O-dibutyryl cAMP Research Articles

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.

Regulation of N‐acetylaspartate and N‐acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.

Peptide Blockers of the Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Amyloid β

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.

CAMP-induced Astrocytic Differentiation of C6 Glioma Cells Is Mediated by Autocrine Interleukin-6

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt(2)AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt(2)AMP/theophylline. Taken together, these results indicated that Bt(2)AMP/theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.

In vitro effects of cAMP-elevating agents and glucocorticoid either alone or in combination on the production of nitric oxide, interleukin-12 and interleukin-10 in IFN-gamma- and LPS-activated mouse peritoneal macrophages.

The effects of cAMP-elevating agents, N6-2'-O-dibutyryl cAMP (Bu2cAMP), and glucocorticoid (dexamethasone) on the production of inflammatory mediators--nitric oxide and interleukin-12 (IL-12) and anti-inflammatory mediator interleukin-10 (IL-10) were demonstrated in murine peritoneal macrophages. Inducible nitric oxide synthase (iNOS) and iNOS mRNA were detected by northern blot and western blot, respectively. The cAMP elevating agents Bu2cAMP and prostaglandin E2 each alone did not show any effect on NO production but along with IFN-gamma and lipolysaccharide (LPS) they slightly enhanced NO production. Dexamethasone inhibited NO production in IFN-gamma- and LPS-treated cells; cAMP elevating agents interfered with the NO production inhibited by dexamethasone. Inhibition was revealed at the mRNA level as well as at protein level. Bu2cAMP or dexamethasone either alone or synergistically inhibited IL-12 production; Bu2cAMP interfered with dexamethasone-mediated inhibition of IL-10 production in IFN-gamma- and LPS-treated macrophages. The use of glucocorticoids along with cAMP elevating agents was beneficial in lowering the level of inflammatory mediator IL-12 and producing high levels of the anti-inflammatory mediator IL-10 active in cell protection. On the other hand, interference of Bu2cAMP with dexamethasone-mediated NO inhibition may have adverse effect. Therefore, adverse effects due to cAMP-mediated interference (inhibition) with NO synthesis may occur in many inflammatory diseases during combined drug therapy by glucocorticoids and cAMP elevating agents.

Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents.

Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N(6),2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.

Human ejaculated spermatozoa contain active P450 aromatase.

The generation of cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) knockout mice has raised new interest in the physiological role of estrogens in male reproduction. Testicular expression of P450arom, the enzyme that converts androgens into estrogens, has been shown in both somatic and germ cell types in several species, whereas in humans, testicular expression is confined to the somatic cells. The aim of this study was to determine whether P450arom is present in human ejaculated spermatozoa. Using RT-PCR and specific primers, we amplified the highly conserved helical, aromatic, and heme-binding sequences of the conventional human P450arom from RNA isolated from human spermatozoa. Employing a rabbit polyclonal antiserum directed against human placental P450arom, immunoblotting analysis demonstrated aromatase protein expression, which was localized primarily to the tail and midpiece of spermatozoa. Measurement of enzymatic activity using a sensitive (3)H(2)O aromatase assay revealed that activity was enhanced by the 2'-O-dibutyryl cAMP and completely inhibited in the presence of the specific aromatase inhibitor, letrozole. These results represent the first demonstration that human spermatozoa are a potential site of estrogen biosynthesis. The physiological relevance of estrogen synthesis in spermatozoa remains to be elucidated and opens a new area of investigation in male fertility.

Human Ejaculated Spermatozoa Contain Active P450 Aromatase

The generation of cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) knockout mice has raised new interest in the physiological role of estrogens in male reproduction. Testicular expression of P450arom, the enzyme that converts androgens into estrogens, has been shown in both somatic and germ cell types in several species, whereas in humans, testicular expression is confined to the somatic cells. The aim of this study was to determine whether P450arom is present in human ejaculated spermatozoa. Using RT-PCR and specific primers, we amplified the highly conserved helical, aromatic, and heme-binding sequences of the conventional human P450arom from RNA isolated from human spermatozoa. Employing a rabbit polyclonal antiserum directed against human placental P450arom, immunoblotting analysis demonstrated aromatase protein expression, which was localized primarily to the tail and midpiece of spermatozoa. Measurement of enzymatic activity using a sensitive 3H2O aromatase assay revealed that activity was enhanced by the 2′-O-dibutyryl cAMP and completely inhibited in the presence of the specific aromatase inhibitor, letrozole. These results represent the first demonstration that human spermatozoa are a potential site of estrogen biosynthesis. The physiological relevance of estrogen synthesis in spermatozoa remains to be elucidated and opens a new area of investigation in male fertility.

Angiotensin II and cyclic adenosine 3',5'-monophosphate induce human steroidogenic acute regulatory protein transcription through a common steroidogenic factor-1 element.

Steroidogenic acute regulatory protein (StAR) synthesis and steroidogenesis are stimulated by activation of divergent signaling pathways in the adrenal cortex. The two major physiological regulators of aldosterone synthesis in the adrenal zona glomerulosa are angiotensin II (AII) and extracellular K+, which both mediate an increase in intracellular calcium levels, although by distinct mechanisms. Previously, we demonstrated that increased mineralocorticoid synthesis by N6,2'-O-dibutyryl cAMP (Bt2cAMP), AII, and K+ treatment is paralleled by an increase in StAR protein in the H295R human adrenocortical cell line. We now show that StAR steady state messenger RNA levels are increased by Bt2cAMP and AII, but not by K+ or 12-O-tetradecanoylphorbol-13-acetate, treatment of H295R cells. Northern analysis detected two major transcripts of 1.7 and 2.7 kb present in H295R cells, with the most prominent effect of agonist treatment on induction of the 1.7-kb messenger RNA. Similarly, StAR promoter activity in transient transfections of H295R cells with a luciferase reporter gene driven by 1.3 kb of the human promoter was increased only by Bt2cAMP and AII treatment. 5'-Deletion analysis of the StAR promoter indicates that both the cAMP- and AII-responsive elements are within 150 bases of the transcriptional start site. Mutation of a steroidogenic factor-1/AdBP4 element localized at -95 abolishes both Bt2cAMP- and AII-induced luciferase activity in these transient transfection assays. Thus, transcriptional activation of the StAR gene by a steroidogenic factor-1-dependent mechanism may represent a common pathway for ACTH (protein kinase A) and AII action in stimulating steroid production in the adrenal fasciculata and glomerulosa.

Protective Effects of the Antiparkinsonian Drugs Talipexole and Pramipexole against 1-Methyl-4-phenylpyridinium-Induced Apoptotic Death in Human Neuroblastoma SH-SY5Y Cells

Treatment of human neuroblastoma SH-SY5Y cells with 1 mM 1-methyl-4-phenylpyridinium (MPP+) for 3 days induced production of reactive oxygen species (ROS), followed by caspase-3 activation, cleavage of poly(ADP-ribose) polymerase (PARP), and apoptotic cell death with DNA fragmentation and characteristic morphological changes (condensed chromatin and fragmented nuclei). Simultaneous treatment with 1 mM talipexole slightly inhibited the MPP+-induced ROS production and apoptotic cell death. In contrast, pretreatment with 1 mM talipexole for 4 days markedly protected the cells against MPP+-induced apoptosis. However, this protective effect might not be mediated by dopamine receptors. The talipexole pretreatment induced an increase in antiapoptotic Bcl-2 protein level but had no effect on levels of proapoptotic Bax, Bak, and Bad. It also inhibited MPP+-induced ROS production, p53 expression, and cleavages of caspase-3 and PARP. Similarly, pramipexole pretreatment increased Bcl-2 and inhibited MPP+-induced apoptosis. Although pretreatment with bromocriptine also had a protective effect against MPP+-induced apoptosis, it had no effect on the protein levels of Bcl-2 family members. On the other hand, N6,2'-O-dibutyryl cAMP or calphostin C induced a decreased Bcl-2 level and enhanced MPP+-induced cell death. These results suggest that talipexole has dual actions: (1) it directly scavenges ROS, affording slight protection against MPP+-induced apoptosis, and (2) it induces Bcl-2 expression, thereby affording more potent protection, if it is administrated before MPP+. Pramipexole has similar effects, whereas bromocriptine seems to exhibit the former but not the latter effect.

Production, Uptake, and Metabolic Effects of Cyclic AMP in the Bivascularly Perfused Rat Liver

Production, uptake, and metabolic effects of cyclic AMP (cAMP) were measured in the bivascularly perfused rat liver in anterograde and retrograde perfusion. Glucagon, cAMP, N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP were infused into the portal vein (anterograde perfusion), the hepatic vein (retrograde perfusion), or the hepatic artery (anterograde and retrograde perfusion) in order to reach different cell populations. The following results were obtained: (1) cAMP release caused by glucagon was directly proportional to the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; since the metabolic effects of glucagon were not proportional to the accessible cell spaces, this observation also implies a disproportion between cAMP release and metabolic effects of the hormone; (2) when cAMP and N 6,2′- O-dibutyryl cAMP were given to all liver cells (e.g. when infused into the portal vein), their metabolic effects were qualitatively and quantitatively the same and qualitatively equal to the effects of glucagon; (3) the changes caused by cAMP were a function of the cell spaces that can be reached via the hepatic artery in anterograde and retrograde perfusion; this behaviour contrasts markedly with that of glucagon, whose metabolic effects were practically independent of the accessible cell spaces; and (4) the effects of N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP were independent of the cell spaces that were accessible via the hepatic artery in anterograde and retrograde perfusion; in this respect their behaviour was equal to that of glucagon. It is apparent that exogenously added cAMP mimicked the metabolic effects of glucagon in the liver only when it was supplied to all liver cells. Since glucagon, N 6,2′- O-dibutyryl cAMP, and N 6-monobutyryl cAMP were able to produce a full response even when given to only 30% of the liver parenchyma, it was concluded that cAMP production under the stimulus of glucagon or in consequence of the metabolic transformation of N 6,2′- O-dibutyryl cAMP and N 6-monobutyryl cAMP occurs in a compartment to which exogenous cAMP has no access. cAMP generated within this compartment is possibly able to diffuse from cell to cell.

Cellular basis for follicle-stimulating hormone-stimulated calcium signaling in single rat Sertoli cells: possible dissociation from effects of adenosine 3',5'-monophosphate.

To study the cellular basis for FSH-stimulated dose-dependent graded increases in intracellular Ca2+ concentrations in populations of Sertoli cells, we investigated the effects of FSH on free Ca2+ ion concentrations ([Ca2+]i) in individual rat Sertoli cells using the Ca(2+)-sensitive dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca2+]i within 20-140 sec, with a peak level 2.7 +/- 0.9-fold greater than the basal value (mean +/- SEM; n = 8) lasting for 4-16 min. The amplitude and kinetics of the FSH-induced [Ca2+]i signal were not dose dependent. Instead, increasing doses of FSH recruited a higher percentage of responding cells. Chelation of extracellular Ca2+ or cotreatment with verapamil or cobalt abolished FSH-induced [Ca2+]i increases. Furthermore, in the presence of extracellular Mn2+, direct evidence for FSH-mediated Ca2+ influx was obtained from the quench of fura-2 fluorescence. Induced Ca2+ increases were mimicked by forskolin or protein kinase-A type I activators [8-(6-amino-hexyl)amino-cAMP and N6-benzoyl-cAMP (N6B)]. However, the cAMP analogs, 8-bromo-cAMP, N6,2'-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N6B), induced [Ca2+]i increases even in the absence of extracellular Ca2+, and the time course of the [Ca2+]i rise induced by cAMP analogs was more rapid than that induced by FSH. Similarly, the uninhibited rise in [Ca2+]i induced by FSH in pertussis toxin-pretreated Sertoli cells suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca2+]i. In summary, we demonstrate that FSH evokes sustained [Ca2+]i increases in single Sertoli cells in a nongraded fashion and recruits increasing numbers of responding cells in a dose-dependent fashion. We also provide explicit evidence that FSH induces Ca2+ influx. Mimicry of the FSH-induced [Ca2+]i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP and N6B; protein kinase-A type I activator] or forskolin suggests that Ca2+ may be part of a dual pathway of cAMP-initiated intracellular signaling.

Calcium influx in platelet activating factor-induced atrial natriuretic peptide release in rat cardiomyocytes.

Atrial natriuretic peptide (ANP) is released from the myocardium after the activation of protein kinase C and/or ischemia, events that are associated with an increase in platelet activating factor (PAF) production in this tissue. In this study we demonstrate that PAF, but not lyso-PAF, induces a concentration-dependent increase in ANP secretion in spontaneously beating neonatal rat cardiomyocytes, a response associated with increases in cellular adenosine 3',5'-cyclic monophosphate (cAMP) formation, calcium influx, and the mobilization of calcium from intracellular stores. cAMP formation and calcium influx appear to play major roles in PAF-induced ANP secretion in this system, insofar as PAF-induced ANP release was substantially reduced in the presence of the (R)-p-diastereoisomer of adenosine 3',5'-cyclic monophosphorothioate (10 microM), whereas both PAF-induced calcium influx and ANP secretion were abolished in the presence of the calcium channel antagonist nifedipine (0.1 microM). Consistent with these results, N6-2'-O-dibutyryl cAMP (DBcAMP, 10 microM) and/or forskolin (0.1 microM) simultaneously increased cAMP production, calcium influx, and ANP release in these cells, with both DBcAMP- and forskolin-induced ANP secretion being fully abolished in the presence of 0.1 microM nifedipine. Taken together, these results suggest that PAF, DBcAMP, and forskolin promote ANP secretion in spontaneously beating cardiomyocytes via the activation of a cAMP-dependent, nifedipine-sensitive myocardial calcium channel and that calcium influx is a major requirement for cAMP-induced ANP secretion in this system.

Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP.

Acetoxymethyl esters of alkyl or aryl phosphates can be prepared by reacting their trialkylammonium or silver salts with acetoxymethyl bromide. Because acetoxymethyl esters are rapidly cleaved intracellularly, they facilitate the delivery of organophosphates into the cytoplasm without puncturing or disruption of the plasma membrane. In addition, acylation of free hydroxyls, for example with butyryl groups, is useful both for synthetic convenience and increased hydrophobicity of the permeant derivatives. The highly polar intracellular messengers cAMP and cGMP were thus converted into uncharged membrane-permeant derivatives. Extracellularly applied N6,2'-O-dibutyryl cAMP acetoxymethyl ester (Bt2cAMP/AM) is shown to simulate intracellular cAMP in three model systems, namely dissociation of cAMP-dependent protein kinase in fibroblasts, activation of Cl- secretion of monolayers of the human colon epithelial cell line T84, and dispersion of pigment granules in angel fish melanophores. Bt2cAMP/AM is effective at concentrations two or three orders of magnitude less than those required for commonly used membrane-permeant cAMP derivatives such as Bt2cAMP, 8-Br-cAMP, and 8-pCPT-cAMP lacking the acetoxymethyl ester. This methodology should be of general utility for the intracellular delivery of phosphate-containing second messengers.

Vasoactive intestinal peptide (VIP) inhibits substrate adherence capacity of rat peritoneal macrophages by a mechanism that involves cAMP.

In this study, vasoactive intestinal peptide (VIP) is shown to inhibit substrate adherence capacity of rat peritoneal macrophages. The inhibitory response occurred in the 0.1-1,000 nM range of VIP concentrations and it was a time-dependent process. At 15 min, half maximal inhibition (IC50) was obtained at 0.37 +/- 0.26 nM and maximal inhibition (53.8%) at 10(-6) M VIP. The inhibitory effect of VIP was correlated with the stimulation by this peptide of cyclic AMP (cAMP) production in rat peritoneal macrophages. Moreover, agents that inhibited VIP-stimulated cAMP production, such as the VIP-antagonist [4-Cl-D-Phe6, Leu17]-VIP and somatostatin, also decreased the inhibitory effect of VIP on substrate adherence capacity of macrophages. On the contrary, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the lipid-soluble derivative of cAMP N6,2'-O-dibutyryl cAMP (Bu-cAMP) inhibited the adherence of macrophages to substrate and potentiated the inhibitory action of VIP. These results demonstrate that VIP inhibits substrate adherence capacity of rat peritoneal macrophages by a mechanism that involves cAMP, and show, for the first time, an action of VIP on the function of peritoneal macrophages.

Differential regulation of multiple c- erbA expression by thyrotropin, insulin and insulin-like growth factor I in rat thyroid FRTL-5 cells

Regulation of multiple c- erbA gene expression was studied in rat thyroid FRTL-5 cells. Two species of erbA α (α1 and α2) mRNA and one species of erbA β (β1) mRNA were identified by Northern blot analysis. Withdrawal of thyrotropin (TSH), insulin and serum from the complete medium resulted in an increase in α1 and α2 erbA mRNA levels without altering the level of erbA β1 mRNA. Readdition of TSH, N 6,2′- O-dibutyryl cAMP or forskolin caused a transient reduction of α1 and α2 mRNA levels (75–90%) at 3–12 h. The α1 and α2 mRNA levels were restored at 24 h. The TSH action was dose-dependent showing the half-maximal effect at around 10 −9 M. Readdition of TSH did not show any effect on β1 mRNA level. The action of TSH was not dependent on ongoing protein synthesis but required ongoing transcription. Inhibitors of thyroid hormone biosynthesis, propylthiouracil and methylmercaptoimidazole, did not show any effect on TSH action. Readdition of insulin or insulin-like growth factor I (IGF-I) caused a dose-dependent reduction of α1 and α2 mRNA levels without any effect on β1 mRNA level. Their action was slower than TSH and persistent. The actions of insulin and IGF-I were dependent on both ongoing translation and transcription. These results indicate that TSH and insulin/IGF-I reduce levels of c- erbA α1 and α2 mRNA possibly by two distinct mechanisms without altering c- erbA β1 mRNA level in FRTL-5 cells.

Multiple changes induced by cholera toxin contribute to the stimulation of aerobic lactate production in rat-1 fibroblasts

Exposure of rat-1 fibroblasts to cholera toxin increased aerobic lactate production 3- to 8-fold with maximal stimulation observed between 1 and 2 h at a concentration of 1–2 μg/ml. Concomitant with this change was a 10- to 40-fold elevation in the intracellular concentration of cAMP. The cell permeable cAMP analogue, N 6 ,2′- O-dibutyryl cAMP and the cyclic nucleotide phosphodiesterase inhibitor RO-20-1724 also increased lactate production and intracellular cAMP levels, although less effectively. Cholera toxin and dibutyryl cAMP induced a 2- to 3-fold elevation of intracellular fructose 2,6-bisphosphate and 2- to 3-fold increases in both 3- O-methylglucose and inorganic phosphate transport. A survey of five additional cell lines revealed striking variabilities in their individual responses to cholera toxin and dibutyryl cAMP. All were observed to be considerably less sensitive to either agent than rat-1 cells. These data suggest that a cooperative effect involving multiple parameters may be responsible for the observed increases in aerobic lactate production in response to cAMP and that these parameters may vary significantly among cell lines.

Effects of Thyrotropin, Carbachol, and Protein Kinase-C Stimulators on Glucose Transport and Glucose Oxidation by Primary Cultures of Dog Thyroid Cells*

Thyroid glucose metabolism can provide NADPH and H2O2 for thyroid hormone synthesis. Several agents stimulate glucose oxidation in thyroid slices, but little is known about glucose transport in this tissue. In the present study, various thyroid stimulators were tested on glucose transport and oxidation using primary cultures of dog thyroid cells. After preincubating the cells with the agonists, glucose uptake was measured by adding 2-deoxy-D-[1-3H]glucose [( 3H]2-DOG) for 5 min, and glucose oxidation was assessed during a single 60-min incubation with agonist and D-[1-14C]glucose. TSH (0.1-10 mU/ml), 12-O-tetradecanoyl phorbol-13-acetate (TPA; 10(-8)-10(-6) M), and carbachol 10(-6)-10(-2) M) stimulated [3H]2-DOG transport and glucose oxidation in a dose-dependent manner. The effect of TSH appears to be mediated by cAMP, since N6,2'-O-dibutyryl cAMP, 8-bromo-cAMP, cholera toxin, and isobutylmethylxanthine also stimulated [3H]2-DOG transport. Norepinephrine, which had no effect by itself on either transport or oxidation, inhibited TSH stimulation of [3H]2-DOG transport via an alpha 2-adrenergic receptor. The mechanism of the TPA and carbachol effect does not involve cAMP. A combination of maximal amounts of TSH or bromo-cAMP and carbachol or TPA produced additive effects on transport, while addition of TPA with carbachol produced no such additive effect. Kinetic analysis of 2-DOG transport indicated that all three agonists reduced the Km and increased the maximum velocity. Basal 2-DOG transport was increased in Ca2+-free medium, with or without EGTA, or in the presence of calcium channel blockers such as La3+ or Mn2+. In the presence of such increased basal glucose transport, TSH further stimulated it when Mn2+ was used, had no effect in Ca2+-free buffer plus EGTA, and caused an inhibition with La3+. Such inhibition was not caused when N6,2'-O-dibutyryl cAMP was used in the presence of La3+. Carbachol and TPA did not stimulate transport when the Ca2+ channel blockers were used, but a small increase was seen in Ca2+-free buffer containing EGTA. TSH stimulation of cAMP production was also diminished in the presence of La3+, but enhanced in the presence of Mn2+. The calmodulin inhibitor W-7 and the intracellular Ca2+ blocker 8-N,N-diethylamino octyl-3,4,5-trimethoxybenzoate hydrochloride diminished the stimulation of [3H]2-DOG transport and glucose oxidation induced by TSH, carbachol, and TPA. These data indicate that thyroid glucose transport and glucose oxidation are regulated by both cAMP-dependent and cAMP-independent systems.

Desensitization of β-receptors on primary astrocyte cultures by norepinephrine but not by tricyclic antidepressants

Primary astrocyte cultures from neonatal rat cerebral hemispheres were treated chronically for up to 3 weeks with the tricyclic antidepressants amitryptyline (AMT) or desipramine (DMI), or acutely with AMT and DMI added at the same time as the agonist, norepinephrine (NE). AMT and DMI were added at concentrations from 10 −9 to 10 −5 M. Both types of treatment did not decrease the increase in cyclic AMP (cAMP) content of these cells in response to a 10 min exposure to 10 −5 M NE. Chronic exposure to the antidepressants also did not affect stimulation of cAMP by isoproterenol (iso) in both rat and mouse primary astrocyte cultures. In contrast to the lack of effect of the tricyclic antidepressants pretreatment of the cultures with 10 −5 M NE resulted in total inhibition of the cAMP response after 2 h, with a 50% decrease occurring in about 45 min. This is similar to the agonist-induced desensitization of the β-receptor-adenylate cyclase system seen in many other cells. This effect could, in part, be a direct response to increased intracellular cAMP since pretreatment with 0.25 and 1.0 mM N 6-2′-O-dibutyryl cAMP (DBcAMP) also resulted in total inhibition of the cAMP response after 4 h. Receptor labelling experiments using [ 125I]cyanopindolol showed no decreases in apparent binding sites up to 3 h after exposure to 10 −5 M norepinephrine, suggesting that the rapid desensitization of the cAMP response was primarily due to an uncoupling of the receptor from the adenyl cyclase. These results suggest that if the desensitization of the β-receptor mediated cAMP increase observed after chronic treatment with antidepressants in vivo is partly due to an effect on β-receptors in astrocytes it is more likely a secondary response, perhaps due to a delayed increase in extracellular NE levels.