Biomedical subjects

K H Jakobs

Publications and source records attributed to K H Jakobs.

At least 37 records · Page 2Linked to original sources

PubMed1999

Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade.

Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade.

Bacterial Proteins↗

PubMed1999

Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor.

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.

Calcium Signaling↗

PubMed1999

Rho-specific binding and guanine nucleotide exchange catalysis by KIAA0380, a dbl family member.

Several guanine nucleotide exchange factors (GEFs) for Rho-GTPases have been identified, all of them containing a Dbl homology (DH) and pleckstrin homology (PH) domain, but exhibiting different specificities to the Rho family members, Rho, Rac and Cdc42. We report here that KIAA0380, a protein with a tandem DH/PH domain, an amino-terminal PDZ domain and a regulator of G protein signalling (RGS) homology domain, is a specific GEF for RhoA, but not for Rac1 and Cdc42, as determined by GDP release, guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) binding and protein binding assays. When expressed in J82 cells, DH/PH domain-containing forms of KIAA0380 induced actin stress fibers, whereas expression of the RGS homology domain prevented lysophosphatidic acid (LPA)-induced stress fiber formation.

Actins↗

PubMed1999

Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.

The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level.

Adenylate Cyclase Toxin↗

PubMed1999

A role for rho-kinase in rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor.

Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway.

Cell Membrane↗

PubMed1999

Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin.

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.

Arrestins↗

PubMed1999

The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D.

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.

ADP-Ribosylation Factors↗

PubMed1999

Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase.

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

Calcium↗

PubMed1999

RhoA-sensitive trafficking of muscarinic acetylcholine receptors.

The clathrin-mediated sequestration pathway is used by non-G protein-coupled receptors (e.g., transferrin receptors) and a large number of G protein-coupled receptors, including beta-2 adrenoceptors and various muscarinic acetylcholine receptor (mAChR) subtypes. Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferrin receptor internalization. Because mAChRs and other G protein-coupled receptors are able to activate RhoA, we investigated in HEK-293 cells whether RhoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectively. Overexpression of wild-type RhoA inhibited agonist-induced sequestration of both m1 and m2 mAChRs by as much as 70%. Inhibition could be reversed by coexpression of Clostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation. Overexpression of C3 transferase alone had no effect on m1 and m2 mAChR sequestration. In addition, overexpression of RhoA inhibited m1 and m2 mAChR transport to the plasma membrane by 60 and 31%, respectively, which was blocked by coexpression of C3 transferase. We conclude that RhoA is not an endogenous regulator of mAChR sequestration, but when overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.

Biological Transport↗

PubMed1998

Tyrosine-phosphorylation-dependent and rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels.

The polyphosphoinositide PtdIns(4,5)P2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes. The aim of this study was to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M3 muscarinic acetylcholine receptor. Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2. 5-fold increase in the total cellular level of PtdIns(4,5)P2, which was both time- and concentration-dependent. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P2 formation. Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in the HEK-293 cell PtdIns(4,5)P2 level, which was not altered by additional genistein treatment. Furthermore, toxin B treatment abolished the pervanadate-induced increase in PtdIns(4,5)P2 levels. As PtdIns(4,5)P2 is an essential stimulatory cofactor for phospholipase D (PLD) enzymes, we finally examined the effects of the agents regulating PtdIns(4,5)P2 levels on PLD activity in HEK-293 cells. Inhibition of tyrosine phosphatases by pervanadate caused an increase in PLD activity, which was susceptible to genistein and tyrphostin 23, and which was abolished by prior treatment with toxin B. In conclusion, the data presented indicate that the cellular level of the multifunctional phospholipid, PtdIns(4,5)P2, in HEK-293 cells is controlled by a tyrosine-kinase-dependent mechanism and that this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation.

ADP-Ribosylation Factors↗

PubMed1998

Discrimination between plasma membrane and intracellular target sites of sphingosylphosphorylcholine.

On the background of the emerging concept of G protein-coupled sphingolipid receptors, Ca2+ mobilization by sphingosylphosphorylcholine (SPPC) in intact cells and SPPC-induced Ca2+ release in permeabilized cells, both occurring at similar, micromolar concentrations, were characterized and compared. In intact human embryonic kidney (HEK-293) cells, SPPC rapidly increased [Ca2+]i by mobilization of Ca2+ from thapsigargin-sensitive stores. In saponin-permeabilized HEK-293 cells, SPPC released stored Ca2+, in a manner similar to but independent of inositol 1,4,5-trisphosphate. Only the action of SPPC on intact cells, but not that in permeabilized cells, was, at least in part, sensitive to pertussis toxin. In addition and most important, Ca2+ release by SPPC in permeabilized cells was not stereoselective, whereas in intact cells only the naturally occurring D-erythro-SPPC, but not L-threo-SPPC, increased [Ca2+]i. Stereoselectivity of SPPC-induced [Ca2+]i increase was also demonstrated in bovine aortic endothelial cells. In conclusion, Ca2+ mobilization by SPPC in intact cells is independent of the previously described SPPC-gated Ca2+ channel on endoplasmic reticulum but probably mediated by a membrane sphingolipid receptor. Thus, SPPC can regulate Ca2+ homeostasis by acting apparently at two cellular targets, which exhibit clearly distinct recognition patterns.

Animals↗

PubMed1998

Receptor subtype-specific regulation of muscarinic acetylcholine receptor sequestration by dynamin. Distinct sequestration of m2 receptors.

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.

Animals↗

PubMed1998

Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors.

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.

Animals↗

PubMed1998

Specific inhibition of phorbol ester-stimulated phospholipase D by Clostridium sordellii lethal toxin and Clostridium difficile toxin B-1470 in HEK-293 cells. Restoration by Ral GTPases.

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.

3T3 Cells↗

PubMed1998

Association of a human G-protein beta3 subunit variant with hypertension.

Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.

Alleles↗

PubMed1998

Guanine nucleotide-sensitive inhibition of L-type Ca2+ current by lysosphingolipids in RINm5F insulinoma cells.

The lysosphingolipids sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) reportedly increase free cytosolic Ca2+ concentration ([Ca2+]i) in a variety of cell types, apparently by activating G protein-coupled plasma membrane receptors. We investigated whether and how sphingolipids modulate Ca2+ homeostasis in the insulinoma cell line RINm5F. The addition of SPPC and glucopsychosine (GPS) did not affect basal [Ca2+]i but inhibited the KCl (30 mM)-induced increase in [Ca2+]i in a pertussis toxin-insensitive and concentration-dependent manner (EC50 approximately 5 micro M). Similar inhibitory effects were observed with dihydro-SPPC and psychosine, whereas SPP and various N-acylated sphingolipids (at 10 micro M each) had little or no effect on the KCl-induced [Ca2+]i increase. Because in RINm5F cells the primary pathway for depolarization-induced [Ca2+]i increase are L-type Ca2+ channels, we studied whether sphingolipids reduce L-type Ca2+ current (ICa.L). When added to the bath, GPS and SPPC, but not SPP (10 micro M each), rapidly reduced maximal ICa.L by approximately 35%, similar to the alpha2-adrenoceptor agonist clonidine (30 micro M). However, when applied internally, GPS had no effect on ICa. L. When the electrode solution contained the stable GDP analog guanosine-5'-O-(2-thio)diphosphate (1 and 10 mM), the inhibitory effect of GPS was abolished. In conclusion, a novel cellular action of lysosphingolipids is observed in RINm5F cells (i.e., a guanine nucleotide-sensitive inhibition of L-type Ca2+ currents). The pharmacological profile of this inhibition is unique and unlike any known lysosphingolipid receptor-mediated action.

Barium↗

PubMed1998

Gi- and protein kinase C-mediated heterologous potentiation of phospholipase C signaling by G protein-coupled receptors.

We recently reported that activation of the highly efficient phospholipase C (PLC) stimulatory m3 muscarinic acetylcholine receptor (mAChR) can induce a long-lasting Gi-mediated heterologous potentiation of PLC stimulation in human embryonic kidney (HEK) 293 cells, which was accompanied by an increased cellular level of the PLC substrate phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Here, we examined whether such a potentiated PLC response is also induced by the rather poorly PLC stimulatory m2 mAChR and the endogenously expressed purinergic and lysophosphatidic acid receptors. Pretreatment of m2 mAChR-expressing HEK 293 cells for 2 min with carbachol, followed by agonist washout and measurement of PLC activity >/=40 min later, caused a long-lasting (up to approximately 90 min) heterologous potentiation of receptor- and G protein-mediated PLC stimulation. A similar heterologous potentiation of receptor-mediated PLC stimulation was induced by short term activation of lysophosphatidic acid and purinergic receptors. Either of the three receptor agonists increased the cellular level of PtdIns(4,5)P2 by approximately 50%. The mAChR-induced PLC potentiation was fully prevented by either pertussis toxin or the protein kinase C (PKC) inhibitors staurosporine and Gö 6976, which did not affect acute PLC stimulation. On the other hand, the rise in PtdIns(4,5)P2 was prevented only by combined treatment of HEK 293 cells with pertussis toxin and PKC inhibitors. In conclusion, we demonstrated that activation of poorly PLC stimulatory receptors can also induce a long-lasting Gi-mediated heterologous potentiation of PLC signaling in HEK 293 cells and that this novel PLC regulatory process is under the control of PKC.

Adenosine Triphosphate↗

PubMed1997

Characteristics of protein-kinase-C- and ADP-ribosylation-factor-stimulated phospholipase D activities in human embryonic kidney cells.

Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbol-ester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently acting via the GTP-binding proteins, ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [3H]oleic acid, GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [3H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD cofactor, phosphatidylinositol 4,5-bisphosphate, decreased basal and GTP[S]- or PMA-stimulated PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[beta-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the nonselective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cytosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1. potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 and bisindolylmaleimide I. Immunoblot analysis demonstrated that both conventional PKC (alpha, beta, gamma) and atypical PKC isozymes (zeta, tau) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S]' action.

ADP-Ribosylation Factor 1↗