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)

Ca2+ fluxes were examined in HEK 293 cells stably expressing the rat or porcine calcitonin receptors (CTRs). Calcitonin (CT) rapidly increased cytosolic Ca2+ ([Ca2+]i) concentrations in these cells in a manner which was sustained in the presence of extracellular Ca2+ ([Ca2+]e). In cells pretreated with CT, elevation of the [Ca2+]e concentration resulted in a further increase in [Ca2+]i which was concentration-dependent with respect to both the concentration of CT and the increment of [Ca2+]e. Untransfected cells, cells transfected with vector alone, and CTR-transfected cells not treated with CT, were unresponsive to [Ca2+]e. The microsomal Ca(2+)-ATPase inhibitor thapsigargin was able to mimic both the acute [Ca2+]i fluxes and responsiveness to [Ca2+]e mediated by CT in these cells. The CT-induced responsiveness to [Ca2+]e was neither mimicked by, nor affected by, activators of the cAMP or protein kinase C pathways. Treatment of cells with pertussis toxin influenced neither the primary Ca2+ fluxes in response to CT or thapsigargin nor the agonist-induced [Ca2+]e influx. Nifedipine failed to block responses to either CT or thapsigargin. These results lead to the important conclusion that the CTR participates in receptor-activated Ca2+ inflow, in which depletion of intracellular Ca2+ pools leads secondarily to influx of extracellular Ca2+.
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PMID:Calcium inflow in cells transfected with cloned rat and porcine calcitonin receptors. 769 52

It is now widely appreciated that G-protein-coupled cell-surface receptors can modulate distinct signal transduction pathways via coupling to different GTP-binding proteins. In the present study, we have used a transient co-expression approach to study the coupling of a single alpha 2-adrenergic receptor (alpha 2AAR) population to three different G protein subtypes (Gi, Gq, and Gs) acting on two different cellular effectors in HEK 293 cells. In all cases, the affinity of the receptor for the alpha 2A-adrenergic agonist, UK14304, is unchanged (KD approximately equal to 670 nM). However, there is a dramatic difference in the EC50 of UK14304 in eliciting inhibition of endogenous adenylyl cyclase via endogenous Gi (0.09 nM) versus activation of phospholipase C via co-transfected Gq (50 nM) or stimulation of endogenous adenylyl cyclase via co-transfected Gs (70 nM) in HEK 293 cells. These findings are consistent with the interpretations that the alpha 2AAR preferentially interacts with Gi rather than Gs or Gq. When the alpha 2AAR was mutated at Asp79, a residue highly conserved among G-protein-coupled receptors, the mutant D79N alpha 2AAR lost the ability to couple to Gq and Gs and, although it was able to couple to inhibition of cyclase via pertussis toxin-sensitive pathways (Gi), it did so with a lower potency than observed for the wild-type alpha 2AAR (EC50 = 7.2 nM). The most straightforward interpretation of these data is that the D79N mutation in the alpha 2AAR reduces the efficiency of coupling of the alpha 2AAR to all G-proteins, thus eliminating signal transduction through those pathways less efficiently coupled to the alpha 2AAR. Since the transient expression assays described permit manipulation of the structure of both the receptor or the G-protein, the present strategies could be exploited to delineate the complementary domains specifying the affinity and/or efficacy of receptor coupling to distinct GTP-binding proteins.
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PMID:Coupling of the alpha 2A-adrenergic receptor to multiple G-proteins. A simple approach for estimating receptor-G-protein coupling efficiency in a transient expression system. 790 86

We have isolated a 5199-nucleotide cDNA from a mouse library containing an open reading frame encoding the 1099-amino acid type VII adenylyl cyclase protein. The type VII protein is most closely related in primary structure to an unpublished human adenylyl cyclase clone (GenBank accession no. D25538) and type II adenylyl cyclase. Northern blot analysis demonstrates that the type VII mRNA is most abundant in mouse heart, spleen, and lung. cAMP content rises rapidly in HEK 293 cells overexpressing type VII adenylyl cyclase following treatment with phorbol ester, peaking by 4 min, while cells expressing the type II adenylyl cyclase reach peak accumulation only after 20 min. Increases in intracellular calcium through treatment of type VII-293 cells with either ATP or A23187 alone failed to increase intracellular cAMP content. Phorbol ester treatment acted synergistically with beta-adrenergic stimulation to increase cAMP content in type VII-transformed cells. Pretreatment of type VII-transformed cells with pertussis toxin fails to prevent phorbol ester potentiation of isoproterenol stimulation. Thus the ability of phorbol ester to increase basal and isoproterenol-stimulated type VII activity appears to be a direct effect on this adenylyl cyclase isoform and not the result of modification of the inhibitory G protein, Gi.
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PMID:Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells. 796 50

The interaction of the SRIF receptor subtype SSTR2 with pertussis toxin-sensitive G proteins was investigated using an immunoprecipitation approach employing peptide-directed antisera against Gi alpha and G(o) alpha. Antisera directed against either the COOH terminus of Gi alpha or Go alpha uncoupled SSTR2-G protein complexes from CHO cells stably expressing the cloned receptor indicating that both G proteins form complexes with SSTR2. Chinese hamster ovary cells primarily express Gi alpha 3 and G(o) alpha 2 immunoreactivity, with much lower levels of the other pertussis toxin-sensitive G proteins. Antiserum against Gi alpha 3 uncoupled SSTR2/G protein complexes to a similar extent as Gi alpha common antiserum while antisera against Gi alpha 1 and Gi alpha 2 did not. These findings indicate that SSTR2 expressed in Chinese hamster ovary cells predominantly associates with Gi alpha 3 and G(o) alpha 2. In HEK 293 cells which endogenously express low densities of SSTR2 and similar levels of Gi alpha 1 and Gi alpha 3 immunoreactivity but no G(o) alpha, only antiserum directed against Gi alpha 3 immunoprecipitated SSTR2-G protein complexes, indicating that in these cells SSTR2 primarily associates with Gi alpha 3. SRIF can not inhibit forskolin-stimulated cAMP formation in wild-type HEK 293 cells nor in HEK 293 cells transfected with SSTR2. In contrast, SRIF can inhibit cAMP formation in HEK 293 cells expressing the cloned SRIF receptor SSTR3, which requires the presence of Gi alpha 1 to functionally couple to adenylyl cyclase. The lack of efficient association of SSTR2 with Gi alpha 1 may be the cause of its inability to mediate inhibition of cAMP formation. Differences in the G protein-coupling domains of the cloned SRIF receptors may be responsible for their differences in G protein association and ability to effect various signaling pathways.
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PMID:Gi alpha 3 and G(o) alpha selectively associate with the cloned somatostatin receptor subtype SSTR2. 809 3

Muscarinic m4 acetylcholine receptors are normally coupled through Gi to inhibition of adenylyl cyclases. In the olfactory bulb and some cultured cells, however, m4 receptors can couple to stimulation of adenylyl cyclase activity. In this study, m4 receptors and specific isozymes of adenylyl cyclases were coexpressed in HEK-293 cells to characterize the mechanism(s) for m4 receptor stimulation of adenylyl cyclases. The calmodulin-sensitive type I and type III adenylyl cyclases were chosen for this study because neither enzyme is stimulated by the beta/gamma complex of G coupling proteins. M4 receptors exhibited either inhibition or stimulation of type I and III adenylyl cyclases depending upon receptor density and agonist concentration. Inhibition of adenylyl cyclase was apparently due to M4 coupling through Gi. Adenylyl cyclase stimulation through m4 receptors was not due to increases in intracellular Ca2+ and stimulation of the calmodulin-sensitive enzymes since it was evident in isolated membranes in the absence of free Ca2+ and with whole cells preloaded with the Ca2+ chelator BAPTA/AM. Stimulation of adenylyl cyclase activities by m4 receptors was apparently mediated via Gs since it was GTP-dependent, was insensitive to pertussis toxin, and was not due to beta/gamma stimulation. Synthetic peptides derived from a G protein activating region of the m4 receptor mimicked the m4-mediated stimulation of adenylyl cyclase activity. These data demonstrate a novel mechanism for muscarinic regulation of adenylyl cyclases that may involve crossover from inhibitory to stimulatory G protein coupling.
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PMID:A novel mechanism for coupling of m4 muscarinic acetylcholine receptors to calmodulin-sensitive adenylyl cyclases: crossover from G protein-coupled inhibition to stimulation. 830 42

We have cloned a human cDNA for a novel CC chemokine receptor (CC CKR) designated CC CKR5 that has 48-75% amino acid identity to other CC CKRs. CC CKR5 mRNA was detected constitutively in primary adherent monocytes but not in primary neutrophils or eosinophils. Macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and RANTES were all potent agonists for CC CKR5 (EC50 = 3-30 nM) when calcium flux was measured in transfected HEK 293 cells, yet the apparent binding affinities of the corresponding iodinated chemokines to intact cells expressing the receptor were low (IC50 approximately 100 nM). The calcium flux responses were completely blocked by treatment of transfected cells with pertussis toxin. These data suggest that CC CKR5 is a G(i)-coupled receptor that may mediate monocyte responses to MIP-1alpha, MIP-1beta, and RANTES.
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PMID:Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1(alpha), MIP-1(beta), and RANTES. 869 19

The introduction of D1A dopamine receptors and mu-opioid receptors into HEK 293 cells that were also transiently transfected with adenylyl cyclase cDNA imparted to dopamine and to mu-opioid receptor agonists the ability to modulate the activity of the expressed adenylyl cyclase. Dopamine added to cells expressing D1A receptors and type V adenylyl cyclase significantly stimulated type V enzyme activity. The concomitant addition of morphine produced a dose-dependent inhibition of dopamine-stimulated type V adenylyl cyclase activity. On the other hand, if the HEK 293 cells were transfected with cDNA for type VII adenylyl cyclase instead of the type V isoform, morphine stimulated this adenylyl cyclase activity beyond the stimulation produced by dopamine. Both the inhibitory and stimulatory effects of morphine were blocked by naloxone or pretreatment of the transfected HEK 293 cells with pertussis toxin. When expressed in the HEK 293 cells, the alpha subunit of transducin, which is considered to be the putative scavenger of the beta gamma subunits of G proteins, suppressed the stimulatory effect of morphine on type VII adenylyl cyclase. We also expressed the adenylyl cyclases in cells that were transfected with D1A receptor and G beta 1 and G gamma 2 cDNAs. Dopamine was more efficacious in stimulating type VII adenylyl cyclase activity in cells concomitantly transfected with the beta gamma subunit cDNAs than in cells not transfected with these G protein subunits. Transfection with beta gamma subunit cDNAs did not affect dopamine stimulation of type V adenylyl cyclase activity, and morphine-induced inhibition of type V adenylyl cyclase activity was still evident in cells cotransfected with the alpha subunit of transducin. These data support the contention that the effects on type VII adenylyl cyclase activity mediated through the G1/G(o) proteins may depend on the actions of the beta gamma subunits. The same is not the case for type V adenylyl cyclase. Our data demonstrate that both qualitative and quantitative responses to mu-opioid receptor stimulation depend on the isoform of adenylyl cyclase expressed in neurons or other cells of the body.
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PMID:mu-Opioid receptors inhibit dopamine-stimulated activity of type V adenylyl cyclase but enhance dopamine-stimulated activity of type VII adenylyl cyclase. 870 Jan 17

Agonist-bound muscarinic receptors open atrial K+ channels through a GTP-dependent pathway mediated by the G protein Gk. However, nucleotides other than GTP are also able to support channel activity, even in the absence of agonists. This process was proposed to be mediated by nucleoside-diphosphate (NDP) kinase, which would transfer phosphate from nucleotide triphosphates to the GDP bound to Gk, producing Gk-GTP without the need for receptor-induced GDP-GTP exchange. We examined the effect of antibodies to NDP kinase on the ATP-supported activity of atrial muscarinic K+ channels and the corresponding GIRK1/CIR channels expressed in HEK 293 cells. Inhibitory antibodies reduced ATP-induced channel openings, but this effect displayed an absolute requirement for agonist and was also seen with antibodies that do not inhibit the enzyme. Both types of antibodies also reduced agonist-dependent channel activity in the presence of GTP, ruling out a role for NDP kinase in GDP rephosphorylation. Channel activity was not affected by the antibodies in preparations where ATP-induced muscarinic channels are not under tight receptor control, namely pertussis toxin-treated atrial patches and membranes from cells expressing KACh channel subunits. Thus, participation of NDP kinase in this pathway requires activated receptors and has a function distinct from phosphate transfer between nucleotides.
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PMID:Participation of nucleoside-diphosphate kinase in muscarinic K+ channel activation does not involve GTP formation. 870 81

We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.
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PMID:Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH/PTHrP receptor stably expressed in a human kidney cell line. 872 98

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.
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PMID:Differential agonist regulation of the human kappa-opioid receptor. 910 9


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