Nicotine-induced up regulation of α4ß2 neuronal nicotinic receptors is mediated by the protein kinase C-dependent phosphorylation of α4 subunits.

Sustained exposure to nicotine is well known to increase the cell surface density of α4ß2* neuronal nicotinic receptors both in vivo and in vitro, but the cellular mechanisms mediating this effect are equivocal. Using a pharmacological approach to investigate the effects of nicotine on receptor subunit expression and phosphorylation in SH-EP1 cells expressing human α4 and ß2 nicotinic receptor subunits, we have demonstrated that incubation with nicotine for 24 h increased the expression of immature and mature forms of both α4 and ß2 subunits in a concentration-dependent manner, and that inhibition of protein kinase C (PKC), but not cAMP-dependent protein kinase (PKA) inhibited the nicotine-induced increased expression of subunits. Incubation of cells with nicotine for 24 h also increased the phosphorylation of immature forms of α4 subunits similar to that induced by activation of either PKC or PKA. When cells were preincubated with nicotine, the PKC-mediated increased phosphorylation was inhibited; the PKA-mediated phosphorylation was unaltered. The phosphopeptide maps for immature α4 subunits following nicotine exposure or PKC activation were identical, and phosphoamino acid analyses indicated phosphorylation on serine residues only. Results indicate that nicotine-induced up regulation of α4ß2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, promoting subunit assembly and receptor maturation. Because up regulation of these receptors has been implicated to mediate tolerance, locomotor sensitization and addiction to nicotine, results identify a potential new target for modulating the effects of nicotine on the brain.

Combined thiazolidinedione-metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle.

AIMS/HYPOTHESES: Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione-metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. METHODS: Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione-metformin therapy for activation of muscle aPKC and PKBbeta and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic-hyperinsulinaemic clamp studies conducted with sub-maximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. RESULTS: Following combined thiazolidinedione-metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBbeta, were observed. Increases in PKBbeta enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. CONCLUSIONS/INTERPRETATION: Combined thiazolidinedione-metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBbeta in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.

Cyclic AMP-dependent protein kinase A and protein kinase C phosphorylate alpha4beta2 nicotinic receptor subunits at distinct stages of receptor formation and maturation.

Neuronal nicotinic receptor alpha4 subunits associated with nicotinic alpha4beta2 receptors are phosphorylated by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human alpha4 and/or beta2 subunits were incubated with (32)Pi, and PKA or PKC was activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing alpha4beta2 receptors or only alpha4 subunits were used to identify free alpha4 subunits, and alpha4 subunits present in immature alpha4beta2 complexes and mature alpha4beta2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of alpha4 subunits associated with mature pentamers was three times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free alpha4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature alpha4beta2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated beta2 subunits associated with mature alpha4beta2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation alpha4beta2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of human alpha4beta2 receptors.

The effects of atrazine on the sexual maturation of female rats.

The mammalian hazard assessment of the herbicide atrazine (ATR) has focused on the induction of mammary tumors and accelerated reproductive aging of adult rats, and the relationship of these effects to the inhibition of leutinizing hormone (LH) release from the pituitary, an effect itself caused by inhibition of GnRH signaling by the adult rat hypothalamus. In earlier studies, Laws et al. (Toxicol. Sci., 58, 366-376, 2000) demonstrated a delay in female rat sexual maturation induced by ATR, effects that could equally have been caused by inhibition of hypothalamic GnRH release. The present studies were designed to compare the doses that interfere with GnRH signaling seen in previous studies in adult Sprague-Dawley (SD) rats (LH surge suppression) with doses that impair GnRH signaling in peripubertal rats, as indicated by delayed sexual maturation. The studies evaluated the effects of ATR treatment on the timing of uterine growth and vaginal opening (VO) in peripubertal female Wistar (Alderley Park, AP) and SD rats. Doses of 10, 30, and 100 mg/kg ATR were administered daily from postnatal day (pnd) 21 to up to pnd 46. Determinations of uterine weight were made at pnd 30, 33, 43 (AP), and 46 (SD) and the timing of VO was also assessed in the last two of these experiments. The centrally acting GnRH antagonist Antarelix (ANT) was used as a positive control agent as it has previously been shown to prevent uterine growth and to delay VO in peripubertal AP rats. Uterine growth and VO were completely prevented in AP rats exposed to ANT. Uterine growth was delayed at pnd 30 and 33 in AP rats exposed to 100 mg/kg ATR, but this growth inhibition had been overcome by pnd 43. VO was significantly delayed in AP rats for the 100 mg/kg ATR dose. By pnd 46, VO was significantly delayed in SD rats exposed to both 30 and 100 mg/kg ATR, but uterine weights were unaffected by that time (as for AP rats). It is concluded that the no-effect level for the effects of ATR on sexually immature rats (10 mg/kg in SD; 30 mg/kg AP) is approximately the same as reported previously by Laws et al. in peripubertal Wistar rats (25 mg/kg). However, the no-effect level in peripubertal female SD rats is nearly an order of magnitude greater than the no-observed effect level observed in female SD rats fed ATR for 6 months (1.8 mg/kg) where LH suppression was used as an indicator of effect on the pituitary/hypothalamic axis (USEPA, Atrazine-DACT Fourth Report of the Hazard Identification and Review Committee, April 5, 2002). These results support the conclusion that the pituitary/hypothalamic axis in peripubertal female SD rats is less sensitive than that in adult female SD rats.

Characterization of human alpha4beta2 neuronal nicotinic receptors stably expressed in SH-EP1 cells.

These studies characterized human alpha4beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EPI-halpha4beta2 cells were functional, as determined by increases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC50 of 2.4 microM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-halpha4beta2 cells expressed a single class of high affinity binding sites for [3H]cytisine with a Kd of 0.63 +/- 0.08 nM and a Bmax of 6,797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-dependent effect with an EC50, of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours. Results indicate that SH-EPI-halpha4beta2 cells may be a good model system to study regulation of human alpha4beta2 receptors, the most abundant nicotinic receptor subtype in brain.

An assessment of fertility in male workers exposed to molinate.

Molinate is a thiocarbamate herbicide used for weed control in rice fields. Since the late 1970s, findings from reproductive toxicology studies of rats have led to concern that molinate might affect human male fertility. Semen samples were collected from 272 formulation and production workers at three US plants. The samples were collected at the end of four alternate monitoring periods of either high or low exposure to molinate. In addition, 222 married workers provided reproductive-history information. Workers' mean exposures to molinate during the monitoring periods ranged from 12.7 micrograms/m3 to 210.9 micrograms/m3. There was no evidence that sperm and serum hormone levels were related to exposure to molinate before the study or exposure during the four monitoring periods. There was also no evidence of a molinate exposure-related effect on the ratio of observed to expected births.

Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans?

The purpose of the workshop "Do Peroxisome Proliferating Compounds Pose a Hepatocarcinogenic Hazard to Humans?" was to provide a review of the current state of the science on the relationship between peroxisome proliferation and hepatocarcinogenesis. There has been much debate regarding the mechanism by which peroxisome proliferators may induce liver tumors in rats and mice and whether these events occur in humans. A primary goal of the workshop was to determine where consensus might be reached regarding the interpretation of these data relative to the assessment of potential human risks. A core set of biochemical and cellular events has been identified in the rodent strains that are susceptible to the hepatocarcinogenic effects of peroxisome proliferators, including peroxisome proliferation, increases in fatty acyl-CoA oxidase levels, microsomal fatty acid oxidation, excess production of hydrogen peroxide, increases in rates of cell proliferation, and expression and activation of the alpha subtype of the peroxisome proliferator-activated receptor (PPAR-alpha). Such effects have not been identified clinically in liver biopsies from humans exposed to peroxisome proliferators or in in vitro studies with human hepatocytes, although PPAR-alpha is expressed at a very low level in human liver. Consensus was reached regarding the significant intermediary roles of cell proliferation and PPAR-alpha receptor expression and activation in tumor formation. Information considered necessary for characterizing a compound as a peroxisome proliferating hepatocarcinogen include hepatomegaly, enhanced cell proliferation, and an increase in hepatic acyl-CoA oxidase and/or palmitoyl-CoA oxidation levels. Given the lack of genotoxic potential of most peroxisome proliferating agents, and since humans appear likely to be refractive or insensitive to the tumorigenic response, risk assessments based on tumor data may not be appropriate. However, nontumor data on intermediate endpoints would provide appropriate toxicological endpoints to determine a point of departure such as the LED10 or NOAEL which would be the basis for a margin-of-exposure (MOE) risk assessment approach. Pertinent factors to be considered in the MOE evaluation would include the slope of the dose-response curve at the point of departure, the background exposure levels, and variability in the human response.

Testis-specific RT7 protein localizes to the sperm tail and associates with itself.

The RT7 gene recently cloned by us is expressed as an abundant RNA in round spermatids. In vitro transcription-translation showed that the RT7 gene encodes a protein of 26-27 kDa on SDS-polyacrylamide gels. Here we report the development of monoclonal antibodies (mAbs) raised against a peptide from the predicted N-terminal amphipathic alpha-helix of the rat RT7 protein. All mAbs recognize RT7 protein or N-terminal parts of it. To investigate RT7 in vivo, mAbs were used in immunofluorescence microscopy and confocal laser immunofluorescence microscopy. Several mAbs recognize RT7 protein in elongating spermatids: the observed staining pattern suggests a nonrandom localization in these cells. Two mAbs recognize the protein only in sperm tails. Using co-immunoprecipitation assays, we found that RT7 can form stable complexes with itself that are associated through a region located in the N-terminal half of RT7. Our results identify the RT7 protein as a major sperm tail component and suggest that it may be a structural component of sperm tail outer dense fibers (ODF).

Case management and plasma half-life in a case of brodifacoum poisoning.

Brodifacoum is a readily available, second-generation anticoagulant rodenticide (superwarfarin) that causes extended depletion of vitamin K1-dependent clotting factors. Brodifacoum ingestions are being reported with increasing frequency. For the first time, we compare plasma brodifacoum concentration to prothrombin levels over time in a case of brodifacoum poisoning. Brodifacoum was eliminated according to a two-compartment model, with an initial half-life of 0.75 days and a terminal half-life of 24.2 days. On admission, the brodifacoum level was 731 micrograms/L and the patient suffered severe urinary tract hemorrhage, requiring transfusion of blood products. Persistently increased prothrombin times necessitated treatment with phytonadione up to 80 mg/d for 4 months, until the brodifacoum level reached 10 micrograms/L. These data may help project the duration of phytonadione treatment required in future cases of brodifacoum poisoning. Superwarfarin exposure must be suspected in an otherwise unexplained vitamin K1-deficient coagulopathy.

Biochemical and morphological studies of ammonium perfluorooctanoate-induced hepatomegaly and peroxisome proliferation.

Ammonium perfluorooctanoate (APFO) is known to induce a striking hepatomegaly in rats. The purpose of these studies was to determine the causes of the hepatomegaly and compare the effect to other liver-enlarging compounds. Since the total hepatic DNA content was similar in control and APFO-treated rats, the hepatomegaly represented a hypertrophic rather than a hyperplastic response. The cytochrome P-450 content and activity of benzphetamine N-demethylase increased in the livers of APFO-treated rats, indicating the proliferation of the smooth endoplasmic reticulum. In contrast to the membrane-bound enzymes, the soluble enzymes glutathione S-transferase and UDPglucuronyltransferase were unaffected by APFO treatment. The activity of carnitine acetyltransferase was disproportionately increased relative to carnitine palmitoyltransferase in the livers of APFO vs that in control rats, confirming the predominant proliferation of peroxisomes vs that of mitochondria. Morphological studies confirmed the proliferation of the endoplasmic reticulum, mitochondria, and peroxisomes in the livers of APFO-treated rats. In contrast to many other peroxisome proliferating agents, APFO did not possess hypolipidemic activity.