Pertussis Toxin-sensitive G-protein Research Articles

Potentiation of nicotinic currents by bradykinin in the paratracheal ganglia neurons of rats

The effects of bradykinin on nicotine-induced responses were investigated in neurons dissociated from rat paratracheal ganglia using the nystatin-perforated patch-clamp recording technique. When bradykinin (10 − 9 to 10 − 8 M) was pretreated and then simultaneously applied with 10 − 5 M nicotine, bradykinin potentiated the nicotine-induced currents. The potentiation was mimicked by [Hyp 3]-bradykinin and inhibited by HOE-140, pertussis toxin, neomycin and U-73122, but not U-73433. These results suggest that bradykinin potentiates nicotinic currents via bradykinin B 2 receptor, pertussis toxin-sensitive G-protein and phospholipase C. Since bradykinin inhibits the M-current via bradykinin B 2 receptor and pertussis toxin-insensitive G-protein [Mochidome, T., Ishibashi, H., Takahama, K., 2001. Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents. Neuroscience 105, 785–791.], it seemed that bradykinin B 2 receptor activated two distinct signal transduction pathways in the paratracheal ganglia neurons. This effect of bradykinin might cause enhanced synaptic transmission in paratracheal ganglia neurons and contribute to the aggravation of pathological conditions of the lower airway via enhanced acetylcholine release from the postganglionic nerve terminals.

Identification of novel gamete receptors that mediate sperm adhesion to the egg coat

Mammalian fertilization is initiated by the species-specific binding of sperm to the zona pellucida, or egg coat. Earlier studies suggested that sperm–egg adhesion in mouse is mediated by the binding of β1,4-galactosyltransferase-I (GalT) on the sperm surface to specific glycoside ligands on the egg coat glycoprotein, ZP3. Binding of multiple ZP3 oligosaccharides induces GalT aggregation, triggering a pertussis toxin-sensitive G-protein cascade leading to induction of the acrosome reaction. Consistent with this, sperm bearing targeted deletions in GalT are unable to bind ZP3 nor undergo ZP3-dependent acrosomal exocytosis; however, GalT-null sperm are still able to bind to the egg coat. This indicates that sperm–egg binding requires at least two independent binding mechanisms: a GalT–ZP3-independent event that mediates initial adhesion, followed by a GalT–ZP3 interaction that facilitates acrosomal exocytosis. During the past few years, novel GalT–ZP3-independent gamete receptors have been identified that appear to participate in initial gamete adhesion. On such receptor is SED1, an EGF repeat and discoidin domain protein that coats sperm as they traverse through the epididymis, and which is required for sperm to bind the egg coat. Similarly, a novel egg coat ligand is present on ovulated oocytes, but not on ovarian eggs, and which also appears to function in initial sperm binding. The identification of novel gamete receptors that are required for sperm–egg binding opens up new avenues for the development of specific contraceptive strategies.

U50488 inhibits outwardly rectifying potassium channel in PC12 cells via pertussis toxin-sensitive G-protein

This study was undertaken to determine the effect of U50488, a κ-opioid receptor agonist, on outwardly rectifying potassium channel (Ik) in undifferentiated PC12 cells. Using whole-cell and on-cell patch–clamp techniques, we found that U50488 decreased Ik amplitude in a time-dependent manner and Ik activation was delayed. Single-channel kinetic analysis provided a two-stage model for us to illuminate the blockage effect induced by U50488. To identify whether U50488 mediates the effect through opioid receptor and G-protein, several specific blockers and activators were used. Not only naloxone but also PTX and GDPβS abolished U50488-induced suppression; however, such effect was not observed when cAMP or other adenylyl cyclase activators were used. It is postulated that κ-opioid receptor and G i/o protein, but not cAMP, are involved in U50488-induced suppression of Ik.

Lysophosphatidylserine Stimulates L2071 Mouse Fibroblast Chemotactic Migration via a Process Involving Pertussis Toxin-Sensitive Trimeric G-Proteins

Lysophosphatidylserine (LPS) may be generated after phosphatidylserine-specific phospholipase A2 activation. However, the effects of LPS on cellular activities and the identities of its target molecules have not been fully elucidated. In this study, we observed that LPS stimulates an intracellular calcium increase in L2071 mouse fibroblast cells, and that this increase was inhibited by 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) but not by pertussis toxin, suggesting that LPS stimulates calcium signaling via G-protein coupled receptor-mediated phospholipase C activation. Moreover, LPS-induced calcium mobilization was not inhibited by the lysophosphatidic acid receptor antagonist, (S)-phosphoric acid mono-{2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl} ester (VPC 32183), thus indicating that LPS binds to a receptor other than lysophosphatidic acid receptors. It was also found that LPS stimulates two types of mitogen-activated protein kinase [i.e., extracellular signal-regulated protein kinase (ERK) and p38 kinase] in L2071 cells. Furthermore, these LPS-induced ERK and p38 kinase activations were inhibited by pertussis toxin, which suggests the role of pertussis toxin-sensitive G-proteins in the process. In terms of functional issues, LPS stimulated L2071 cell chemotactic migration, which was completely inhibited by pertussis toxin, indicating the involvement of pertussis toxin-sensitive G(i) protein(s). This chemotaxis of L2071 cells induced by LPS was also dramatically inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and by 2'-amino-3'-methoxyflavone (PD98059). This study demonstrates that LPS stimulates at least two different signaling cascades, one of which involves a pertussis toxin-insensitive but phospholipase C-dependent intracellular calcium increase, and the other involves a pertussis toxin-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and ERK.

Neuropeptide Y as a partial agonist of the Y1 receptor

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC 50 ∼ 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu 31Pro 34) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu 31Pro 34) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

Internalization of cloned pancreatic polypeptide receptors is accelerated by all types of Y4 agonists

Internalization of cloned rat or human Y4 receptors expressed in Chinese hamster ovary (CHO) cells increased with concentration of all types of Y4 agonists, including human and rat pancreatic polypeptides, the Y1 receptor group co-agonists possessing C-terminal TRPRY.NH 2 pentapeptide, and a C-terminally amidated dimeric nonapeptide related to neuropeptide Y, GR231118. These peptides also inhibited forskolin-stimulated adenylyl cyclase activity in Y4 receptor-expressing cells, and stimulated the binding of 35S-labeled GTP-γ-S to pertussis toxin-sensitive G-proteins in particulates from these cells. Peptide VD-11 (differing from GR231118 only by C-terminal oxymethylation) acted as a competitive antagonist in all of the above processes. Agonist-induced stimulation of the Y4 receptor internalization persisted in the presence of allosteric inhibitors of hPP binding, N5-substituted amilorides, which also were relatively little active in G-protein stimulation and cyclase inhibition by Y4 agonists. Acceleration of Y4 receptor internalization by agonists apparently is related to relaxation of allosteric constraints to ligand attachment and sequestration of the receptor–ligand complex.

The role of CCL21 in recruitment of T-precursor cells to fetal thymi

AbstractDuring embryonic development, T-lymphoid precursor cells colonize the thymus. Chemoattraction by the fetal thymus is thought to mediate T-precursor cell colonization. However, the molecules that attract T-precursor cells to the thymus remain unclear. By devising time-lapse visualization in culture, the present results show that alymphoid fetal thymus lobes attract T-precursor cells from fetal liver or fetal blood. CD4–CD8–CD25–CD44+ fetal thymocytes retained the activity to specifically re-enter the thymus. The attraction was predominantly due to I-A–expressing thymic epithelial cells and was mediated by pertussis toxin-sensitive G-protein signals. Among the chemokines produced by the fetal thymus, CCL21, CCL25, and CXCL12 could attract CD4–CD8–CD25–CD44+ fetal thymocytes. However, fetal thymus colonization was markedly diminished by neutralizing antibodies specific for CCL21 and CCL25, but not affected by anti-CXCL12 antibody. Fetal thymus colonization was partially defective in CCL21-deficient plt/plt mice and was further diminished by anti-CCL25 antibody. These results indicate that CCL21 is involved in the recruitment of T-cell precursors to the fetal thymus and suggest that the combination of CCL21 and CCL25 plays a major role in fetal thymus colonization.

Dynamics of fast synaptic excitation during trains of stimulation in myenteric neurons of guinea-pig ileum

Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A 1, opioid, α 2-adrenergic and 5-HT 1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K + (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter.

THE JANUS* FACES OF ADRENOCEPTORS: FACTORS CONTROLLING THE COUPLING OF ADRENOCEPTORS TO MULTIPLE SIGNAL TRANSDUCTION PATHWAYS

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val- d-Met-induced calcium influx

Intracellular calcium (Ca 2+) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca 2+ signaling. The addition of exogenous LPA induced dramatic Ca 2+ influx but not intracellular Ca 2+ release in U937 cells. LPA-induced Ca 2+ influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val- d-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A 2 and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A 2-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca 2+ influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca 2+ influx.

C-peptide induces chemotaxis of human CD4-positive cells: involvement of pertussis toxin-sensitive G-proteins and phosphoinositide 3-kinase.

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. The present study examined the effect of C-peptide on CD4(+) lymphocyte migration, an important process in early atherogenesis. C-peptide stimulated CD4(+) cell chemotaxis in a concentration-dependent manner. This process involves pertussis toxin-sensitive G-proteins as well as activation of phosphoinositide 3-kinase (PI 3-K). Biochemical analysis showed that C-peptide induced recruitment of PI 3-K to the cell membrane as well as PI 3-K activation in human CD4(+) cells. In addition, antidiabetic peroxisome proliferator-activated receptor gamma-activating thiazolidinediones inhibited C-peptide-induced CD4(+) cell chemotaxis as well as PI 3-Kgamma activation. Finally, immunofluorescence staining of thoracic artery specimen of diabetic patients showed intimal CD4(+) cells in areas with C-peptide deposition. Thus, C-peptide might deposit in the arterial intima in diabetic patients during early atherogenesis and subsequently attract CD4(+) cells to migrate into the vessel wall.

Rapid effects of androgens in macrophages

We investigated the existence of membrane receptors for testosterone (mAR) in mouse macrophages of the cell lines IC-21 and RAW 264.7 as well as their roles in nongenomic pathways, gene expression and cell functioning. Both cell lines lack intracellular androgen receptors (iARs) and respond to testosterone with rapid rises in [Ca 2+] i. These rises in [Ca 2+] i can neither be inhibited by iAR- nor by iER blockers, but are rather mediated through mAR. Pharmacological approaches suggest that the mAR belongs to the class of membrane receptors which are coupled to phospholipase C via pertussis toxin (PTX) sensitive G-proteins. The mAR can be localized as specific surface binding sites for testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM)and flow cytometry, and are characterized by their agonist-sequestrability. In order to examine a possible role of the testosterone-induced rise in [Ca 2+] i on gene expression, a c- fos promoter reporter gene construct was transfected into RAW 264.7 macrophages. The increase in [Ca 2+] i induced by testosterone cannot significantly activate the c- fos promoter directly. Also, no significant activation of ERK1/2, JNK/SAPK and p38 can be observed following testosterone-stimulation alone. However, testosterone-induced rises in [Ca 2+] i do have specific effects on gene expression in context with lipopolysaccharide (LPS)-induced genotropic signaling: testosterone specifically down-regulates LPS-induced activation of c- fos promoter, p38 MAPK and NO production. In fetal calf serum (FCS)-induced genotropic signaling, the situation is reversed, i.e. testosterone augments the activation of c- fos promoter and ERK1/2. Our studies demonstrate a cross-talk between the testosterone-induced nongenomic Ca 2+ signaling and the genotropic signaling induced by LPS and FCS in macrophages.

In vivo activation of insulin receptor tyrosine kinase by melatonin in the rat hypothalamus.

Melatonin is the pineal hormone that acts via a pertussis toxin-sensitive G-protein to inhibit adenylate cyclase. However, the intracellular signalling effects of melatonin are not completely understood. Melatonin receptors are mainly present in the suprachiasmatic nucleus (SCN) and pars tuberalis of both humans and rats. The SCN directly controls, amongst other mechanisms, the circadian rhythm of plasma glucose concentration. In this study, using immunoprecipitation and immunoblotting, we show that melatonin induces rapid tyrosine phosphorylation and activation of the insulin receptor beta-subunit tyrosine kinase (IR) in the rat hypothalamic suprachiasmatic region. Upon IR activation, tyrosine phosphorylation of IRS-1 was detected. In addition, melatonin induced IRS-1/PI3-kinase and IRS-1/SHP-2 associations and downstream AKT serine phosphorylation and MAPK (mitogen-activated protein kinase) phosphorylation, respectively. These results not only indicate a new signal transduction pathway for melatonin, but also a potential cross-talk between melatonin and insulin.

Analysis of calcium responses mediated by the A 3 adenosine receptor in cultured newborn rat cardiac myocytes

Intracellular calcium signaling cascade induced by adenosine A 3 receptor activation was studied in this work. It was found that adenosine A 3 receptor activation (and not A 1 or A 2A adenosine receptors activation) leads to an increase in cytosolic calcium and its further extrusion. A selective A 3 agonist Cl-IB-MECA (2-chloro- N 6-(3-iodobenzyl)adenosine-5′- N-methyluronamide) induced an increase in cytoplasmic calcium in a dose-dependent manner, and was independent on extracellular calcium. The Ca 2+ signal in newborn cardiomyocytes, induced by A 3 receptor activation, is dependent on a pertussis toxin-sensitive G-protein. The action of Cl-IB-MECA was not inhibited by an inhibitor of phospholipase C (PLC), and by antagonists to inositol 1,4,5-trisphosphate (IP 3) receptor. In contrast, inhibition of ryanodine receptor prevented calcium elevation induced by this agonist. It was shown that extrusion of the elevated cytosolic Ca 2+ was achieved via activation of sarcoplasmic reticulum (SR) Ca 2+-reuptake and of sarcolemmal Na +/Ca 2+ exchanger (NCX). The increase in the SR Ca 2+-uptake and NCX Ca 2+ efflux were sufficient not only for compensation of Ca 2+ release from SR after A 3 receptor activation, but also for an effective prevention of extensive increase in intracellular Ca 2+ and may provide mechanism against cellular Ca 2+ overload. In cells with elevated [Ca 2+] i (due to increase of [Ca 2+] o), adenosine or Cl-IB-MECA decreased the [Ca 2+] i toward diastolic control level, whereas agonist of A 1 receptor was ineffective. The protective effect of A 3 receptor agonist was abolished in the presence of selective A 3 receptor antagonist MRS1523.

The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8.

Neutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial-derived formylated peptides, to dominate over endogenous attractants, such as interleukin-8 (IL-8). Here we show that triggering of the formyl peptide receptor (FPR) with f-Met-Leu-Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL-8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so-called end-type (i.e. fMLF) and weak or intermediate-type (i.e. IL-8) chemoattractants, is also operating during activation of the NADPH-oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide-like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL-8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor-alpha and platelet-activating factor. Moreover, the cytoskeleton-disrupting substance, cytochalasin B, had no effect either on IL-8-triggered oxidase activation or on CXCR reactivation. A pertussis toxin-sensitive G-protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3-kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH-oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively.

ACTH stimulates the release of alkaline phosphatase through Gi-mediated activation of a phospholipase C and the release of inositolphosphoglycan

We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.

Dual effects of ATP on rat hippocampal synaptic plasticity.

Although ATP is reported to modulate synaptic plasticity, the mechanism of action of ATP on synaptic transmission is not fully understood. Here we show that ATP enhances long-term potentiation (LTP), and P2X receptor antagonists inhibit this ATP effect, but do not affect paired pulse facilitation (PPF) in rat hippocampal slices. ATP rapidly increases intracellular calcium, and P2X receptor antagonists inhibit this increase in cultured dissociated neurons. These results indicate that ATP enhances LTP via activation of postsynaptic P2X receptors. A pertussis toxin-sensitive G-protein inhibitor significantly attenuates PPF, although it does not affect LTP, indicating that presynaptic P2Y receptors also play an important role in neuronal plasticity. We conclude that ATP modulates synaptic plasticity via dual effects on pre- and post-synaptic mechanisms.

C-Peptide Colocalizes with Macrophages in Early Arteriosclerotic Lesions of Diabetic Subjects and Induces Monocyte Chemotaxis In Vitro

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. This study tested the hypothesis that C-peptide might participate in atherogenesis in these patients. We demonstrate significantly higher intimal C-peptide deposition in thoracic aorta specimens from young diabetic subjects compared with matched nondiabetic controls as determined by immunohistochemical staining. C-peptide colocalized with monocytes/macrophages in the arterial intima of artery specimen from diabetic subjects. In vitro, C-peptide stimulated monocyte chemotaxis in a concentration-dependent manner with a maximal 2.3+/-0.4-fold increase at 1 nmol/L C-peptide. Pertussis toxin, wortmannin, and LY294002 inhibited C-peptide-induced monocyte chemotaxis, suggesting the involvement of pertussis toxin-sensitive G-proteins as well as a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In addition, C-peptide treatment activated PI3K in human monocytes, as demonstrated by PI3K activity assays. C-peptide accumulated in the vessel wall in early atherogenesis in diabetic subjects and may promote monocyte migration into developing lesions. These data support the hypothesis that C-peptide may play an active role in atherogenesis in diabetic patients and suggest a new mechanism for accelerated arterial disease in diabetes.

Celecoxib simulates respiratory burst through pertussis toxin-sensitive G-protein, a possible signal for β 2-integrin expression on human neutrophils

The superoxide anion-generating effect of celecoxib (4-[5-(4-methylpheny)-3-(trifluoromethyl)-1 H-pyrazol-1-yl]benzenesulfonamide); SC58633), a selective cyclooxygenase-2 inhibitor, on human neutrophils was evaluated in this study. Celecoxib induced superoxide anion generation in a concentration-dependent manner in human neutrophils. The EC 50 value of celecoxib on superoxide anion generation was 15.5±2.5 μM. A NADPH oxidase inhibitor, diphenyliodonium (20 μM), and superoxide dismutase (150 U/ml) completely inhibited the free radical generation caused by celecoxib, indicating that the respiratory burst was activated by celecoxib. 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM;10 μM) and staurosporine (200 nM) completely inhibited the superoxide anion release caused by celecoxib, respectively. These data indicated that celecoxib increased superoxide anion release by increasing intracellular calcium and protein kinase C activation. Moreover, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5 H-indolo(2,3-a)pyrrolo(3,4-C)-carbazole (Go-6976; 1 μM) and 3-[1-[3-(amidinothio)propyl-1 H-indol-3-yl]-3-(1-methyl-1 H-indol-3-yl)maleimide, methane sulfate (Ro-31-8220; 0.5 μM), specific inhibitors of conventional protein kinase C isotypes (α, β I and β II), significantly inhibited superoxide anion release caused by celecoxib. Rottlerin (5 μM), a protein kinase C δ inhibitor, did not affect the free radical generation caused by celecoxib. Celecoxib caused translocation of protein kinase C α, β I and β II from the cytosol to the cellular membrane. 2-[2-amino-3-methoxyphenyl]-4 H-1-benzopyran-4-one (PD98059; 20 μM) and wortmannin (100 nM) did not decrease the superoxide anion generation caused by celecoxib, indicating that Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3 kinase) were not involved in the respiratory burst induced by celecoxib. Pertussis toxin (2 μg/ml), a Gi-protein sensitive inhibitor, significantly inhibited superoxide anion release. Moreover, pertussis toxin significantly inhibited intracellular calcium mobilization and protein kinase C α, β I and β II translocation from the cytosol to the membrane. Celecoxib increased β 2-integrin expression on human neutrophils and this effect was inhibited by BAPTA/AM (10 μM), superoxide dismutase (150 U/ml), genistein (25 μM) and PD98059 (20 μM). This information indicated that intracellular calcium, superoxide anion, tyrosine kinase and MAP kinase are involved in β 2-integrin expression. Furthermore, BAPTA/AM, superoxide dismutase and genistein inhibited celecoxib-increased MAP kinase activity, indicating that MAP kinase is a downstream signal for β 2-integrin expression. In conclusion, celecoxib stimulates superoxide anion release from human neutrophils by activating pertussis toxin sensitive G-protein. An increase in intracellular calcium and protein kinase C α, β I and β II is involved in this process. Celecoxib also regulates β 2-integrin expression through superoxide anion release, tyrosine kinase and p42/p44 MAP kinase on human neutrophils.

Differential coupling of m-cholinoceptors to Gi/Go-proteins in failing human myocardium

Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor–G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [ 32P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [ 3H]QNB, was significantly higher in right-atrial (RA ± SEM, 959 ± 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 ± 53 fmol/mg, n = 6). Standardized immunoblots revealed that Giα-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goα (Goα-1 and/or Goα-3) was almost exclusively detectable in RA. Levels of Giα-3 and a 39-kDa splice variant of Goα (Goα-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Giα/Goα subtypes. The carbachol (10 μmol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goα-1/3 (336 ± 95% of LV, n = 4) and for Giα-3 (211 ± 83%), lower for Giα-2 (42 ± 5%), and was similar in both regions for Goα-2 (130 ± 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor–G-protein level.