Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Whole-cell potassium current in rabbit corneal epithelium activated by fenamates

Summary Rabbit corneal epithelium contains a large-conductance, potassium-selective channel, which is a major contributor to the whole-cell current. In perforated-patch recordings of the macroscopic current, the isolated cells studied had resting voltages of –41±20 mV and capacitances of 5.8±2.6 pF (mean + sd for n=255). Activation of the channels was weakly voltage dependent. They opened at about –100 mV and reached an open probability of about 0.2 at +100 mV. The current was blocked by millimolar concentrations of external Ba²⁺ and quinidine. Diltiazem also blocked when applied to the external surface of the membrane. Nonsteroidal anti-inflammatory agents of the fenamate group were powerful activators of the channel at submillimolar concentrations when applied either to the inside or the outside of the channels. The mechanism of action which leads to his activation is not yet known.We are grateful to Erika Wohlfiel for secretarial help, Helen Hendrickson for cell preparation, and Joan Rae for software development. This work was supported by NIH grants EY06005 and EY03282 and an unrestricted award from Research to Prevent Blindness.     

Plasma fractionation issues

Procurement and processing of human plasma for fractionation of therapeutic proteins or biological medicines used in clinical practice is a multi-billion dollar international trade. Together the private sector and public sector (non-profit) provide large amounts of safe and effective therapeutic plasma proteins needed worldwide. The principal therapeutic proteins produced by the dichotomous industry include gamma globulins or immunoglobulins (including pathogen-specific hyperimmune globulins, such as hepatitis B immune globulins) albumin, factor VIII and Factor IX concentrates. Viral inactivation, principally by solvent detergent and other processes, has proven highly effective in preventing transmission of enveloped viruses, viz. HBV, HIV, and HCV.     

Confirmation of quantitative trait loci affecting fatness in chickens

In this report we describe the analysis of an advanced intercross line (AIL) to confirm the quantitative trait locus (QTL) regions found for fatness traits in a previous study. QTL analysis was performed on chromosomes 1, 3, 4, 15, 18, and 27. The AIL was created by random intercrossing in each generation from generation 2 (G₂) onwards until generation 9 (G₉) was reached. QTL for abdominal fat weight (AFW) and/or percentage abdominal fat (AF%) on chromosomes 1, 3 and 27 were confirmed in the G₉ population. In addition, evidence for QTL for body weight at the age of 5 (BW5) and 7 (BW7) weeks and for the percentage of intramuscular fat (IF%) were found on chromosomes 1, 3, 15, and 27. Significant evidence for QTL was detected on chromosome 1 for BW5 and BW7. Suggestive evidence was found on chromosome 1 for AFW, AF% and IF%, on chromosome 15 for BW5, and on chromosome 27 for AF% and IF%. Furthermore, evidence on the chromosome-wise level was found on chromosome 3 for AFW, AF%, and BW7 and on chromosome 27 for BW5. For chromosomes 4 and 18, test statistics did not exceed the significance threshold.     

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues

The gaseous molecule hydrogen sulfide (H₂S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H₂S. This production occurs through the activity of two enzymes, cystathionine-γ-lyase and cystathionine-β-synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H₂S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.     

广义隐Markov模型在基因识别中的应用

广义隐Markov模型(GHMM)是基因识别的一种重要模型,但是其计算量比传统的隐Markov模型大得多,以至于不能直 接在基因识别中使用。根据原核生物基因的结构特点,提出了一种高效的简化算法,其计算量是序列长度的线性函数。在此 基础上,构建了针对原核生物基因的识别程序GeneMiner,对实际数据的测试表明,此算法是有效的。     

Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca(2+) channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba(2+) as the charge carrier. Maximal peak Ba(2+) current (I(Ba)) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 +/- 3%). The NO donor S-nitroso-N-acetylpenicillamine also increased I(Ba), and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in I(Ba) was blocked when cells were pretreated with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 microM) and milrinone (3 microM), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on I(Ba). Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca(2+) channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO.