Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NIH-3T3 cells, a nontransformed murine fibroblast cell line previously found to be unresponsive to 5-hydroxytryptamine (5-HT) when cultured in 5-HT-free medium, became responsive to 5-HT, which induced an increase in intracellular calcium concentration. Pharmacological and ligand binding studies showed that NIH-3T3 cells endogenously express a 5-HT2A receptor that, when activated, mobilizes calcium from ionomycin-sensitive intracellular stores via coupling to a pertussis toxin-insensitive pathway. Using reverse transcriptase-PCR cloning and northern blot analysis, the presence of 5-HT2A receptor RNA with a similar nucleotide sequence (99% identity) and molecular size to that of murine brain was detected in NIH-3T3 cells. Responsiveness of the endogenous 5-HT2A receptor in nontransfected cells was completely desensitized after chronic treatment (half-time = 2 h) with 1 microM 5-HT and resensitized on removal of 5-HT. In contrast to NIH-3T3 cells transfected with 5-HT2A receptor cDNA under control of a viral promoter, the long-term agonist-induced functional desensitization in nontransfected NIH-3T3 cells was paralleled by a decrease in both 5-HT2A receptor density and RNA level. These results show that NIH-3T3 cells express an endogenous 5-HT2A receptor that is desensitized by agonist via down-regulation of both receptor number and mRNA. The NIH-3T3 cells provide a novel system for understanding 5-HT2A receptor regulation.
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PMID:Identification of an endogenous 5-hydroxytryptamine2A receptor in NIH-3T3 cells: agonist-induced down-regulation involves decreases in receptor RNA and number. 910 26

Smooth muscle cell-like mesangial cells play an important role in the regulation of glomerular blood flow and are involved in renal inflammatory reactions, thereby interacting with circulating cells. The platelet products serotonin (5-HT) and ATP induce similar, e.g. mitogenic, effects in mesangial cells, but differentially activate and induce inflammation-related genes. To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca2+ signal elicited by 5-HT and ATP in rat mesangial cells was investigated. Both phases of the Ca2+ signal, release from internal stores as well as influx of extracellular Ca2+, were dependent on phospholipase C activation as shown by the specific inhibitor U73122 (complete inhibition at 10 microM U73122). There was no evidence for voltage-gated L-type channels in these cells, suggesting that Ca2+ influx was mediated by Ca2+ release-activated channels. The L-type channel blocker verapamil, however, dose-dependently (0.1-10 microM) and specifically inhibited 5-HT-elicited Ca2+ signals by interference with binding of 5-HT to 5-HT2A receptors. 5-HT-mediated Ca2+ release was reduced by 80% when protein kinase C was activated by the phorbolester TPA (0.1 microM). Interaction of 5-HT2A receptors with phospholipase C was also inhibited by genistein (30% at 5 microM; 100% at 50 microM), an inhibitor of tyrosine kinases. Binding of 5-HT to its receptor reduced subsequent ATP-mediated Ca2+ signaling. The cross talk between the receptors was sensitive to genistein. ATP-mediated Ca2+ signaling was attributed to different types of P2y receptors and/or multiple G-proteins coupled, because the signal was partially inhibited by pertussis toxin (50%). In accordance, modulation of the ATP-mediated signaling by phosphorylation was less tightly controlled than 5-HT-mediated Ca2+ release. These data indicate that although the Ca2+ responses elicited by the two stimuli are comparable, interactions between receptors, G-proteins and target enzymes are regulated differentially.
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PMID:Mechanisms of serotonin-induced Ca2+ responses in mesangial cells. 927 31

Mechanisms underlying the 5-HT2A receptor induction of intracellular Ca2+ mobilization and Ca2+ influx in type I astroglial cells in primary culture from newborn rat cerebral cortex were evaluated. The 5-HT-evoked Ca(2+)-transients, inhibited by the 5-HT2A antagonists ketanserin or 4-(4-fluorobenzoyl)-1-(4-phenylbutyl) piperidine oxalate, consisted of an initial peak caused by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from internal stores, and a second sustained part which was due to Ca2+ transport over the plasma membrane. The responses were pertussis toxin-insensitive, suppressed by the phospholipase C inhibitor neomycin and were inhibited by the Ca(2+)-ATPase inhibitor thapsigargin. Furthermore, the responses were inhibited by the IP3 receptor antagonist heparin. When the second sustained part of the 5-HT-evoked response was studied, it was concluded that Ca2+ influx was not a result of opening of voltage operated calcium channels of either L, N or T-type. Instead it appeared that Ca2+ entered the cells through specialized voltage independent Ca2+ channels which were dependent of the IP3 production and subsequent Ca2+ release from internal stores. From this, we conclude that 5-HT opens Ca2+ channels in astrocytes which closely resemble depletion-operated Ca2+ channels (DOCCs).
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PMID:Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca2+ channels. 954 27

Signaling pathways responsible for serotonin (5-HT)-mediated induction of early response genes prostaglandin G/H synthase-2 (PGHS-2, cyclooxygenase-2) and egr-1 were investigated in rat mesangial cells. Gene induction by 5-HT was dependent on 5-HT2A receptors that were pertussis toxin insensitive indicating coupling to a G-protein of the Gq family. Binding of 5-HT to this receptor activates phosphatidylinositol-specific phospholipase C (PLC) and release of Ca2+ from internal stores, but this activation was not related to PGHS-2 mRNA expression. Similarly, PI-3 kinase was not involved in 5-HT signaling. Instead, inhibition of phosphatidylcholine-specific PLC interfered with PGHS-2 and egr-1 mRNA induction, suggesting this enzyme as a link between 5-HT2A receptors and protein kinase C, an essential part of 5-HT-mediated signaling. The MAP kinase pathway was identified as common signaling pathway of 5-HT or phorbol ester-induced gene expression. Increase of intracellular cAMP by forskolin or dibutyryl cAMP did not induce PGHS-2 or egr-1 mRNA expression by itself, but strongly inhibited 5-HT-mediated mRNA induction. PGHS-2 mRNA and protein induction by 5-HT was also abolished by chelation of Ca2+ ions by EGTA, suggesting involvement of Ca2+-dependent enzymes. In contrast, egr-1 mRNA expression was superinduced in the presence of EGTA. Induction of Egr-1 protein was not changed by EGTA hinting to Ca2+-sensitive posttranscriptional steps. Activation of the Gq-coupled 5-HT2A receptor thus leads to the expression of the early response genes PGHS-2 and egr-1, using common as well as differing signaling elements that allow differential regulation of the expression of these genes that are functionally related to renal hemodynamics and proliferation of mesangial cells, respectively.
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PMID:Signaling pathways mediating induction of the early response genes prostaglandin G/H synthase-2 and egr-1 by serotonin via 5-HT2A receptors. 957 79

We studied the endogenous expression of the serotonin-2A (5-hydroxytryptamine2A, 5-HT2A) 5-HT2C, and a splice-variant of the 5-HT2C receptor in murine Balb/c-3T3 fibroblast cells that is revealed when these cells are maintained in medium containing 5-HT-free serum. RNA editing of the 5-HT2C receptor was exclusively at a single brain-specific site. Addition of 5-HT (EC50 = 23 +/- 2.9 nM) induced an immediate release of calcium from an ionomycin-sensitive intracellular store by coupling to a pertussis toxin-insensitive pathway. The 5-HT-induced calcium mobilization displayed a 5-HT-2-like pharmacology, and ligand binding analyses indicated the presence of specific binding sites (27.5 +/- 2 fmol/mg protein) with a 5-HT2A-like pharmacology. Although the 5-HT2A receptor site was predominant, the smaller component of 5-HT2C receptors alone was sufficient to mediate a maximal calcium response. The 5-HT-induced increase in [Ca2+]i was reversibly inhibited by >75% following a 12-hr pretreatment (T1/2 = 2 hr) with 5-HT (EC50 = 400 nM). Extended treatment (24-96 hr) with 5-HT induced a complete functional desensitization that was associated with a partial (60%) reduction in 5-HT2 receptor number, implicating both receptor down-regulation and post-receptor mechanisms in 5-HT-induced desensitization. Long-term (hours to days) treatment with 5-HT did not modulate DNA synthesis, cell proliferation, or transformation in Balb/c-3T3 cells. These results demonstrate that Balb/c-3T3 cells express endogenous 5-HT2 receptors that are desensitized by the 5-HT present in normal serum, illustrating the importance of growth conditions in the identification of receptor responsiveness. The lack of proliferative response to 5-HT in Balb/c-3T3 suggests a putative role of desensitization as a "safety valve" to prevent abnormal cell growth during sustained 5-HT2 receptor activation.
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PMID:Endogenous serotonin-2A and -2C receptors in Balb/c-3T3 cells revealed in serotonin-free medium: desensitization and down-regulation by serotonin. 982 34

The G protein coupling of human 5-hydroxytryptamine5A (h5-ht5A) receptors was investigated in stably transfected human embryonic kidney (HEK) 293 cells, using radioligand and guanosine-5'[gamma-35S]thiotriphosphate binding to membranes and cyclic adenosine monophosphate measurements in cells. 5-Carboxamido[3H]tryptamine bound to high- and low-affinity sites on h5-ht5A-HEK 293 cell membranes. Guanylyl-imidodiphosphate addition and pertussis toxin pre-treatment abolished high-affinity binding, indicating coupling to G proteins of the Gi/Go family. [N-methyl-3H]Lysergic acid diethylamide bound to a single site; guanylyl-imidodiphosphate and pertussis toxin did not alter lysergic acid diethylamide affinity. 5-Hydroxytryptamine stimulated guanosine-5'[gamma-35S]thiotriphosphate binding to 130% over basal and this effect was completely abolished by pertussis toxin. Various 5-hydroxytryptamine receptor ligands were tested for inhibition of 5-carboxamido[3H]tryptamine binding and in guanosine-5'[gamma-35S]thiotriphosphate binding assays. 5-Hydroxytryptamine consistently inhibited forskolin-induced cyclic adenosine monophosphate formation by 25% in h5-ht5A-HEK 293 cells; no effect was detected on basal cyclic adenosine monophosphate levels, on intracellular Ca2+ concentration or arachidonic acid release. Our studies demonstrate functional coupling of the h5-ht5A receptor to pertussis toxin-sensitive G proteins and to inhibition of adenylate cyclase activity.
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PMID:The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells. 986 21

5-Hydroxytryptamine (5-HT, 'serotonin') is a potent inducer of the early response gene cyclo-oxygenase 2 (Cox-2; prostaglandin G/H synthase) in mesangial cells. Protein kinase C (PKC), Ca2+-dependent enzymes and mitogen-activated protein kinase (p42/44 MAPK) have previously been shown to be essential modules of the signalling pathway leading from the pertussis-insensitive 5-HT2A receptor to the induction of Cox-2 mRNA expression. In the present study, PKC activation was linked to the 5-HT-mediated phosphorylation and thus the activation of p42/44 MAPK: the inhibition of PKC by the specific inhibitor GF109203x prevented p42/44 MAPK activation. Ca2+/calmodulin-dependent (CaM) kinase II delta2 was detected in mesangial cells by Western blot analysis. The inhibition of CaM kinase by the inhibitors KN62 or KN93 led to a partial inhibition of 5-HT-induced Cox-2 mRNA expression and decreased basal, but not PMA-mediated, Cox-2 expression. The 5-HT-mediated activation of MAPK was not decreased by KN62 or KN93, excluding CaM kinase as a signalling module upstream of p42/44 MAPK. Taken together, these results indicate a modulatory involvement of CaM kinase in the regulation of 5-HT-mediated Cox-2 mRNA expression in addition to the main pathway that consists of the activation of PKC and p42/44 MAPK.
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PMID:Independent regulation of cyclo-oxygenase 2 expression by p42/44 mitogen-activated protein kinases and Ca2+/calmodulin-dependent kinase. 1019 Dec 63

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.
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PMID:5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells. 1037 10

Monocyte chemotactic protein 1 (MCP-1), which is synthesized by vascular cells, is a chemoattractant for monocytes and has been implicated in a wide range of acute and chronic inflammatory processes characterized by monocyte infiltration, including atherosclerosis. However, it is unclear whether MCP-1 is able to modulate vascular smooth muscle cell (VSMC) proliferation. We assessed the effect of MCP-1 on VSMC proliferation and its interaction with serotonin (5-HT), a mitogen for VSMCs. Growth-arrested VSMCs were stimulated with different concentrations of MCP-1 (25-200 ng/ml) and 5-HT (5 and 50 microM) in serum-free medium. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. 5-HT at concentrations of 5 and 50 microM significantly stimulated DNA synthesis by 1.8- and 2.1-fold over the control value, respectively (p < 0.0001). However, MCP-1 at the concentrations tested did not have any significant effect on DNA synthesis. Even though MCP-1 (50 ng/ml) by itself is not mitogenic, when added to 5-HT, it significantly amplified the mitogenic effect of 5-HT compared with that of 5-HT alone (p < 0.0001). The 5-HT2A receptor antagonist sarpogrelate (10 microM) and its major metabolite M-1 (0.1 microM), pertussis toxin (10 ng/ml), Src family protein tyrosine kinase (PTK) inhibitor PP2 (1 microM), protein kinase C (PKC) inhibitor Ro31-8220 (0.1 microM) and mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 microM) significantly inhibited the mitogenic effect of 5-HT and its interaction with MCP-1. Anti-MCP-1 antibody (2 microg/ml) and the Janus kinase 2 (JAK2) inhibitor AG490 (10 microM) significantly inhibited the interaction of MCP-1 with 5-HT. Further, the amplified mitogenic effect of 5-HT with MCP-1 was completely reversed by the combined use of sarpogrelate with anti-MCP-1 antibody. Our results suggest that MCP-1 amplifies the mitogenic effect of 5-HT on VSMCs. The mitogenic effect of 5-HT may be mediated by the G protein-Src family PTK-PKC-MAPK pathway. The activation of the JAK2/signal transducer and activator of transcription 3 pathway by MCP-1 in addition to the MAPK pathway by 5-HT may explain the potentiating effect of MCP-1 on 5-HT-induced mitogenesis.
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PMID:Monocyte chemotactic protein 1 amplifies serotonin-induced vascular smooth muscle cell proliferation. 1145 5

Previous studies in our laboratory have shown that in NIH3T3-5HT2A cells, 5-HT-induced AA release is PLA2-coupled and independent of 5-HT2A receptor-mediated PLC activation. Although 5-HT2A receptor-mediated PLC activation is known to be Galphaq-coupled, much less is understood about 5-HT2A receptor-mediated PLA2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT2A receptor to PLA2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT2A receptor can couple to PLA2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Galphai/o, as well as pertussis toxin-insensitive G proteins, namely Galpha12/13, in 5-HT-induced AA release. Furthermore, inactivation of both Gbetagamma heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT2A receptor expressed in NIH3T3 cells can couple to PLA2 activation though a complex signaling mechanism involving both Galphai/o-associated Gbetagamma-mediated ERK1,2 activation and Galpha12/13-coupled, Rho-mediated p38 activation.
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PMID:A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases. 1288 95


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