Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Sphingosine 1-phosphate and control of vascular tone

Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC(50) 10 +/- 3 nM); maximal vasodilation (55 +/- 8%) is seen approximately 2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOS(null) mice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor N(omega)-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K(+) channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser(1179). Maximum S1P-induced eNOS Ser(1179) phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser(1179) dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.     

Effects of thiol antioxidant on reduced nicotinamide adenine dinucleotide phosphate oxidase in hypertensive Dahl salt-sensitive rats

Recent studies implicate of reactive oxygen species (ROS) in hypertension; however, whether reactive oxygen species promote hypertensive derangements is not fully clear. We thus investigated the effects of an antioxidant, N-acetyl-L-cysteine, on hypertensive Dahl salt-sensitive rats. High-salt intake for 4 weeks markedly elevated systolic arterial pressure, urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the enzyme activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with the elevated expression of its subunits gp91phox and p47phox at the levels of mRNA and protein. Supplement with N-acetyl-L-cysteine reduced the increase in systolic arterial pressure and counteracted the elevation of urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the increases in NADPH oxidase activity/expression in high-salt-loaded Dahl salt-sensitive rats. N-acetyl-L-cysteine supplement ameliorated plasma and urinary levels of thromboxane B(2) (an end metabolite of thromboxane A(2)), associated with improvement of both the abnormal contraction and the impaired nitric oxide-dependent relaxation in renal arteries. These results revealed that oxidative stress mediates hypertensive changes in Dahl salt-sensitive rats, because thiol antioxidant N-acetyl-L-cysteine attenuated the augmentation of local ROS production by diminishing the elevation of NADPH oxidase expression and ameliorated renal/vascular hypertensive changes.     

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.     

Suspension culture of drosophila cells employing a gyratory shaker

Summary To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM₂ and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and grew normally. Lower speed of gyration caused adhesion of the cells to a substratum. Furthermore, size of the culture vessels was found to affect the pattern of cell growth. Five- or 10-ml Erlenmeyer flasks gave satisfactory results, but the growth curves in 30-ml flasks differed from flask to flask and the saturation level was lower. Besides, the growth curves in the latter case were quite different depending on the volume of the medium. A preliminary experiment showed that the type of flask might affect the pattern of a growth curve. Initial cell densities has to be more than 6×10⁴ cells per ml. Lower densities resulted in the longer doubling time or no increase in the cell number. Therefore the following conditions are recommended as a standard for gyration culture ofD. melanogaster cell, GM₂: speed of gyration, 180 rpm; culture vessel, 5- or 10-ml Erlenmeyer flask of a certain type; initial cell density, 1 to 5×10⁵ per ml. Both D20 and modified Schneider’s medium could be utilized as the medium.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Induction of Functional Interleukin-2 Receptor in Mouse Microglia

Abstract: Interleukin (IL)-2, initially discovered for its mitogenic activity on T cells, also acts on monocytes, resulting in the activation of cytokine production, superoxide production, and tumoricidal activity. Because severe brain damage was observed in IL-2-transgenic mice, this cytokine may have some influence(s) on the cells of the CNS. We investigated IL-2 receptor-bearing cells in the CNS and found that activated microglia expressed α-chain mRNA and immunoreactive IL-2 receptor β-chain protein in culture. Although microglia did not express IL-2 receptors under normal culture conditions, they were induced to express these receptors by lipopolysaccharide (LPS) in a time-dependent manner. The IL-2 receptors were found to be functional because the viability and growth activity of LPS-treated microglia, but not untreated controls, increased in response to recombinant mouse IL-2 as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay and bromodeoxyuridine uptake experiment, respectively. These effects of recombinant IL-2 were blocked by pretreatment with anti-mouse IL-2 receptor β-chain antibody. Our findings suggest that activated microglia in the CNS can respond to this T cell-derived factor regulating their growth, which may be an important mechanism of communication between nervous and immune systems in physiological and pathological conditions.     

Notch2 expression negatively correlates with glial differentiation in the postnatal mouse brain

Notch family molecules are thought to be negative regulators of neuronal differentiation in early brain development. After expression in the embryonic period, Notch2 continues to be expressed postnatally in the specific regions in the rodent brain. Here, we examined Notch2 expression in the postnatal mouse brain using lacZ knockin animals at the Notch2 locus. Notch2 expression was observed in the developing cerebellum and hippocampus, characteristic regions where neurogenesis persists after birth. Double staining of sections revealed that Notch2 was expressed by Bergmann glia in the cerebellum, radial glia in the hippocampus, and some astrocytes in both regions. Notch2 expression by glial cells was clearly confirmed in dissociated cell cultures. Interestingly, neocortical glia, many of which did not express Notch2 in vivo, did express Notch2 in a dissociated culture condition. The triple staining of dissociated cell cultures revealed that stronger Notch2 expression correlated with the immature type of glial gene expressions: stronger vimentin and weaker glial fibrillary acidic protein expressions. In addition, Notch2 expression correlated with the incorporation of bromodeoxyuridine both in vivo and in vitro. Thus, these findings demonstrate that Notch2 is expressed not only by neuronal cells in the embryonic brain, but also by glial cells in the postnatal brain, and that its expression negatively correlates with glial differentiation, proposing its novel function as a negative regulator of glial differentiation in mammalian brain development.     

CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.