Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. This effect was prevented by 5-HT2 specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT2 receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.     

Phosphorylation of cockroach antennal polypeptides: effects of second messengers and pheromonal blend

In insect antennal extracts, Schleicher et al.¹ showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol trisphosphate responses, which suggests that pheromonal signals act on phosphorylation of specific proteins. To confirm this hypothesis, we studied the effects of second messengers and a pheromonal blend on phosphorylation of antennal proteins in the cockroachPeriplaneta americana. Proteins from adult male antennae were phosphorylated in vitro in the presence of [³²P] triphosphate, then separated by SDS-polyacrylamide gel electrophoresis. Numerous phosphopolypeptides were visualized. The presence of Ca⁺⁺/calmodulin in the incubation medium resulted in increased phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa. Stimulation of PKC by addition of Ca⁺⁺ phosphatidylserine (PS)/phorbol myristate acetate (PMA) resulted in the appearance of three phosphopolypeptides of 36, 70 and 120 kDa. In the presence of cyclic adenosine monophosphate, two new major polypeptides of 46 and 42 kDa appeared; the latter polypeptide also appeared in the presence of cyclic guanosine monophosphate. Comparison with polypeptide composition of tissue from the cerci, leg, brain and fat body showed that the 36 and 48 kDa polypeptides were specific to antennae, whereas the 120 kDa polypeptide was also present in the adult brain. When antennae are subjected to pheromonal stimulation for 16 seconds prior to homogenization, in vitro phosphorylation of the 120, 70, 64 and 38 kDa polypeptides was inhibited, whereas phosphorylation of the 58, 54, 51 and 48 kDa polypeptides was strongly stimulated. It is noteworthy that a 107 kDa polypeptide was observed only after pheromonal stimulation by Ca⁺⁺/PS/PMA. Our findings suggest that Ca⁺⁺-and PKC-dependent protein phosphorylation systems play an important role in the transduction of pheromonal signals in antennae of male cockroachP. americana. We speculate that specific phosphoproteins may modulate sensitivity and signal amplification during the olfactory transduction process.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Effects of a new cholecystokinin antagonist (GE 410) on the smooth muscle of the guinea pig ileum

Summary Suc-Tyr-(SE)-Met-Gly-Trp-Met-Asp--phenethylamide (GE 410) competitively antagonized the contractions of smooth muscle strips from guinea pig ileum (pA₂=7.6, n=0.95) induced by cholecystokinin-octapeptide (CCK8). GE 410 inhibited the electrically-induced cholinergically mediated contractile responses and the [³H]ACh release in the ileum, as well as the CCK-stimulated electrical contractile responses and the [³H]ACh release in the cholinergic nerve terminals. The results suggest the existence of CCK-receptors not only in the smooth muscles but also on the neurons.     

Nitroglycerin inhibits the phosphorylation of intermediate filament proteins rather than myosin light chain on porcine coronary artery sustained contraction

The smooth muscle relaxation induced by nitroglycerin is hypothesized to be mediated by an increase in the cytoplasmic concentration of guanosine 3′,5′-monophosphate (cGMP) and subsequent dephosphorylation of the 20-kilodalton myosin light chain (MLC). We investigated this hypothesis in procine coronary arterial smooth muscle stimulated with histamine (3 μM) or K⁺ (30 mM). Stimulation of [³²P]Pi-labeled muscle with histamine or K⁺ for 2 min resulted in a four- or 6.2-fold increase, respectively, in the incorporation of³²P into MLC. After 48 min of exposure to histamine. MLC phosphorylation decreased to the basal level and the phosphorylation of desmin, synemin, and of three unidentified cytosolic proteins was increased. K⁺ stimulation resulted in a sustained increase of MLC phosphorylation but had no effect on the phosphorylation of desmin, synemin, or the three unidentified cytosolic proteins. Application of nitroglycerin (1 μM) 48 min after histamine stimulation inhibited the phosphorylation of desmin, synemin, and the three cytosolic proteins. The sustained phase of histamine-induced contraction was also inhibited to a greater extent then the acute phase of histamine-induced contraction and both the acute and sustained phases of K⁺-induced contraction. These results suggest that MLC phosphorylation is required for both phases of K⁺-induced contraction, whereas phosphorylation of intermediate filament proteins is required for the sustained phase of histamine-induced contraction. Intermediate filament proteins, rather than MLC, may also be the target for the relaxant action of nitroglycerin during histamine-induced sustained contraction.     

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca²⁺ mobilization and Ca²⁺ sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca²⁺]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca²⁺]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.     

Characterization of the 5-HT1A receptor of the honeybee (Apis mellifera) and involvement of serotonin in phototactic behavior

Serotonin plays a key role in modulating various physiological and behavioral processes in both protostomes and deuterostomes. The vast majority of serotonin receptors belong to the superfamily of G-protein-coupled receptors. We report the cloning of a cDNA from the honeybee (Am5-ht1A) sharing high similarity with members of the 5-HT₁ receptor class. Activation of Am5-HT_1A by serotonin inhibited the production of cAMP in a dose-dependent manner (EC₅₀ = 16.9 nM). Am5-HT_1A was highly expressed in brain regions known to be involved in visual information processing. Using in vivo pharmacology, we could demonstrate that Am5-HT_1A receptor ligands had a strong impact on the phototactic behavior of individual bees. The data presented here mark the first comprehensive study—from gene to behavior—of a 5-HT_1A receptor in the honeybee, paving the way for the eventual elucidation of additional roles of this receptor subtype in the physiology and behavior of this social insect.     

Interaction of galectin-1 with caveolae induces mouse embryonic stem cell proliferation through the Src, ERas, Akt and mTOR signaling pathways

Galectins have the potential to provide a promising alternative for unveiling the complexity of embryonic stem (ES) cell self-renewal, although the mechanism by which galectins maintain ES cell self-renewal has yet to be identified. Galectin-1 increased [³H]-thymidine incorporation as well as cyclin expression and decreased p27^kip1 expression. Src and caveolin-1 phosphorylation was increased by galectin-1, and phospho-caveolin-1 was inhibited by PP2. In addition, inhibition of caveolin-1 by small interfering RNA and methyl-β-cyclodextrin (Mβ-CD) decreased galectin-1-induced cyclin expression and [³H]-thymidine incorporation. Galectin-1 caused Akt and mTOR phosphorylation, which is involved in cyclin expression. Galectin-1-induced phospho-Akt and -mTOR was inhibited by PP2, ERas siRNA, caveolin-1 siRNA and Mβ-CD. Furthermore, mTOR phosphorylation was decreased by LY294002 and Akt inhibitor. Galectin-1-induced increase in cyclin expression and decrease in p27^kip1 was blocked by Akt inhibitor and rapamycin. In conclusion, galectin-1 increased DNA synthesis in mouse ES cells via Src, caveolin-1 Akt, and mTOR signaling pathways. Received 30 October 2008; received after revision 18 February 2009; accepted 24 February 2009     

Phospholipase C activation via a GTP-binding protein in tumoral islet cells stimulated by carbamylcholine

Summary Carbamylcholine and GTP act synergistically in stimulating the production of [³H]inositol-1-phosphate by digitonized tumoral islet cells (RINm5F line) prelabeled with myo-[2-³H(N)]inositol. The response to these two agents is similar to that evoked by GTPS. These findings suggest that a GTP-binding regulatory protein couples the occupancy of muscarinic receptors to activation of phospholipase C in pancreatic islet cells.This work was supported by grants from the Belgian Foundation for Scientific Medical Research.     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE₂ increased [³H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE₂ increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE₂ obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE₂-induced cell cycle regulatory protein expression and thymidine incorporation. PGE₂ caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE₂-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells. Received 30 January 2009; received after revision 03 March 2009; accepted 10 March 2009     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group

Summary (1 R) [1-³H,²H₁] 3-Phenylpropanol, the key intermediate in the synthesis of (4 R) [4-³H,²H₁] D, L-homoserine and of the (4 S)-isomer, is obtained from (1 S) [1-²H₁] 3-phenylpropanol and (1 RS) [1-³H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD⁺; under similar conditions 2-phenylethanol undergoes very small exchange with [1-²H₂] ethanol.     

Calyculin a increases voltage-dependent inward current in,smooth muscle cells isolated from guinea pig taenia coli

The effects of a potent phosphatase inhibitor, calyculin A (CL-A), on inward currents in guinea pig taenia coli smooth muscle cells were examined. CL-A increased the inward current, and this effect of CL-A was inhibited by a protein kinase C inhibitor, H-7, and by nifedipine. Phorbol 12,13-dibutyrate, an activator of protein kinase C, also increased the inward current and this effect was antagonized by H-7. These results suggest that in guinea pig taenia coli smooth muscle cells CL-A may facilitate the opening of thel-type Ca²⁺ channels through the protein kinase C-dependent phosphorylation system.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Insulin-producing cells in the brain of adult Drosophila are regulated by the serotonin 5-HT1A receptor

Insulin signaling regulates lifespan, reproduction, metabolic homeostasis, and resistance to stress in the adult organism. In Drosophila, there are seven insulin-like peptides (DILP1–7). Three of these (DILP2, 3 and 5) are produced in median neurosecretory cells of the brain, designated IPCs. Previous work has suggested that production or release of DILPs in IPCs can be regulated by a factor secreted from the fat body as well as by neuronal GABA or short neuropeptide F. There is also evidence that serotonergic neurons may regulate IPCs. Here, we investigated mechanisms by which serotonin may regulate the IPCs. We show that the IPCs in adult flies express the 5-HT_1A, but not the 5-HT_1B or 5-HT₇ receptors, and that processes of serotonergic neurons impinge on the IPC branches. Knockdown of 5-HT_1A in IPCs by targeted RNA interference (RNAi) leads to increased sensitivity to heat, prolonged recovery after cold knockdown and decreased resistance to starvation. Lipid metabolism is also affected, but no effect on growth was seen. Furthermore, we show that DILP2-immunolevels in IPCs increase after 5-HT_1A knockdown; this is accentuated by starvation. Heterozygous 5-HT_1A mutant flies display the same phenotype in all assays, as seen after targeted 5-HT_1A RNAi, and flies fed the 5-HT_1A antagonist WAY100635 display reduced lifespan at starvation. Our findings suggest that serotonin acts on brain IPCs via the 5-HT_1A receptor, thereby affecting their activity and probably insulin signaling. Thus, we have identified a second inhibitory pathway regulating IPC activity in the Drosophila brain.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Summary Rat brain homogenate was preloaded with [³H]noradrenaline or [³H]GABA and stimulated with high K⁺. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [³H]noradrenaline release in the same range of toxin concentrations starting below 10^–10M. In contrast, release of -amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Serum-induced stimulation of snRNA synthesis in mouse 3T3 fibroblasts

Summary Small nuclear RNAs (snRNAs) from quiescent and serum-stimulated 3T3 cultures, labeled with [³H]uridine ([³H]U), were electrophoresed in polyacrylamide-urea slab gels and revealed by staining with ethidium bromide and by fluorography, Judged by labeling with [³H]U, synthesis of 7S and U1-U6 RNAs was very low or absent in quiescent cultures. The serum-induced transition of 3T3 cells from a resting to a growing state was accompanied by an early, apparently sequential stimulation of snRNA synthesis; stimulated synthesis of 7S, U1, U2, U3, U4 and U6 RNAs coincided in time with serum-induced stimulation of 45S pre-ribosomal RNA (pre-rRNA) and heterogeneous nuclear RNA (hnRNA) synthesis.