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Dean D Schwartz - One of the best experts on this subject based on the ideXlab platform.
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activation of alpha 2 adrenergic receptors inhibits norepinephrine release by a Pertussis Toxin insensitive pathway independent of changes in cytosolic calcium in cultured rat sympathetic neurons
Journal of Pharmacology and Experimental Therapeutics, 1997Co-Authors: Dean D SchwartzAbstract:Whole-cell electrophysiological studies suggest that sympathetic nerve alpha-2 adrenergic receptors are coupled to voltage-dependent N-type calcium channels through the Gi family of proteins to inhibit neurotransmitter release. Because most nerve terminals are too small for direct electrophysiological recordings, the aim of this study was to examine the relationship between alpha-2 adrenergic receptor-mediated inhibition of norepinephrine release and the rise in cytosolic calcium in neurites from cultured sympathetic neurons. In cultured rat superior cervical ganglion neurons, the alpha-2 adrenergic receptor agonists, UK-14304 (0.01-10 microM) and oxymetazoline (0.1-10 microM), and the N-type calcium channel blocker, omega-conoToxin GVIA (0.1-10 nM), inhibited the release of tritiated norepinephrine in response to electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V). The inhibitory effect of the alpha-2 adrenergic receptor agonists was not altered by pretreatment with Pertussis Toxin (200 ng/ml, 18 h), although Pertussis Toxin blocked the inhibition of forskolin-stimulated cAMP accumulation by UK-14304. In fura-2 loaded cells, electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V) increased cytosolic calcium in sympathetic neuronal processes. Blockade of N-type calcium channels with omega-conoToxin (1 and 10 nM) reduced the rise in cytosolic calcium by 25 +/- 3% and 52 +/- 6%, respectively, whereas UK-14304 and oxymetazoline did not alter the electrically stimulated rise in cytosolic calcium. These data suggest that blockade of N-type calcium channels with omega-conoToxin GVIA inhibits stimulated norepinephrine release and cytosolic calcium measured with fura-2 at similar concentrations, whereas activation of alpha-2 adrenergic receptor inhibits norepinephrine release by a pathway that is insensitive to Pertussis Toxin and changes in cytosolic calcium in neurites from cultured rat superior cervical ganglion cells.
Katsutoshi Goto - One of the best experts on this subject based on the ideXlab platform.
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orexin receptor type 1 couples exclusively to Pertussis Toxin insensitive g proteins while orexin receptor type 2 couples to both Pertussis Toxin sensitive and insensitive g proteins
Journal of Pharmacological Sciences, 2003Co-Authors: Yoshihiro Miwa, Megumi Shibahara, Toshihiko Yada, Takeshi Sakurai, Akihiro Yamanaka, Katsutoshi GotoAbstract:Abstract Signal transduction pathways of orexin receptors were examined using a nerve-like cell line transfected with orexin receptor type-1 (OX1R) and orexin receptor type-2 (OX2R). Forskolin-stimulated cyclic adenosine 3,5-monophosphate (cAMP) accumulation in OX2R-expressing cells was inhibited by orexin in a dose-dependent manner, and the effect was abolished by pretreatment with Pertussis Toxin (PTX). The inhibitory effect of orexin on forskolin-stimulated cAMP accumulation was not observed in OX1R-expressing cells. Administration of orexin to these cells resulted in a transient increase of intracellular calcium concentration ([Ca2+]i). Orexin-stimulated increases in [Ca2+]i in OX1R- or OX2R-expressing cells were not affected by the PTX pretreatment. These observations suggest that OX1R couples exclusively to PTX-insensitive G-proteins, while OX2R couples to both PTX-sensitive and -insensitive G-proteins. To examine the relative contributions of these G-proteins in OX2R-mediated activation of neurons, we used histaminergic tuberomammillary nucleus neurons, in which OX2R is abundantly expressed. We found that a phospholipase C (PLC)-inhibitor, U73122, inhibits orexin-mediated neuronal activation, but PTX showed no effect on it. This suggests that although OX2R couples to multiple G-proteins, activation of neurons by orexins through OX2R is mediated via a PTX-insensitive, PLC dependent pathway.
Toshiro Fujita - One of the best experts on this subject based on the ideXlab platform.
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a newly identified peptide proadrenomedullin n terminal 20 peptide induces hypotensive action via Pertussis Toxin sensitive mechanisms
Hypertension, 1997Co-Authors: Tatsuo Shimosawa, Katsuyuki Ando, Toshiro FujitaAbstract:Abstract Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) are novel hypotensive peptides. Although they are derived from the same gene product, proadrenomedullin, their hypotensive mechanisms are different; PAMP inhibits the release of norepinephrine from the peripheral sympathetic nerve endings, whereas AM fosters vasodilation by elevating intracellular cAMP, possibly via activation of cholera Toxin–sensitive G proteins. In PC12 cells, PAMP inhibited N-type calcium channel via activation of Pertussis Toxin–sensitive mechanisms. To clarify the relationship between the hypotensive effect of PAMP and Pertussis Toxin−sensitive mechanisms, we administered Pertussis vaccine intraperitoneally into rats for 3 consecutive days. By using mesenteric artery preparation, we showed that PAMP’s ability to decrease norepinephrine overflow was significantly attenuated in Pertussis Toxin−treated rat (−18.5±6.9%; P <.05 versus control rats). In electrically stimulated pithed rat, PAMP (20 and 40 nmol/kg) showed a hypotensive effect (−13±5 and −18±7 mm Hg, respectively; P <.05, P <.01), whereas in Pertussis vaccine−treated rat it did not (−2±3 and −8±9 mm Hg, respectively; P =NS). Also, in pithed rat, plasma norepinephrine level was significantly elevated by electrical stimulation in both control (0.323±0.035 ng/mL) and Pertussis vaccine− treated groups (0.355±0.079 ng/mL). After injection of PAMP (40 nmol/kg), plasma norepinephrine level significantly decreased in the control group (0.225±0.044 ng/mL; P <.01) but not in the Pertussis vaccine−treated group (0.392±0.021 ng/mL; P =NS). Moreover, in conscious rats, intravenous administration of PAMP (40 nmol/kg) did not evoke hypotension after Pertussis vaccine treatment, although untreated controls had significantly decreased arterial pressure (−5±2 versus −20±3 mm Hg; P <.01). In contrast to PAMP, the administration of AM (1 nmol/kg) significantly reduced the blood pressure of Pertussis vaccine−treated as well as control rats (−20±5 versus −18±7 mm Hg; P =NS). These results demonstrate that the ability of PAMP to inhibit norepinephrine release from peripheral sympathetic nerve endings and to decrease blood pressure is Pertussis Toxin sensitive. Our findings thus suggest that despite being derived from the same gene, PAMP and AM apparently produce hypotension by activating different signaling pathways.
Raimundo Goberna - One of the best experts on this subject based on the ideXlab platform.
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pancreastatin activates Pertussis Toxin sensitive guanylate cyclase and Pertussis Toxin insensitive phospholipase c in rat liver membranes
Journal of Cellular Biochemistry, 1994Co-Authors: Victor Sanchezmargalet, Raimundo GobernaAbstract:We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of Pertussis Toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10−7 M pancreastatin (EC50 = 3 nM). This stimulation was partially inhibited by treatment of the membranes with Pertussis Toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30°C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10−7 M pancreastatin at 10 min (EC50 = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with Pertussis Toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2–5 min) at 30°C by 10−7 M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10−7 M pancreastatin, maximal stimulation was obtained (EC50 = 3 nM). GTP (10−5 M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10−5 M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with Pertussis Toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas GMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with Pertussis Toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin. In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, Pertussis Toxin-sensitive, and -insensitive, respectively. © 1994 Wiley-Liss, Inc.
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pancreastatin increases free cytosolic ca2 in rat hepatocytes involving both Pertussis Toxin sensitive and insensitive mechanisms
Biochemical Journal, 1993Co-Authors: Victor Sanchezmargalet, Miguel Lucas, Raimundo GobernaAbstract:Freshly isolated rat hepatocytes, loaded with the Ca2+ probe Fluo-3, responded to homologous pancreastatin with a sudden increase in free cytosolic Ca2+ ([Ca2+]i) as well as glucose release. Addition of rat pancreastatin (0.1 microM) to hepatocytes resulted in an increase in [Ca2+]i from 150 nM to 700 nM, which declined back to nearly basal values within 2-3 min. Half-maximal and maximal effects were observed at 0.3 and 100 nM pancreastatin respectively. The increase in [Ca2+]i induced by vasopressin and noradrenaline was very similar in extent (from 150 to 800 nM) to that produced by pancreastatin. Neither the alpha 1-adrenergic blocker prazosin nor the vasopressin antagonist V1 modified the increase in [Ca2+]i induced by pancreastatin. Pig pancreastatin and its 33-49 C-terminal fragment produced about 65 and 75% of the effect of homologous pancreastatin respectively. Glucose production correlated with changes in [Ca2+]i in the same order of potency: vasopressin > rat pancreastatin > pig 33-49 pancreastatin > pig 1-49 pancreastatin. The effect of pancreastatin on [Ca2+]i was decreased by 50% when Ca2+ was omitted from the medium, and totally abolished when hepatocytes were depleted of internal Ca2+ stores by preincubation without Ca2+ and with 2 mM EGTA. When hepatocytes were preincubated for 5 min with PMA, the effects of ATP and noradrenaline were prevented, and those of vasopressin and pancreastatin remained unchanged. The pretreatment of hepatocytes with Pertussis Toxin diminished the response to pancreastatin and vasopressin. These results suggest that pancreastatin is a new Ca(2+)-mobilizing glycogenolytic hormone acting through a specific receptor which may involve both Pertussis-Toxin-sensitive and -insensitive GTP-binding regulatory proteins.
Toshiaki Katada - One of the best experts on this subject based on the ideXlab platform.
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Pertussis Toxin catalyzed adp ribosylation of gtp binding proteins with digoxigenin conjugated nad identification of the proteins in plasma membranes and nuclei
FEBS Letters, 1994Co-Authors: Yoshinori Takei, Katsunobu Takahashi, Yasunori Kanaho, Toshiaki KatadaAbstract:Abstract ADP-ribose moiety containing digoxigenin was transferred by Pertussis Toxin (IAP) to the α subunit of Gi (Giα) from digoxigenin-conjugated NAD (DIG-NAD) in a βγ subunit-dependent manner. ADP-ribosylation of Giα with DIG-NAD plus IAP was inhibited by native NAD. These results indicate that nonradiolabeled DIG-NAD also serves as the substrate for IAP-catalyzed ADP-ribosylation of G proteins. Using DIG-NAD and fluorescein isothiocyanate-labeled anti-digoxigenin antibody, IAP-sensitive G protein(s) was found to be exist in nuclei as well as plasma membranes of rat liver and HeLa cells. Thus, DIG-NAD is useful to identify Pertussis Toxin-substrate G proteins.