this reaction initiates the acceleration of phosphatidylinositol turnover. The specificity of hormonally regulated phospholipase C reaction for polyphosphoinositides has important implications for the potential role of the phospholipid response as a mechanism of membrane signal transduction.     

Effects of lithium on angiotensin-stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship

Turnover of 32P-labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P]phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin-stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.     

Effect of dodecylsulfate binding on the activity of creatine kinase

The number of lithium dodecylsulfate (LDS) molecules tightly bound to creatine kinase has been found by isotachophoresis to be 87 at 25°C upon saturation of the enzyme dimer with LDS. The binding shows positive cooperativity by both the Hill and Scatchard plots. The enzyme is completely inactivated when its high-affinity sites are fully occupied with LDS. However, at partial LDS saturation the activity remaining is definitely higher than can be accounted for by the amount of free enzyme left, showing the presence of species of active molecules with the tight LDS sites only partly saturated. The presence of ATP leads to a decrease in detergent bound at the high-affinity sites with partial restoration of activity.     

Lithium and hormesis: Enhancement of adaptive responses and biological performance via hormetic mechanisms

Biomedical and consumer interest in the health-promoting properties of pure single entities of known or unknown chemical constituents and mixtures has never been greater. Since its “rediscovery” in the 1950s, lithium is an example of such a constituent that represents an array of scientific and public health challenges and medical potentials that may now be understood best when seen through the lens of the dose-response paradigm known as hormesis. The present paper represents the first review of the capacity of lithium to induce hormetic dose responses in a broad range of biological models, organ systems, and endpoints. Of significance is that the numerous hormetic findings occur with extensive concentration/dose response evaluations with the optimal dosing being similar across multiple organ systems. The particular focus of these hormetic dose-response findings was targeted to research with a broad spectrum of stem cell types and neuroprotective effects. These findings suggest that lithium may have critically valuable systemic effects with respect to those therapeutically treated with lithium as well as for exposures that may be achieved via dietary intervention.     

Evidence for dietary essentiality of lithium in the rat

The purpose of this study was to determine the dietary essentiality of lithium (Li) in rats. In three experiments, two types of diets were fed during growth, reproduction, and lactation. In the first experiment, dams were maintained on a corn-based diet containing 2 ng or 500 ng (controls) Li/g through five successive periods of pregnancy and lactation. The offspring of dams fed the low-Li diet had significantly lower weaning weights (p=0.011), and the percent weaned was lower (p=0.094) than that of controls. In the second experiment, rats were maintained through three generations on a rice-based diet containing 0.6 ng Li/g, or the control (500 ng/g) diet. There was a significant effect of Li level on litter size (p=0.017) and litter wt at birth (p=0.006) in the third generation. The overall effect through three generations on litter wt at birth approaches statistical significance (p=0.086). In the third experiment, third-generation rats were continued on the respective rice-based diets with three levels of dietary sodium, the normal level, one-half, and four times that level. The litter size and birth wt were significantly lower (p=0.0030 and 0.0038, respectively) among the low-Li dams that consumed the normal and high-sodium levels compared to those that consumed the low-sodium diets. The interaction of Li and sodium as regards litter wt at birth approached significance (p=0.083). Various tissues of the rats in the third experiment were analyzed for Li. It seems likely that Li exerts an essential nutrient role for the rat. Contribution of the Missouri Agricultural Experiment Station, Journal Series No. 11,327.     

Lithium Reduqes the Accumulation or Inositol Polyphosphate Second Messengers Following Cholinergic Stimulation of Cerebral Cortex Slices

Abstract: The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [³H]inpsitol monophosphate but suppressed the accumulation of the putative second messengers [³H]inositol 1,4,5-trisphosphate ([³H]Ins(1,4,5)P₃) and f³H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P₃showed similar inhibitory effects, which could be prevented by preincubation with myo -inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-midiated neurotransmission in brain.