Altered TRPC7 gene expression in bipolar-I disorder.

BACKGROUND: As altered storage-operated calcium (Ca(2+)) entry (SOCE) may affect Ca(2+) homeostasis in bipolar disorder (BD), we determined whether changes occur in the expression of TRPC7 and SERCA2s, proteins implicated or known to be involved in SOCE, in B lymphoblast cell lines (BLCLs) from BD-I patients and comparison subjects. METHODS: mRNA levels were determined in BLCL lysates from BD-I, BD-II, and major depressive disorder patients, and healthy subjects by comparative reverse transcriptase-polymerase chain reaction, and BLCL basal intracellular Ca(2+) concentration ([Ca(2+)]B) was determined by ratiometric spectrophotometry using Fura-2, in aliquots of the same cell lines, at 13-16 passages in culture. RESULTS: TRPC7 mRNA levels were significantly lower in BLCLs from BD-I patients with high BLCL [Ca(2+)]B compared with those showing normal [Ca(2+)]B (-33%, p =.017) and with BD-II patients (-48%, p =.003), major depressive disorder patients (-47%, p =.049) and healthy subjects (-33%, p =.038). [Ca(2+)]B also correlated inversely with TRPC7 mRNA levels in BLCLs from the BD-I group as a whole (r = -.35, p =.027). CONCLUSIONS: Reduced TRPC7 gene expression may be a trait associated with pathophysiological disturbances of Ca(2+) homeostasis in a subgroup of BD-I patients.

Altered IMPA2 gene expression and calcium homeostasis in bipolar disorder.

Reduced inositol monophosphatase (IMPase) activity and elevated basal intracellular calcium levels ([Ca(2+)](B)) have been reported in B lymphoblast cell lines (BLCLs) from bipolar I affective disorder (BD-I) patients, which may reflect cellular endophenotypes of this disorder. As the PI cycle couples to intracellular Ca(2+) mobilization, these two putative endophenotypes may be related. Using an RT-PCR assay, mRNA levels were estimated for IMPA1 and 2 genes encoding human IMPase 1 and 2, respectively, in BLCLs phenotyped on [Ca(2+)](B), from patients with a DSM-IV diagnosis of BD-I (n = 12 per phenotype) and from age- and sex-matched healthy subjects (n = 12). IMPA2 mRNA levels were significantly lower in BLCLs from male BD-I patients with high [Ca(2+)](B) (n = 6) compared with healthy male subjects (n = 5) (-52%, P = 0.013), male BD-I patients with normal BLCL [Ca(2+)](B) (n = 8) (-42%, P = 0.003) and female BD-I patients with high [Ca(2+)](B) (n = 6) (-59%, P = 0.0004). A significant negative correlation was observed between IMPA2 mRNA levels and [Ca(2+)](B) in BLCLs from male (P = 0.046), but not female BD-I patients. Sex-dependent differences were also evident in postmortem temporal cortex IMPA2 mRNA levels which, in contrast to BLCLs, were significantly higher in male BD-I subjects compared with male controls (P = 0.025, n = 4/group). Collectively, these observations suggest a potential sex-dependent link between abnormalities in IMPA2 expression and calcium homeostasis in the pathophysiology of BD.

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.

Lack of effect of antidepressants on mononuclear leukocyte G-protein levels or function in depressed outpatients.

Evidence from studies in animal and cultured cell models suggests that antidepressants (ADs) may enhance postreceptor signalling through the G protein coupled adenylyl cyclase (AC) pathways. To test whether this also occurs in patients receiving AD treatment, G-protein-activated-AC activity and the levels of alpha s and alpha i were measured in mononuclear leukocytes (MNLs) from 12 subjects with major depressive disorder (MDD) at baseline and after a 5 week trial of AD treatment. Although no differences were found in GTP gamma S-and forskolin-stimulated AC activity or the levels of alpha s and alpha i in MDD subjects compared with age- and sex-matched healthy subjects, pretreatment basal AC activity was significantly lower in treatment responders compared with healthy subjects. No significant changes were evident in any of these biochemical measures following 5 weeks of AD treatment in the patient group as a whole or stratified by response. These findings do not support an effect of ADs on the G-protein AC pathway, at least in MNLs. Lower pretreatment basal AC activity in responders suggests some change(s) in post-receptor signalling processes may be associated with an increased likelihood of therapeutic response to ADs.

Mononuclear leukocyte levels of G proteins in depressed patients with bipolar disorder or major depressive disorder.

Stimulatory (Gs) and inhibitory (G(i)) guanine nucleotide binding protein alpha subunit levels were measured in mononuclear leukocytes from 22 drug-free depressed patients (eight with bipolar disorder, 14 with major depressive disorder) and a comparison group of 17 age- and sex-matched healthy subjects. The levels of Gs alpha and G(i) alpha were significantly higher (160% and 114%, respectively) in the bipolar patients, but not the patients with major depressive disorder, than in the healthy subjects. These data add to the evidence for abnormalities in G protein levels and function in the pathophysiology of bipolar disorder.

Cerebral cortex Gs alpha protein levels and forskolin-stimulated cyclic AMP formation are increased in bipolar affective disorder.

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 Gs alpha 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 (Gs alpha, Gi alpha, Go alpha, and G beta) 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 Gs alpha levels, basal and GTP gamma S- and forskolin-stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, Gs alpha (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 (Gi alpha, Go alpha, G beta) 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 GTP gamma S-stimulated cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated stimulatory and reduced inhibitory G protein alpha subunits in cerebellar cortex of patients with dominantly inherited olivopontocerebellar atrophy.

Although guanine nucleotide binding proteins (G proteins) are one of the critical components of signal transduction units for various membrane receptor-mediated responses, little information is available regarding their status in brain of patients with neurodegenerative illnesses. We measured the immunoreactivity of G protein subunits (Gs alpha, Gi alpha, Go alpha, Gq/11 alpha, and G beta) in autopsied cerebellar and cerebral cortices of 10 end-stage patients with dominantly inherited olivopontocerebellar atrophy (OPCA) who all had severe loss of Purkinje cell neurons and climbing fiber afferents in cerebellar cortex. Compared with the controls, the long-form Gs alpha (52-kDa species) immunoreactivity was significantly elevated by 52% (p < 0.01) in the cerebellar cortex of the OPCA patients, whereas the Gi1 alpha concentration was reduced by 35% (p < 0.02). No statistically significant differences were observed for Go alpha, Gi2 alpha, G beta 1, G beta 2, or Gq/11 alpha in cerebellar cortex or for any G protein subunit in the two examined cerebral cortical subdivisions (frontal and occipital). The cerebellar Gs alpha elevation could represent a compensatory response (e.g., sprouting, reactive synaptogenesis) by the remaining cerebellar neurons (granule cells?) to neuronal damage but also might contribute to the degenerative process, as suggested by the ability of Gs alpha, in some experimental preparations, to promote calcium flux. Further studies will be required to determine the actual functional consequences of the G protein changes in OPCA and whether the elevated Gs alpha is specific to OPCA cerebellum, because of its unique cellular pattern of morphological damage, or is found in brain of patients with other progressive neurodegenerative disorders.

Regional distribution of guanine nucleotide binding proteins (Gs and Gi alpha) in human brain: correlation with adenylyl cyclase activity.

Gs and Gi alpha immunoreactivities and adenylyl cyclase activity were measured in autopsied human brain. Highest levels of Gs 52 kDa and Gi alpha subunits occurred in frontal cortex, and highest Gs 45 kDa levels in caudate. Although basal adenylyl cyclase activity was similar in all regions, GTP gamma S-stimulated adenylyl cyclase activity and forskolin-stimulated activity showed specific brain regional patterns which correlated with Gs 45 kDa and Gi alpha levels underscoring an important functional relationship between these measures.

Maturational and aging effects on guanine nucleotide binding protein immunoreactivity in human brain.

Age-related changes in transmembrane signal transduction have been reported for a number of hormonal receptors in human tissues. Guanine nucleotide binding proteins (G-proteins) are major regulatory components in the signal transduction processes for numerous receptors. Developmental changes in the abundance of specific G-protein alpha subunits, especially for Go, have already been shown in rat brain and in neuronal cell lines. In this study, immunoblotting analyses were performed with specific rabbit polyclonal antisera to Gs alpha, Gi alpha, Go alpha, and G beta subunits to estimate semi-quantitatively these G-protein subunits in samples of parietal cortex obtained postmortem from 18 subjects free of neurologic or psychiatric disease whose ages ranged from 3 days to 92 years. The Gs and Gi alpha subunit immunoreactivities were correlated significantly and inversely with age. The Gi alpha immunoreactivity declined markedly (46%) after the age of 40. As other G-protein subunit concentrations showed no age-dependent changes, the observed relationship between Gs and Gi alpha subunits and age is not likely due to cell loss occurring with advancing age. Of particular interest, the ratio of 52 to 45 kDa Gs alpha subunit immunoreactivities was significantly higher in infants than that found for adult parietal cortex. Given that changes in G-protein subunit abundance directly affect receptor-G-protein-effector functionality and response, these age-related alterations may be of importance in cerebral dysfunction and the development of neuropsychiatric disease in the later years of life.

Postmortem cerebral cortex Gs alpha-subunit levels are elevated in bipolar affective disorder.

We examined the relative abundance of G-protein subunits in postmortem brain obtained from 7 patients with bipolar affective disorder (BAD) compared with 7 age- and sex-matched controls. G-protein subunit immunoreactivities were determined in membranes prepared from postmortem prefrontal cortex using SDS-PAGE and immunoblotting with specific polyclonal antisera against selected G-protein subunits: Gsa, Gi(1&2) alpha, Go alpha and G beta(1&2). Of these G-protein subunits, only Gs alpha immunoreactivity was found to be significantly elevated in frontal (+ 34%), and occipital (+ 80%) cortex (P less than 0.05) in BAD compared with control subjects. Smaller increments (+ 22%) in cerebellar Gs alpha immunoreactivity were also found but were not statistically significant. On the basis that increased Gs alpha immunoreactivity may reflect enhanced functional responsiveness of the receptor-effector units to which this coupling protein is integral, the present findings suggest that disturbances in Gs-mediated signal transduction may be involved in the pathophysiology of BAD.

Evidence for presence of proinsulin-immunoreactive glycoprotein(s) in fetal bovine pancreatic extracts.

A large-molecular-weight proinsulin-immunoreactive protein fraction was obtained from an extract of fetal bovine pancreases by gel filtration in 6 M guanidine-1 M acetic acid. Concanavalin A-Sepharose-affinity column chromatography of the large-molecular-weight fraction yielded a discrete alpha-methyl-mannoside-displaceable immunoreactive peak that also displayed N-acetylglucosamine-specific binding to wheat germ lectin-Sepharose. Chemically tritiated and radioiodinated lectin-reactive proteins interacted specifically with antibodies to insulin and bovine proinsulin. Immunochemically purified (reaction with antibodies followed by separation of antigen-antibody complexes on protein A-Sepharose) radiolabeled lectin-reactive proteins were analyzed by gel filtration in guanidine-acetic acid and by sodium dodecyl sulfate polyacrylamide gel electrophoresis after disulfide bond-cleavage treatments. Results from these studies suggest the existence of an approximately 67,000-Mr glycoprotein that contains antigenic domains common to proinsulin and insulin.

Characterization of a chemically tritiated large molecular weight glucagon immunoreactive protein species by lectin-affinity column chromatography and reaction with anti-glucagon antibodies.

High molecular weight glucagon immunoreactive material, obtained by gel-filtration (in the presence of 6 M guanidine hydrochloride) of fetal bovine pancreatic extracts, was tritiated by reductive methylation. Concanavalin-A-Sepharose column chromatography of the radiolabeled preparation yielded a discrete Concanavalin-A-reactive, alpha-methyl-mannoside-displaceable radioactive peak, coinciding with the glucagon immunoreactive peak. Submission of the Con-A-reactive material to wheat germ agglutinin-Sepharose column chromatography yielded a lectin-reactive, N-acetyl-glucosamine-displaceable radioactive peak, coinciding with the glucagon immunoreactive peak. The tritiated Con-A-reactive component interacted specifically with anti-glucagon antibodies. Sephacryl S-200 gel-filtration (in the presence of guanidine hydrochloride) dissociated a approximately 40 kDa radioactive species from the antibody-antigen complex. These data provide direct evidence for the existence of a large molecular weight glycosylated glucagon-related protein species from the fetal bovine pancreas.

Glycoprotein-like large glucagon immunoreactive species in extracts of the fetal bovine pancreas.

Large glucagon immunoreactive substances, extracted from the fetal bovine pancreas and separated by gel filtration in the presence of 6 M guanidinium-hydrochloride, were submitted to lectin-sepharose affinity column chromatograph. Gel-filtered peak I (approximately 45 K delta) and peak II (approximately 10 K delta) interacted biospecifically with concanavalin-A- and wheat-germ-lectin-sepharoses, suggesting glycoproteins as possible constituents of large glucagon immunoreactive substances in extracts of the fetal bovine pancreas. The glucagon-like immunochemical identity of the lectin-sepharose-bound substances was further substantiated by binding to antiglucagon antibodies-sepharose and by characteristic proportional dilutions in the glucagon radioimmunoassay.

Ileal excretion and bacterial modification of bile acids and cholesterol in patients with continent ileostomy.

Bile acid (acidic sterol) and neutral steroid excretion were determined in 15 patients, five with conventional ileostomy, five with continent ileostomy, and five with continent ileostomy and an ileal resection. Acidic sterol losses were normal in conventional ileostomy patients and not significantly increased in those with continent ileostomy alone. Bile acid excretion rates were significantly increased in patients with a continent ileostomy and an ileal resection. Neutral steroid excretion was similar in all groups and not different from normal. Deoxycholic acid was not detected in ileal effluent of patients with conventional ileostomy and less than 2% of neutral steroid excreted was in the form of bacterial metabolites of cholesterol. The same was true of six of the 10 patients with continent ileostomies; in the other four patients at least 10% of acidic or neutral steroids were excreted as secondary bile acids or as a coprostanol. Modification of steroids was not related to ileal resection. Continent ileostomy was associated with a significant increase in percentage water content and a reduction in the pH of ileal effluent.

Toxic nephropathy after low-dose methoxyflurane anesthesia: drug interaction with secobarbital?

Vasopressin-resistant nonoliguric renal insufficiency developed in a 57-year-old man after 2 1/2 hours of low-dose methoxyflurane anesthesia. Secobarbital, 100 mg daily, had been taken for 1 month before. Of 13 patients in whom the influence of methoxyflurane on renal function was being studied, he was the only one to have taken a drug that induces microsomal enzymes. Blood values of methoxyflurane in this patient were lower than group means on all five occasions during anesthesia. Postoperatively his serum inorganic fluoride value reached 114 mumol/l -- more than two standard deviations greater than the group mean. Peak values for serum urea nitrogen, creatinine and uric acid and postvasopressin urine osmolality, and the lowest creatinine clearance in this patient also differed by more than 2 SD from the group mean, and the peak amount of oxalate excreted in his urine was double the group mean. Pretreatment with the barbiturate appears to have altered methoxyflurane metabolism and led to toxic concentrations of metabolites in the blood.