Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na(+)/H(+) exchangers (NHE) and Cl(-)/OH(-) exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 muM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca(2+) and PKCalpha inhibition (Go6976). In vitro assays revealed that PKCalpha interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca(2+) and PKCalpha-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.     

Endothelial cell junctional integrity modulation by serotonin: an ultrastructural analysis

We have reported previously that exogenous serotonin (5-hydroxytryptamine, 5-HT) alters cultured bovine aortic endothelial cell (BAEC) structural integrity by modulating the assembly of stress fibers. In the present study a 5-HT stimulus-coupled change in BAEC junctional integrity was quantitated by determining the width and percentage of intercellular openings in a monolayer. BAEC treated with 5-HT at concentrations of 10(-9) M to 10(-3) M caused a significant dose-dependent decrease in interendothelial cell junctional openings compared to controls, with the greatest reduction induced at 10(-6) M (92% from control). Treatment of BAEC with histamine (10(-4) M) increased the junctional openings by 82% when compared to controls. This change could be prevented by either pretreatment of the monolayers with 5-HT or by adding 5-HT in conjunction with the histamine. To assess a direct interaction of 5-HT with actin filaments, cultured BAEC monolayers were extracted, treated with 5-HT, and processed for immunocytochemical localization of 5-HT using the Avidin-Biotin method. Electron microscopy revealed 5-HT antibody bound to actin filaments and dense in areas of filament intersection, which implies a role for internalized 5-HT in stimulating the assembly of an actin filament network. Collectively, these results suggest that 5-HT helps to regulate the endothelial junctional barrier by promoting actin filament formation and stability, which may in turn increase the junctional apposition between endothelial cells.     

Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium

Cultured bovine aortic endothelial cells were fixed and stained with NBD-phallicidin and quantitated with a digital image analyzer for changes in actin cables and surface area. Serotonin (5-HT), norepinephrine (NE), dopamine and histamine (all at 10(-4)M concentrations) were tested for their ability to induce cytoskeletal changes. Only 5-HT and NE increased actin cables significantly (p less than 0.01), 80.7% and 97.9%, respectively. Dopamine and histamine treated cells showed a 67.4% and 80.8% decrease in actin cables respectively (p less than 0.01). Stimulated increases of actin cables by 5-HT were inhibited by Ketanserin, and propranolol inhibited NE stimulation of actin cables. Treatment of cells with these blockers alone also decreased actin cables below control values (p less than 0.01). Pretreatment of cells with diphenhydramine, but not cimetidine, inhibited histamine-induced decreases in actin cables. Stimulation of surface area by 5-HT and NE was also observed, with 40.8% and 80.7% increases respectively, when compared with controls (p less than 0.01). The increases in actin cables were associated with a lack of ruffled edges that are indicative of motile cells. In contrast, induced decreases in actin cables resulted in cells with ruffled edges. Exogenous 5-HT and NE have been shown to prevent the increased permeability visible as extravasation of red blood cells from postcapillary venules in thrombocytopenic animals. The present data suggest that 5-HT and NE may be involved in maintaining the endothelial barrier function by a receptor-mediated stimulation of actin cables. Also, histamine-induced decreases in actin cables may be correlated with the amine's action in vivo as a mediator of increased inflammatory permeability.     

Involvement of contractile elements in control of bioluminescence in Northern krill, <Emphasis Type="Italic">Meganyctiphanes norvegica</Emphasis> (M. Sars)

Inside the light organs of the bioluminescent (light-producing) crustacean Meganyctiphanes norvegica (krill), numerous capillaries drain haemolymph into the light-producing structure (lantern). We have investigated the arrangement and function of filamentous material found around the opening of the capillaries. These have been suggested to work as sphincters, controlling the haemolymph (i.e. oxygen) supply to the lantern and thereby the production of light. Electron microscopy shows that the filamentous material consists of thick and thin muscle filaments arranged in perpendicular blocks around the opening of each capillary. The actin probe rhodamine phalloidin has revealed that one component is filamentous actin. Clusters of vesicle-dense nerve profiles surround the cells containing filamentous material and antibodies against 5-hydroxytryptamine (5-HT) reveal that 5-HT containing nerves lead to the filamentous area. When exposed to the muscle-relaxing substances papaverine and verapamil, krill respond with luminescence, suggesting that the sphincter structures are functionally involved in the control of light production. Treatment with the muscle-contracting drugs Bay K8544 and thapsigargin gives no light response. Thus, 5-HT stimulates light production in krill; however, a combination of 5-HT and the muscle-relaxing drugs or Bay K8544 potentiates the effect of 5-HT. Thapsigargin quenches the response to 5-HT. Our results corroborate speculations of earlier authors who have suggested that the sphincter structures are of a muscular nature and important in controlling light production in krill. However, other parameters in addition to the oxygen supply to the lantern are involved in controlling bioluminescence in the light organs of M. norvegica. We acknowledge financial support from the Biodiversity Program of the Swedish Science Research Council and from the Royal Swedish Academy of Science for supporting work at the Kristineberg Marine Research Center. Electron microscopy equipment was granted by the Lundberg Research Foundation.     

Stiffness changes in cultured airway smooth muscle cells

Airwaysmooth muscle (ASM) cells in culture stiffen when exposed tocontractile agonists. Such cell stiffening may reflect activation ofthe contractile apparatus as well as polymerization of cytoskeletalbiopolymers. Here we have assessed the relative contribution of thesemechanisms in cultured ASM cells stimulated with serotonin(5-hydroxytryptamine; 5-HT) in the presence or absence of drugs thatinhibit either myosin-based contraction or polymerization offilamentous (F) actin. Magnetic twisting cytometry was used to measurecell stiffness, and associated changes in structural organization ofactin cytoskeleton were evaluated by confocal microscopy. We found that5-HT increased cell stiffness in a dose-dependent fashion and alsoelicited rapid formation of F-actin as marked by increased intensity ofFITC-phalloidin staining in these cells. A calmodulin antagonist (W-7),a myosin light chain kinase inhibitor (ML-7) and a myosin ATPaseinhibitor (BDM) each ablated the stiffening response but not theF-actin polymerization induced by 5-HT. Agents that inhibited theformation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, andY-27632) attenuated both baseline stiffness and the extent of cellstiffening in response to 5-HT. Together, these data suggest thatagonist-evoked stiffening of cultured ASM cells requires actinpolymerization as well as myosin activation and that neitheractin polymerization nor myosin activation by itself is sufficient toaccount for the cell stiffening response.

     

Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.     

A new signaling paradigm for serotonin: use of Crk-associated substrate in arterial contraction

Crk-associated substrate (CAS), a 130-kDa adaptor protein, was discovered as a tyrosine kinase substrate of Src that was important to cellular motility and actin filament formation. As the tyrosine kinase Src is utilized by the 5-hydroxytryptamine (5-HT)(2A) receptor in arterial contraction, we tested the hypothesis that CAS was integral to 5-HT(2A) receptor-mediated vasoconstriction. Rat thoracic aorta was used as a model of the arterial 5-HT(2A) receptor. Western and immunohistochemistry analyses validated the presence of CAS in the aorta, and tissue bath experiments demonstrated reduction of contraction to 5-HT (13.5 +/- 5% control maximum) and the 5-HT(2) receptor agonist alpha-methyl-5-HT (6 +/- 2% maximum) by latrunculin B (10(-6) mol/l), an actin disruptor. In aorta contracted with 5-HT (10(-5) mol/l), tyrosine phosphorylation (Tyr410) of CAS was significantly increased (approximately 225%), and both contraction and CAS phosphorylation were reduced by the 5-HT(2A/2C) receptor antagonist ketanserin (3 x 10(-8) mol/l). Src is one candidate for 5-HT-stimulated CAS tyrosyl-phosphorylation as 5-HT promoted interaction of Src and CAS in coimmunoprecipitation experiments, and the Src tyrosine kinase inhibitor PP1 (10(-5) mol/l) abolished 5-HT-induced tyrosyl-phosphorylation of CAS and reduced 5-HT- and alpha-methyl-5-HT-induced contraction. Antisense oligodeoxynucleotides delivered to the aorta reduced CAS expression (33% control) and arterial contraction to alpha-methyl-5-HT (45% of control), independent of changes in myosin light chain phosphorylation. These data are the first to implicate CAS in the signal transduction of 5-HT.     

Modulation of 5-HT3 receptor-mediated response and trafficking by activation of protein kinase C

Modulation of neurotransmitter-gated membrane ion channels by protein kinase C (PKC) has been the subject of a number of studies. However, less is known about PKC modulation of the serotonin type 3 (5-HT3) receptor, a ligand-gated membrane ion channel that can mediate fast synaptic transmission in the central and peripheral nervous system. Here, we show that PKC potentiated 5-HT3 receptor-mediated current in Xenopus oocytes expressing 5-HT3A receptors and mouse N1E-115 neuroblastoma cells. In addition, using a specific antibody directed to the extracellular N-terminal domain of the 5-HT3A receptor, treatment with the PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA), significantly increased surface immunofluorescence. PKC also increased the amount of 5-HT3A receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3A receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. PMA potentiation is unlikely to occur via direct phosphorylation of the 5-HT3A receptor protein since the potentiation was not affected by point mutation of each of the putative sites for PKC phosphorylation. However, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibited PMA potentiation of 5-HT-activated responses in both N1E-115 cells and oocytes expressing 5-HT3A receptors. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhanced the PMA potentiation of 5-HT3A receptors. The observations suggest that PKC can modulate 5-HT3A receptor function and trafficking through an F-actin-dependent mechanism.     

Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton

Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction of CTGF by transforming growth factor beta (TGF-beta) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediated CTGF induction was due to activation of 5-HT(2A) receptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-beta. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction of CTGF by LPA and TGF-beta. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulated CTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGF expression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants of CTGF expression induced by LPA and 5-HT, and also by TGF-beta.     

A Highlights from MBoC Selection: Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT–dependent neurite outgrowth in Aplysia bag cell neurons

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca²⁺; however, mechanistic roles for Ca²⁺ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca²⁺ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca²⁺ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.     

5-hydroxytryptamine induces mast cell adhesion and migration

The neurotransmitter serotonin (5-hydroxytryptamine (5-HT)) is implicated in enhancing inflammatory reactions of skin, lung, and gastrointestinal tract. To determine whether 5-HT acts, in part, through mast cells (MC), we first established that mouse bone marrow-derived MC (mBMMC) and human CD34(+)-derived MC (huMC) expressed mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mouse and human MC degranulation, adhesion, and chemotaxis. We found no evidence that 5-HT degranulates MC or modulates IgE-dependent activation. 5-HT did induce mBMMC and huMC adherence to fibronectin; and immature and mature mBMMC and huMC migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin, we identified 5-HT(1A) as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT(1A) receptor knockout mouse (5-HT(1A)R(-/-)) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT(1A)R(+/+) mice, but not in 5-HT(1A)R(-/-) mice. These studies are the first to demonstrate an effect of 5-HT on MC. Furthermore, both mouse and human MC respond to 5-HT through the 5-HT(1A) receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.     

Regulation of Serotonin-Induced Trafficking and Migration of Eosinophils

Association of the neurotransmitter serotonin (5-HT) with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos) in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos) were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM)-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not G_αi-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca²⁺. Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca²⁺ levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI)/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway).     

Localization of serotonin and its possible role in early embryos of Tritonia diomedea(Mollusca: Nudibranchia)

A classical neurotransmitter serotonin (5-HT) was detected immunochemically using laser scanning microscopy at the early stages of Tritonia diomedea development. At the one- to eight-cell stages, immunolabeling suggested the presence of 5-HT in the cytoplasm close to the animal pole. At the morula and blastula stages, a group of micromeres at the animal pole showed immunoreactivity. At the gastrula stage no immunoreactive cells were detected, but they arose again at the early veliger stage. Antagonists of 5-HT(2) receptors, ritanserin and cyproheptadine, as well as lipophilic derivatives of dopamine blocked cleavage divisions or distorted their normal pattern. These effects were prevented by 5-HT and its highly lipophilic derivates, serotoninamides of polyenoic fatty acids, but not by the hydrophilic (quaternary) analog of 5-HT, 5-HTQ. The results confirm our earlier suggestion that endogenous 5-HT in pre-nervous embryos acts as a regulator of cleavage divisions in nudibranch molluscs.     

Cutting edge: serotonin is a chemotactic factor for eosinophils and functions additively with eotaxin

Elevated levels of serotonin (5-hydroxytryptamine, 5-HT) are observed in the serum of asthmatics. Herein, we demonstrate that 5-HT functions independently as an eosinophil chemoattractant that acts additively with eotaxin. 5-HT2A receptor antagonists (including MDL-100907 and cyproheptadine (CYP)) were found to inhibit 5-HT-induced, but not eotaxin-induced migration. Intravital microscopy studies revealed that eosinophils roll in response to 5-HT in venules under conditions of physiological shear stress, which could be blocked by pretreating eosinophils with CYP. OVA-induced pulmonary eosinophilia in wild-type mice was significantly inhibited using CYP alone and maximally in combination with a CCR3 receptor antagonist. Interestingly, OVA-induced pulmonary eosinophilia in eotaxin-knockout (Eot-/-) mice was inhibited by treatment with the 5-HT2A but not CCR3 receptor antagonist. These results suggest that 5-HT is a potent eosinophil-active chemoattractant that can function additively with eotaxin and a dual CCR3/5-HT2A receptor antagonist may be more effective in blocking allergen-induced eosinophil recruitment.     

Serotonylation of Vascular Proteins Important to Contraction

Background

Serotonin (5-hydroxytryptamine, 5-HT) was named for its source (sero-) and ability to modify smooth muscle tone (tonin). The biological effects of 5-HT are believed to be carried out by stimulation of serotonin receptors at the plasma membrane. Serotonin has recently been shown to be synthesized in vascular smooth muscle and taken up from external sources, placing 5-HT inside the cell. The enzyme transglutaminase uses primary amines such as 5-HT to covalently modify proteins on glutamine residues. We tested the hypothesis that 5-HT is a substrate for transglutaminase in arterial vascular smooth muscle, with protein serotonylation having physiological function.

Methodology/Principal Findings

The model was the rat aorta and cultured aortic smooth muscle cells. Western analysis demonstrated that transglutaminase II was present in vascular tissue, and transglutaminase activity was observed as a cystamine-inhibitable incorporation of the free amine pentylamine-biotin into arterial proteins. Serotonin-biotin was incorporated into α -actin, β-actin, γ-actin, myosin heavy chain and filamin A as shown through tandem mass spectrometry. Using antibodies directed against biotin or 5-HT, immunoprecipitation and immunocytochemistry confirmed serotonylation of smooth muscle α–actin. Importantly, the α-actin-dependent process of arterial isometric contraction to 5-HT was reduced by cystamine.

Conclusions

5-HT covalently modifies proteins integral to contractility and the cytoskeleton. These findings suggest new mechanisms of action for 5-HT in vascular smooth muscle and consideration for intracellular effects of primary amines.     

5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK

The purpose of this study was to elucidate the mechanism of 5-hydroxytryptamine (5-HT, serotonin) action on migration of vascular smooth muscle cells. Migration of cultured human aortic smooth muscle cells (HASMCs), evaluated using time-lapse microscopy, was significantly enhanced by 5-HT at concentrations of 1-100 nM. The enhancing effect of 5-HT on cell migration was markedly inhibited in the presence of ketanserin, a 5-HT2 receptor antagonist, but not by GR 55562, a 5-HT1 receptor antagonist. Activities of RhoA and ERK were increased by 5-HT, and the increase in cell migration by 5-HT was abolished in the presence of U0126, a MEK1/2 inhibitor, or Y-27632, a Rho-kinase inhibitor. Activation of ERK was strongly inhibited by Y-27632. 5-HT-induced formation of stress fiber and detachment of uropod (trailing edge) were abolished by Y-27632. Thus, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation by 5-HT2 receptor stimulation followed by activation of the Rho-kinase and ERK pathways.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Fluorescence digital image analysis of serotonin-induced calcium oscillations in single blood platelets

The intracellular concentration of Ca2+ [( Ca2+]i) was monitored continuously in single rabbit blood platelets by digital imaging microscopy in conjunction with Fura-2, a specific Ca(2+)-indicator dye. Ionomycin as well as aluminium fluoride caused sustained increase in [Ca2+]i in the platelet, but oscillations of [Ca2+]i were not observed. Serotonin (5-HT) induced oscillatory increases in [Ca2+]i in the presence of 1 mM CaCl2; these had not been detectable in cell populations because the oscillations were not in synchrony. This effect of 5-HT was diminished when CaCl2 was omitted from the medium, and was antagonized by 1 microM ketanserin, a specific 5-HT2 receptor antagonist. Furthermore, DOI, a specific 5-HT2 agonist, had the same effect as 5-HT at lower concentration. A specific effector mechanism, not fully understood at present, therefore appears to mediate 5-HT2 receptors thereby allowing rabbit platelets to generate [Ca2+]i oscillations. It is suggested that protein kinase C in platelets might play a key role in the regulation of [Ca2+]i, and possibly in [Ca2+]i oscillations.     

Reduced microfilament organization in adenovirus type 5-infected rat embryo cells: a function of early region 1a.

The actin microfilament organization in rat embryo cells was examined by fluorescence microscopy with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and by electron microscopy, after mock infection or infection with adenovirus type 5 (Ad5). Infected cells showed severely reduced numbers of actin microfilaments and stress fibers, detectable early after infection. Mutants defective in Ad5 early genes were used to show that reduced microfilament organization was a function of the Ad5 transformation early gene 1a (E1a) and did not require expression of any other viral gene. The product of the E1a 13s mRNA was essential for the effect, although the 12s mRNA product appeared to contribute. Ad5 infection of the cells had no observable effect on total cell actin levels or on the ratio of monomeric to polymeric actin. E1a, therefore, affected only the higher-order organization of actin.     

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.