Pertussis toxin-sensitive G proteins mediate carbachol-induced REM sleep and respiratory depression

1995 ◽  
Vol 269 (2) ◽  
pp. R308-R317 ◽  
Author(s):  
S. L. Shuman ◽  
M. L. Capece ◽  
H. A. Baghdoyan ◽  
R. Lydic
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Respiratory Depression ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Rem Sleep ◽  
State Dependent ◽  
Transduction Mechanisms ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins

Microinjecting cholinomimetics into the medial pontine reticular formation (mPRF) of conscious cats causes a rapid eye movement (REM) sleep-like state and state-dependent respiratory depression. Muscarinic receptors within the mPRF have been shown to mediate this state-dependent respiratory depression, but the specific signal transduction mechanisms remain poorly understood. This study tested the hypothesis that the cholinergically induced REM sleep-like state and state-dependent respiratory depression are mediated by guanine nucleotide binding proteins (G proteins). Cholera toxin, pertussis toxin, 5'-guanylylimidodiphosphate, and forskolin were microinjected alone and in combination with carbachol into the mPRF of intact unanesthetized cats. All of the G protein-altering compounds significantly reduced the ability of carbachol to produce the REM sleep-like state. Pertussis toxin caused the greatest decrease in the percent of time spent in the carbachol-evoked REM sleep-like state, showing for the first time mediation by a pertussis toxin-sensitive (Gi- or G(o)-like) G protein. Cholera toxin blocked the carbachol-induced respiratory depression, indicating mediation by a Gs-like G protein. Forskolin significantly decreased carbachol-evoked REM sleep. These data provide the first demonstration that adenylyl cyclase within the mPRF contributes to the carbachol induction of REM sleep and respiratory depression.

Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells.

1996 ◽  
Vol 199 (10) ◽  
pp. 2153-2160
Author(s):  
S M Wilson ◽  
S Rakhit ◽  
R Murdoch ◽  
J D Pediani ◽  
H Y Elder ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
G Protein ◽  
G Proteins ◽  
Sweat Gland ◽  
Purine Receptors ◽  
Cyclic Amp Accumulation ◽  
Guanine Nucleotide Binding ◽  
Purinergic Nerves ◽  
Nucleotide Binding Proteins

Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx-resistant (alpha q/11) G-proteins were expressed. P2U receptors may, therefore, normally activate both of these G-protein families. Ptx-sensitive, alpha i2/3 subunits permit inhibitory control of adenylate cyclase, and UTP was shown to cause Ptx-sensitive inhibition of adrenaline-evoked cyclic AMP accumulation, suggesting that the receptors activate Gi2/3. Experiments using cells grown on permeable supports suggested that P2U receptors became essentially confined to the apical membrane in post-confluent cultures. Polarised epithelia may, therefore, express apical P2U receptors which influence two centrally important signal transduction pathways. It is highly improbable that these receptors could be activated by nucleotides released from purinergic nerves, but they may be involved in the autocrine regulation of epithelial function.

Is Receptor Cross-Regulation in Human Heart Caused by Alterations in Cardiac Guanine Nucleotide-Binding Proteins?

1993 ◽  
Vol 85 (4) ◽  
pp. 393-399 ◽  
Author(s):  
A. Ferro ◽  
C. Plumpton ◽  
M. J. Brown
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Atrial Appendage ◽  
Right Atrial ◽  
Α Subunit ◽  
Guanine Nucleotide Binding ◽  
Quantitative Changes ◽  
Right Atrial Appendage ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

1. Guanine nucleotide-binding proteins (G-proteins) play a central role in signal transduction between a wide variety of cell-surface receptors and intracellular second messenger systems. Recently, we and others have demonstrated that cross-regulation can occur between a variety of G-protein-linked receptors in human heart. Chronic β1-adrenoceptor blockade gives rise to sensitization of β2-adrenoceptor and of 5HT4-receptor responses, both of which are mediated via stimulation of adenylate cyclase through stimulatory G-proteins (Gs), and also gives rise to desensit-ization of muscarinic M2-receptor responses, which inhibit adenylate cyclase through inhibitory G-proteins (Gi). 2. In order to investigate whether these effects are due to quantitative changes in cardiac G-protein isoforms, we measured their abundance in right atrial appendage from patients taking or not taking β1-adrenoceptor antagonists, by immunoblotting. 3. Samples of right atrial appendage homogenate were subjected to SDS/PAGE, and proteins were electroblotted on to nitrocellulose membranes. These were then probed with specific anti-G protein anti-sera, and binding was revealed by means of a secondary antibody labelled with alkaline phosphatase and using a chromogenic substrate. The resulting bands were quantified by laser densitometry. 4. No quantitative differences were detected, between these two groups of patients, in the amounts of α-subunit of ‘long’ or ‘short’ Gs isoforms (GsαL and GsαS), or in the amounts of Gi 1 + 2 α-subunit (Giα1 + 2). Nor was any difference found in the abundance of the β-subunit of G-proteins. No ‘other’ G-protein (Go) was detectable in these samples by immunoblotting. 5. We conclude that the phenomenon of receptor cross-regulation which we have previously observed in human right atrial appendage is unlikely to be explained by quantitative changes at the G-protein level.

G-protein Activation Decreases Isoflurane Inhibition of N-type Ba2+Currents

2003 ◽  
Vol 99 (2) ◽  
pp. 392-399 ◽  
Author(s):  
Igor M. Nikonorov ◽  
Thomas J. J. Blanck ◽  
Esperanza Recio-Pinto
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Volatile Anesthetic ◽  
Cell Voltage ◽  
Dependent Manner ◽  
Protein Activation ◽  
G Protein Activation ◽  
Voltage Dependent

Background G-protein activation mediates inhibition of N-type Ca2+ currents. Volatile anesthetics affect G-protein pathways at various levels, and activation of G-proteins has been shown to increase the volatile anesthetic potency for inhibiting the electrical-induced contraction in ileum. The authors investigated whether isoflurane inhibition of N-type Ba2+ currents was mediated by G-protein activation. Methods N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method. Results Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 approximately 0.28 mm) in the absence and presence of GDPbetaS (a nonhydrolyzable GDP analog). Interestingly, GTPgammaS (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 microm GTPgammaS completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by approximately 2.4 times (IC50 approximately 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTPgammaS-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin. Conclusions It was found that activation of G-proteins in a neuronal environment dramatically reduced the isoflurane potency for inhibiting N-type Ba2+ currents and, in turn, isoflurane affected the G-protein regulation of N-type Ba2+ currents.

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone

1999 ◽  
Vol 276 (1) ◽  
pp. F10-F17 ◽  
Author(s):  
Sarah Sariban-Sohraby ◽  
Michal Svoboda ◽  
Frédérique Mies
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Mrna Levels ◽  
Short Term ◽  
Gtp Binding ◽  
Epithelial Membranes ◽  
Cultured Renal Epithelia ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPγS revealed a class of binding sites with an apparent K d value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Gαi-3, Gα0, Gαs, and Gαq were not increased after aldosterone. The patterns of small M r G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [α-32P]GTP, in control membranes, showed either no labeling or a faint band of M r 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of M r 59.5 kDa.

scholarly journals Inhibition by pertussis toxin of the activation of Na+-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells

1991 ◽  
Vol 280 (2) ◽  
pp. 515-519 ◽  
Author(s):  
J A Sokoloski ◽  
A C Sartorelli ◽  
R E Handschumacher ◽  
C W Lee
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Dimethyl Sulphoxide ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dependent Transport

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).

scholarly journals Differential regulation of amounts of the guanine-nucleotide-binding proteins Gi and Go in neuroblastoma x glioma hybrid cells in response to dibutyryl cyclic AMP.

1988 ◽  
Vol 256 (2) ◽  
pp. 649-656 ◽  
Author(s):  
I Mullaney ◽  
A I Magee ◽  
C G Unson ◽  
G Milligan
Keyword(s):  
Cyclic Amp ◽  
Cell Line ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Guanine Nucleotide ◽  
Dibutyryl Cyclic Amp ◽  
Differentiated Cells ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the ‘differentiated’ cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the ‘differentiated’ cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the ‘differentiated’-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.

scholarly journals Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides

1990 ◽  
Vol 267 (3) ◽  
pp. 795-802 ◽  
Author(s):  
R Seifert ◽  
G Schultz ◽  
M Richter-Freund ◽  
J Metzger ◽  
K H Wiesmüller ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Human Neutrophils ◽  
Guanine Nucleotide ◽  
Chemotactic Peptide ◽  
Chemotactic Peptides ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.

scholarly journals Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3

1989 ◽  
Vol 262 (2) ◽  
pp. 403-408 ◽  
Author(s):  
F M Mitchell ◽  
S L Griffiths ◽  
E D Saggerson ◽  
M D Houslay ◽  
J T Knowler ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Alpha Subunits ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.

Regulation of renal cortical Na-HCO3 cotransporter. II. Role of G proteins

1995 ◽  
Vol 268 (3) ◽  
pp. F461-F467 ◽  
Author(s):  
O. S. Ruiz ◽  
Y. Y. Qiu ◽  
L. J. Wang ◽  
J. A. Arruda
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inhibitory Effect ◽  
Adp Ribosylation ◽  
Protein Activator ◽  
The Difference ◽  
22Na Uptake ◽  
Gamma S

We examined the regulation of the renal cortical basolateral Na-HCO3 cotransporter by G proteins. Na-HCO3 cotransporter activity was measured in highly purified rabbit renal cortical basolateral membranes (BLMV) as the difference in 22Na uptake in presence of HCO3- and gluconate. HCO(3-)-dependent 22Na uptake was significantly inhibited by 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a G protein activator. In contrast, addition of 50 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), an inhibitor of G protein, prevented the inhibition of the Na-HCO3 cotransporter activity by GTP gamma S. AlF4-, another G protein activator, also inhibited the activity of the Na-HCO3 cotransporter. This inhibitory effect of G protein on the Na-HCO3 cotransporter activity was not prevented by dideoxyadenosine, an adenylate cyclase inhibitor, or by the protein kinase A inhibitor, suggesting a direct effect of G protein on the cotransporter. To identify the G proteins that mediate the regulation of the Na-HCO3 cotransporter, purified BLMV were ADP ribosylated in presence of cholera toxin or pertussis toxin. Autoradiograms of BLMV incubated with [32P]NAD showed that cholera and pertussis toxins caused ADP ribosylation of 42- and 41-kDa G proteins, respectively. To determine whether the ADP ribosylation by cholera or pertussis toxin was associated with alterations of the Na-HCO3 cotransporter activity, we measured HCO(3-)-dependent 22Na uptake in BLMV treated with 20 micrograms/ml cholera toxin or with 100 ng/ml pertussis toxin. Na-HCO3 cotransporter activity was significantly decreased by both cholera and pertussis toxins.(ABSTRACT TRUNCATED AT 250 WORDS)

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