IAP-sensitive GTP-binding Protein Research Articles

Alpha 2 adrenoceptor potentiates glycine receptor-mediated taurine response through protein kinase A in rat substantia nigra neurons.

1. The modulatory effect of alpha 2 adrenoceptor on the taurine response was investigated in substantia nigra (SN) neurons acutely dissociated from the rat using a nystatin perforated-patch recording mode under voltage-clamp conditions. 2. Complete cross-desensitization was observed between 10(-3) M glycine and 3 x 10(-3) M taurine-induced currents. Both currents were antagonized by 10(-6) M strychnine, thus indicating that taurine acts on strychnine-sensitive glycine receptor on the SN neurons. 3. The simultaneous application of norepinephrine (NE) with prazosin (10(-5) M) and propranolol (10(-5) M) potentiated the taurine response (Itau) in an NE concentration-dependent manner at a holding potential (VH) of -40 mV. Clonidine mimicked the NE effect on the Itau, thus indicating the involvement of alpha 2 adrenoceptor activation in the potentiation of Itau. 4. Alpha 2 adrenoceptor activation by NE with prazosin and propranolol significantly potentiated the peak amplitude of Itau without shifting the taurine concentration-response relationships either to left or right side. The respective concentrations of taurine for the threshold, half maximal and maximal responses in the presence of 10(-4) M NE with prazosin (10(-5) M) and propranolol (10(-5) M) were 3 x 10(-5) M, 3.1 x 10(-4) M, and 3 x 10(-3) M. The same concentrations in the absence of NE were 3 x 10(-5) M, 3.2 x 10(-4) M, and 3 x 10(-3) M, respectively. 5. The reversal potentials of Itau with and without NE were very close to the theoretical Cl- equilibrium potential, thus indicating that the potentiation of Itau by alpha 2 adrenoceptor activation was due to an increase in the taurine-induced Cl- currents. 6. Forskolin (3 x 10(-5) M) and isobutylmethylxanthine (3 x 10(-5) M) suppressed the peak amplitude of Itau. In the presence of dibutyryl cyclic AMP (10(-4) M), which also suppressed Itau, alpha 2 adrenoceptor activation failed to potentiate Itau. 7. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89) mimicked the effect of alpha 2 adrenoceptor activation on Itau. In addition, the potentiation of Itau by alpha 2 adrenoceptor was not observed in the presence of 10(-6) M H-89. 8. The treatment of SN neurons with pertussis toxin (500 ng/ ml) for 18 h completely abolished the facilitatory effect of alpha 2 adrenoceptor on Itau. 9. These results suggest that the activation of alpha 2 adrenoceptor coupled with IAP-sensitive GTP binding protein decreases the intracellular cyclic AMP and cyclic AMP-dependent protein kinase activity, thus resulting in the potentiation of glycine receptor-mediated taurine response in rat SN neurons.

GTP-Binding protein regulates the contractile response elicited by the phorbol ester (PMA)-induced activation of protein kinase C in the isolated rat aorta

1. 1. The aim of the present study was to investigate the involvement of GTP-binding protein in the contractile response induced by activation of protein kinase C (PKC) in isolated rat aorta. The rats were treated with islet-activating protein (IAP) for 4 days prior to the experiments. 2. 2. In the aorta from control rats, phorbol 12-myristate 13-acetate (PMA) produced biphasic contractions; twitch contraction superimposed on the slowly developing contraction. The twitch contraction was abolished by the removal of external Ca 2+ or by treatment with nicardipine. In the aorta pretreated with IAP, PMA produced only a slowly developing contraction, and no twitch contraction was induced. 3. 3. The application of Ca 2+ to aortic strips in a Ca 2+-free solution, that had been treated with 10 −6 M PMA caused concentration-dependent contraction, and the contraction was completely inhibited by IAP. 4. 4. Pretreatment with IAP inhibited Ca 2+-induced contraction of the aorta in Ca 2+-free medium in the presence of 10 −6M clonidine, but did not affect the Ca 2+-induced contraction in the medium treated with 10 −6M phenylephrine and 10 −7M nicardipine. 5. 5. These results suggest that the activation of PKC by PMA produces biphasic contractions in the rat aorta. The twitch contraction may be induced by the activation of voltage-dependent Ca 2+-channels and the activation may be regulated by IAP-sensitive GTP-binding protein.

709 Facilitatory effect of collapsin on axoplasmic transport in mouse DRG neurons

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca2+-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca2+ concentration. ACh also suppressed the axoplasmic transport in Ca2+-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca2+ ions.

Mechanism of the effects of acetylcholine on the contractile properties and Ca2+ transients in ferret ventricular muscles.

1. We investigated the mechanism of signal transduction during the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity and the cross-bridge cycling rate (CCR). 2. Membrane-permeable derivatives of cyclic GMP (8-bromo-cyclic GMP and dibutyryl cyclic GMP) did not cause any significant changes in the peaks of Ca2+ transients and tension and the time courses of either signal modulated by isoprenaline (Iso) (0.1 microM). 3. Nitroprusside (0.1-1 mM) likewise did not change the peaks or the time courses of Ca2+ transients and tension in the Iso-treated preparations. 4. In papillary muscles excised from ferrets treated with pertussis toxin (islet-activating protein, IAP), which is known to abolish the function of GTP-binding proteins (Gi, Go and Gt), similar changes in Ca2+ transients and tension produced by treatment with Iso (0.1 microM) were noted as in non-IAP-treated preparations. However, no effects of acetylcholine (ACh; 1 microM) on either signal were observed. 5. The relation between [Ca2+]i and tension measured during the steady state of tetanic contraction was shifted to the right by Iso (0.1 microM), and cyclic GMP derivatives (1 mM) did not change the altered relation. In the IAP-treated preparations, ACh (1 microM) did not influence the relation altered by Iso (0.1 microM). 6. Cyclic GMP derivatives (1 mM) did not alter the Iso (0.1 microM)-increased CCR measured by perturbation analysis. ACh (1 microM) did not restore the Iso-increased CCR in the IAP-treated preparations. 7. These results suggest that signal transduction in muscarinic receptor stimulation is primarily mediated by inhibition of adenylate cyclase via IAP-sensitive GTP-binding proteins, and that cyclic GMP does not play an important role in the effect of muscarinic receptor stimulation on Ca2+ transients, tension, Ca2+ sensitivity or CCR.

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca 2+-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca 2+ concentration. ACh also suppressed the axoplasmic transport in Ca 2+-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca 2+ ions.

Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.

Change of intracellular calcium of neural cells induced by extracellular ATP

Exposure of various neural cells to ATP increased intracellular Ca 2+ and the production of inositol trisphosphate. The Ca 2+ responses were also observed in the absence of extracellular Ca 2+, suggesting that a part of Ca 2+ mobilization took place from cytosolic storage. Since adenosine had no effect on intracellular Ca 2+ increment, ATP appears to act through a P 2-purinergic receptor. Islet-activating protein or pertussis toxin pretreatment hardly influenced the increase in intracellular Ca 2+ and inositol trisphosphate production induced by ATP, suggesting that IAP-sensitive GTP-binding proteins do not play a practical role in this reaction.

Adenosine 3′:5′-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 ± 5% of control value after 15 min exposure to 10 −7M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 μg/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22,4 ng/ml and 46,5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein.

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.

Effects of pertussis toxin on the affinity of exocytosis for Ca 2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K +. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca 2+-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca 2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca 2+-triggered process in the machinery of exocytosis by modulating the affinity for Ca 2+ of its unknown target.

Functional Coupling of the γ‐Aminobutyric AcidB Receptor with Calcium Ion Channel and GTP‐Binding Protein and Its Alteration Following Solubilization of the γ‐Aminobutyric AcidB Receptor

The coupling mechanism of the gamma-aminobutyric acid (GABA)B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP-binding protein was examined using a crude synaptic membrane (P2) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P2 fraction, [3H]GABA binding to the GABAB receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, did not affect on the calcium ion-induced enhancement of GABAB receptor binding. These results suggest that the GABAB receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABAB receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (-)-baclofen. Although the affinity of (-)-baclofen for the GABAB receptor was decreased in the presence of GTP, pretreatment of the P2 fraction with islet-activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (-)-baclofen induced an increase of GTPase activity in the P2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABAB receptor may also be functionally coupled with IAP-sensitive GTP-binding protein. Treatment of the P2 fraction with sodium deoxycholate resulted in the highest solubilization of GABAB receptor among various detergents examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and characterization of leukotriene B 4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B 4 (LTB 4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with K d and B max values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB 4, and K i values for 20-hydroxy- and 20-carboxy-LTB 4, both inactive metabolites of LTB 4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 μM GTPγS, a low affinity binding site appeared with a K d value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTPγS on LTB 4 binding, indicating that the LTB 4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.

Negative coupling of γ-aminobutyric acid (GABA)B receptor with phosphatidylinositol turnover in the brain

Possible coupling between the ?-aminobutyric acid (GABA)(B) receptor, one of the GABA receptor subtypes, and phosphatidylinositol turnover, known to be a potent intracellular signal transducing system, has been examined in the rat brain. (?)Baclofen, an agonist of the GABA(B) receptor, significantly inhibited the accumulation of inositol-1-phosphate and inositol-1,4,5-triphosphate and these inhibitions were counteracted by the addition of phaclofen, a GABA(B) antagonist, as well as by treatment with islet-activating protein (IAP). These results suggest that the cerebral GABA(B) receptor may be coupled negatively with phosphatidylinositol turnover, and this negative coupling may also be mediated by the action of an IAP-sensitive GTP-binding protein.

FMRFamide-induced K +-current is mediated by IAP-sensitive GTP-binding protein

FMRFamide-induced K +-current is mediated by IAP-sensitive GTP-binding protein

Coupling of adrenal medullary opioid receptors to islet-activating protein-sensitive GTP-binding proteins

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin) -sensitive GTP-binding proteins was investigated by studying effects of guanyl-5′-y1 imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [ 3H]D-Ala 2-D-Leu 5 -enkephalin ([ 3H]DADLE) binding by increasing the dissociation constant of [ 3H]DADLE and membranes, and enhanced slightly [ 3H]diprenorphine binding. IAP treatment of membranes reduced [ 3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [ 3H]DADLE binding. Treatment of membranes with IAP and [ 32P]NAD resulted in radiolabeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, β-endorphin, levorphanol and dynorphin A (1–13) did not show any significant inhibitory action on bovine adrenal medulary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.

Islet activating protein-sensitive guanosine triphosphate-binding protein regulates K+-channels coupled with FMRFamide receptors.

A neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) induces K+-dependent outward current in Aplysia neurons. Intracellular application of islet activating protein (IAP) irreversibly and selectively blocked this outward current without affecting resting membrane conductance. An injection of GTP gamma S, a nonhydrolyzable analogue of guanosine triphosphate (GTP), caused very slow irreversible increase in K+-conductance of the resting membrane. However, repetitive applications of FMRFamide significantly expedited the effect of GTP gamma S. These results strongly suggest that K+-channel opening induced by FMRFamide is regulated by an IAP-sensitive GTP-binding protein.