Reduced CHRNA7 expression in C3H mice is associated with increases in hippocampal parvalbumin and glutamate decarboxylase-67 (GAD67) as well as altered levels of GABA(A) receptor subunits.

Decreased expression of CHRNA7, the gene encoding the α7(∗) subtype of nicotinic receptor, may contribute to the cognitive dysfunction observed in schizophrenia by disrupting the inhibitory/excitatory balance in the hippocampus. C3H mice with reduced Chrna7 expression have significant reductions in hippocampal α7(∗) receptor density, deficits in hippocampal auditory gating, increased hippocampal activity as well as significant decreases in hippocampal glutamate decarboxylase-65 (GAD65) and γ-aminobutyric acid-A (GABAA) receptor levels. The current study investigated whether altered Chrna7 expression is associated with changes in the levels of parvalbumin, GAD67 and/or GABAA receptor subunits in the hippocampus from male and female C3H Chrna7 wildtype, C3H Chrna7 heterozygous and C3H Chrna7 knockout (KO) mice using quantitative Western immunoblotting. Reduced Chrna7 expression was associated with significant increases in hippocampal parvalbumin and GAD67 and with complex alterations in GABAA receptor subunits. A decrease in α3 subunit protein was seen in both female C3H Chrna7 Het and KO mice while a decrease in α4 subunit protein was also detected in C3H Chrna7 KO mice with no sex difference. In contrast, an increase in δ subunit protein was observed in C3H Chrna7 Het mice while a decrease in this subunit was observed in C3H Chrna7 KO mice, with δ subunit protein levels being greater in males than in females. Finally, an increase in γ2 subunit protein was found in C3H Chrna7 KO mice with the levels of this subunit again being greater in males than in females. The increases in hippocampal parvalbumin and GAD67 observed in C3H Chrna7 mice are contrary to reports of reductions in these proteins in the postmortem hippocampus from schizophrenic individuals. We hypothesize that the disparate results may occur because of the influence of factors other than CHRNA7 that have been found to be abnormal in schizophrenia.

Quantification of major classes of Xenopus phospholipids by high performance liquid chromatography with evaporative light scattering detection.

Lipid signaling has become a major research area of cell biology and there is a need for methods that accurately and easily measure substrates and products of lipases involved in cell signaling. In this report, we provide new methodology for separation of more than 10 lipids in one chromatographic run by high pressure liquid chromatography (HPLC) and detection with an evaporative light scattering detector (ELSD). There is no significant loss of sphingomyelin and no large baseline change, no peak obscures another, and acidic phospholipids are cleanly separated. We have optimized the procedure for a two-pump HPLC, an inexpensive silica column without the use of a column heater jacket and for low grade nitrogen. An application of the procedure separates lipids from Xenopus laevis cells. These cells are commonly used in the study of various lipid signaling paths in cell division, fertilization, and after expression of exogenous membrane receptors.

Nongenomic action of progesterone: activation of Xenopus oocyte phospholipase C through a plasma membrane-associated tyrosine kinase.

Using a plasma membrane-cortex preparation (wherein the nucleus and >90% of the total cell protein are removed), progesterone stimulated tyrosine kinase activity that stimulated phospholipase C. Although it has been known for over 20 yr that progesterone acts at the plasma membrane of Xenopus oocytes to induce oocyte maturation, this is the first report that progesterone stimulates this tyrosine kinase activity that is associated with the oocyte plasma membrane and cortex. A tyrosine kinase inhibitor (tyrphostin B46) inhibited steroid stimulation of tyrosine kinase and phospholipase C (PLC) activities, but did not block lipase C stimulation by G protein activators. A fusion protein that contains tandem N- and C-terminal SH2 domains of PLCgamma also blocked progesterone stimulation of PLC (a fusion protein with the SH2 domain from Shc was ineffective). Lowering the Ca2+ concentration in the medium inhibited progesterone, but not guanosine 5'-O-(3-thiotriphosphate), stimulation of PLC, and the effects of progesterone and a G protein agonist were additive. However, neither progesterone nor insulin increased phosphotyrosine on PLCgamma. To evaluate another tyrosine kinase path involving phosphatidylinositol 3-kinase, we added wortmannin to our membrane preparation, but wortmannin did not inhibit progesterone's ability to activate PLC.

Stimulation of the intracellular portion of the human insulin receptor by the antidiabetic drug metformin.

Our prior work suggested that the antidiabetic metformin must enter the cell to act and that the drug stimulates tyrosine kinase activity. We now report that therapeutic concentrations (approximately 1 microg/mL) of metformin stimulated the tyrosine kinase activity of the intracellular portion of the beta-subunit of the human insulin receptor (IPbetaIRK), the intracellular portion of the epidermal growth factor receptor and pp60-src, but not cAMP-dependent protein kinase. A derivative of metformin unable to lower glucose was ineffective in stimulating IPbetaIRK. Two derivatives more effective than metformin in patients were also more effective than metformin in stimulating IPbetaIRK. Higher levels (10-100 microg/mL) of metformin or methylglyoxyl bis(guanylhydrazone) inhibited the tyrosine kinases, and this inhibition may be responsible for the ability of these two drugs to block cell proliferation.

sn-1,2-diacylglycerol and choline increase after fertilization in Xenopus laevis.

sn-1,2-Diacylglycerol (DAG) mass and translocation of protein kinase C alpha and beta to a membrane fraction increased approximately 7 min after insemination of Xenopus laevis eggs. The DAG mass increase of 48 pmol (from 62 to 110 pmol/cell) was greater than that for inositol 1,4,5-trisphosphate (IP3; an increase of approximately 170 fmol or approximately 280-fold smaller than the DAG increase), and DAG peaks approximately 5 min after IP3. Choline mass (a measure of phosphatidyl choline-specific phospholipase D) also peaked before DAG and the choline increase (134 pmol/cell) was greater than that of DAG. There was no detectable change in phosphocholine mass (a measure of phosphatidylcholine-specific phospholipase C). During first cleavage, DAG decreased, PKC translocation was low, and choline increased and peaked (whereas published work shows an increase in IP3 mass). Artificial elevation of intracellular calcium ([Ca2+]i) increased DAG levels but prevention of the [Ca2+]i increase after fertilization did not block DAG production. Thus, sperm stimulate production of DAG and choline through [Ca2+]i-independent and [Ca2+]i-dependent paths.

The antidiabetic drug metformin elevates receptor tyrosine kinase activity and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

Although metformin is an important antidiabetic, its mechanism of action is still unknown. To study its mechanism, we examined metformin stimulation of insulin action on the Xenopus oocyte. Similar to therapeutic concentrations, maximal stimulation of insulin-induced meiotic cell division was achieved at about 1-10 microg/ml (or 7.7-77 /microM) metformin. An equivalent concentration of metformin was required to elevate receptor tyrosine kinase activity (in whole cells or a membrane-cortex preparation) and, through this tyrosine kinase activation, inositol 1,4,5-trisphosphate (IP3) production. With whole cells, the preincubation time for metformin stimulation of insulin action (approximately 1 h) was equivalent to the time required for metformin to maximize tyrosine phosphorylation and raise IP3, levels. With the membrane-cortex preparation, metformin was active within minutes; thus, metformin may act at an intracellular site. Since metformin can increase IP3, mass, we prevented elevation of calcium by prior microinjection of a calcium chelator or heparin (a drug that inhibits IP3 binding to the IP3 receptor). Both the chelator and heparin blocked metformin stimulation of insulin action on whole cells. Since microinjection of IP3, also stimulates insulin action, metformin may stimulate insulin action by elevation of intracellular calcium in addition to activation of the receptor tyrosine kinase.

Sperm increase inositol 1,4,5-trisphosphate mass in Xenopus laevis eggs preinjected with calcium buffers or heparin.

Although it is known that all fertilization events are due to elevated intracellular calcium ([Ca2+]i), it is not known how sperm induce an increase in zygote [Ca2+]i. We report that sperm can increase inositol 1,4,5-trisphosphate in Xenopus laevis eggs that have been preinjected with calcium buffers to prevent the increase in [Ca2+]i (both the initial increase and the subsequent wave) after fertilization. After buffering [Ca2+]i to levels well below basal, IP3 production was not blocked, whereas IP3 metabolism may be inhibited. Also, heparin (an inhibitor of inositol 1,4,5-trisphosphate action and the fertilization response) does not prevent a normal increase in IP3 after fertilization. Conditions that produce various levels of polyspermy are associated with IP3 increases similar to those noted after monospermic fertilization. These data suggest a specific order of fertilization events: sperm utilize an initial production of inositol 1,4,5-trisphosphate to produce the [Ca2+]i increase at fertilization.

Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis.

After fertilization in Xenopus laevis, inositol 1,4,5-trisphosphate (IP3) mass increased from 53 to 261 fmol/cell and returned to near basal by 10 min after insemination. IP3 was also elevated over control egg levels during first mitosis and first cleavage. Because IP3 levels and the fertilization calcium wave decline at about the same time and because calcium ionophore or pricking the egg increased IP3, the fertilization calcium wave may be due to calcium-induced IP3 production. In addition, the onset of sperm motility was associated with an increase, whereas the acrosomal reaction was accompanied by a decrease in IP3 mass. Combining our published data with this report, the first chronology of the levels of IP3 from the induction of meiosis (maturation) through fertilization and cleavage in one cellular system is summarized. These data suggest an in vivo dose response for IP3 and calcium release. A small (17 fmol/cell) IP3 change during the induction of meiosis may not be associated with a calcium change. Larger IP3 changes at cleavage (40 fmol/cell) and mitosis (125 fmol/cell) are associated with localized small calcium increases, whereas the largest IP3 change (208 fmol/cell) is associated with the large calcium increase at fertilization.

Protein kinase C initially inhibits the induction of meiotic cell division in Xenopus oocytes.

We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and in vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.

Insulin and progesterone increase 32PO4-labeling of phospholipids and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

After a 4-6 h induction period, insulin or progesterone induces Xenopus oocytes to enter prophase of meiosis. During the period of induction, both insulin and progesterone induced an increase in 32PO4 labeling of phosphatidylcholine and phosphatidylinositol. Through a mass assay, we found that insulin and progesterone increase inositol 1,4,5-trisphosphate (IP3) at about 15-30 s, 15 min and at about 2-3 h (0.5 GVBD50) after hormone addition. Since IP3 increases were small (from a basal of 66 to 104 nM), the results agree with prior conclusions that progesterone does not induce a large, cytosolic calcium elevation. Insulin is probably acting through the insulin-like growth factor-1 receptor as insulin concentrations greater than about 50 nM are required to increase IP3.

Insulin-like growth factor 1, insulin, and progesterone induce early and late increases in Xenopus oocyte sn-1,2-diacylglycerol levels before meiotic cell division.

After a 3 to 6 hour incubation, addition of progesterone (the most effective), insulin-like growth factor 1 (IGF-1; the second most effective), or insulin induces meiotic cell division in Xenopus oocytes. Measurement of an endogenous activator of protein kinase C, sn-1,2-diacylglycerol (DAG), by an enzymatic method recording mass demonstrates that all three hormones alter DAG levels. Five seconds after addition, only progesterone transiently reduces DAG levels by about 25%. At 15 minutes after addition, all three hormones produce a peak of DAG (115% to 160% of control values), with the more effective hormones producing a larger increase in DAG. Insulin produces the smallest DAG increase, but the DAG release is longer lasting. Finally, all three hormones induce a second peak in DAG levels just before white spot appearance (at 0.85 GVBD50, where 1.0 GVBD50 is when 50% of the cells have divided). With these data and since an activator of protein kinase C, a phorbol ester, has been found to induce meiosis, the kinase may play a role in early proliferative events at the plasma membrane and in late events at the nucleus.

Microinjection of inositol 1,2-(cyclic)-4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium.

Inositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium-regulated chloride channels but do not have a calcium-induced calcium release system. Microinjection of inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,2-(cyclic)-4,5-trisphosphate (cIP3), inositol 1,4,5-trisphosphate (IP3), or inositol 4,5-bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1-phosphate (IP1), inositol 1,3,4,5,6-pentakisphosphate (IP5), inositol 1,4-bisphosphate, or inositol 3,4-bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half-maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono- or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long-term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 much greater than IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate.

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes.

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.

Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes.

An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mare's serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis.

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis.

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.