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.

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.

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.

scholarly journals G-domain prediction across the diversity of G protein families

2021 ◽  
Author(s):  
Hiral Sanghavi ◽  
Richa Rashmi ◽  
Anirban Dasgupta ◽  
Sharmistha Majumdar
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Guanine Nucleotide ◽  
Protein Families ◽  
Web Based ◽  
Spacer Length ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Abstract Guanine nucleotide binding proteins are characterized by a structurally and mechanistically conserved GTP-binding domain (G domain), indispensable for binding GTP. The G domain comprises five adjacent consensus motifs called G boxes, which are separated by amino acid spacers of different lengths. Several G proteins, discovered over time, are characterized by diverse function and sequence. This sequence diversity is also observed in the G box motifs (specifically the G5 box) as well as the inter-G box spacer length. The Spacers and Mismatch Algorithm (SMA) introduced in this study can predict G-domains in a given protein sequence, based on user-specified constraints for approximate G-box patterns and inter-box gaps in each G protein family. The SMA parameters can be customized as more G proteins are discovered and characterized structurally. Family-specific G box motifs including the less characterized G5 box were predicted with higher accuracy. Overall, our analysis suggests the possible classification of G protein families based on family-specific G box sequences and lengths of inter-G box spacers. SMA can be implemented via a web-based server at https://labs.iitgn.ac.in/datascience/gboxes/

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C451-C456 ◽  
Author(s):  
E. M. Schwiebert ◽  
D. C. Gruenert ◽  
W. B. Guggino ◽  
B. A. Stanton
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
G Protein ◽  
G Proteins ◽  
Airway Epithelial Cells ◽  
Protein G ◽  
Airway Epithelial ◽  
Human Airway ◽  
Cl Channels ◽  
Human Airway Epithelial Cells

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

Control of Exocytosis in Single Cells

Physiology ◽  
1989 ◽  
Vol 4 (2) ◽  
pp. 53-56 ◽  
Author(s):  
Y Maruyama
Keyword(s):  
Membrane Fusion ◽  
G Proteins ◽  
Secretory Granule ◽  
Single Cells ◽  
Guanine Nucleotide ◽  
Signal Transducers ◽  
Granule Membrane ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Guanine Nucleotide Binding Proteins

Exocytosis can be quantified by measuring changes in membrane capacitance in single internally perfused cells. Exocytosis is controlled by guanine nucleotide-binding proteins (G proteins) acting as key signal transducers. Different G proteins mediate receptor signaling and secretory granule-membrane fusion.

Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle

1995 ◽  
Vol 268 (2) ◽  
pp. L221-L229 ◽  
Author(s):  
H. Kume ◽  
K. Mikawa ◽  
K. Takagi ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Isometric Tension ◽  
Guanine Nucleotide Binding Protein ◽  
Kca Channels ◽  
Stimulatory G Protein ◽  
Guanine Nucleotide Binding

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.

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