scholarly journals Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain

1995 ◽  
Vol 305 (1) ◽  
pp. 277-283 ◽  
Author(s):  
H Boudin ◽  
A Grauz-Guyon ◽  
M P Faure ◽  
P Forgez ◽  
A M Lhiaubet ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Molecular Mass ◽  
Cho Cells ◽  
Insect Cells ◽  
Rat Cerebral Cortex ◽  
Molecular Forms ◽  
Sf9 Cells ◽  
Neurotensin Receptor ◽  
Sf9 Insect Cells ◽  
Peptide Antibody

In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.

scholarly journals Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand

1996 ◽  
Vol 314 (2) ◽  
pp. 603-611 ◽  
Author(s):  
Xilin CHEN ◽  
Karen EARLEY ◽  
Weiping LUO ◽  
Sue-Hwa LIN ◽  
William P. SCHILLING
Keyword(s):  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Sustained Response ◽  
Thrombin Receptor ◽  
Sf9 Cells ◽  
Thrombin Receptors ◽  
Sf9 Insect Cells ◽  
Human Thrombin ◽  
Hel Cells ◽  
Tethered Ligand

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 μM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 μM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 μM to 20 and 7 μM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

scholarly journals Neuropeptide FF (FLFQPQRF-NH2) and its Fragments Bind to α2δ Subunit of Voltage-Gated Calcium Channels

2019 ◽  
Vol 22 ◽  
pp. 292-300
Author(s):  
Hanna Skubatz
Keyword(s):  
Cerebral Cortex ◽  
Amino Acid ◽  
Cho Cells ◽  
New Drugs ◽  
Rat Cerebral Cortex ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Neuropeptide Ff ◽  
Α2δ Subunit ◽  
Ca2 Channels

Purpose: Gabapentin, a drug for neuropathic pain, exerts its therapeutic effect via inhibition of the a2d subunit of N-type Ca2+ channels. Thus, finding peptides that specifically displace gabapentin from its binding site may lead to the development of new drugs. Methods: Displacement of bound [3H]-gabapentin in membrane preparations of rat cerebral cortex and of human Cav2.2/β3/α2δ1 expressed in CHO cell line. Results: Neuropeptide FLFQPQRF-NH2 specifically displaced bound [3H]-gabapentin in membrane preparations from rats and CHO cells. Truncation of the C-terminus of FLFQPQRF-NH2 by three amino acid residues to produce FLFQP-NH2 improved the displacement of gabapentin. FLFQP-NH2 displaced bound  [3H]-gabapentin with IC50 and Ki values of 2.7 µM and 1.7 µM, respectively. Deletion of two amino acid residues (FQ) in the middle of the FLFQP-NH2 sequence yielded FLP-NH2 that displaced bound [3H]-gabapentin with a lower affinity.  IC50 and Ki values were 11.9 µM and 7.8 µM, respectively. Neutral binding cooperativity existed when of FLFQP-NH2, FLP- NH2 and gabapentin when incubated together. FLFQPQRF-NH2 but not FLFQP-NH2 displaced bound [3H]-gabapentin to membrane preparations of human Cav2.2/b3/a2d1 expressed in CHO cells. Conclusion: FLFQPQRF-NH2, FLFQP-NH2 and FLP-NH2 displace bound gabapentin in membrane preparations of rat cerebral cortex. Binding cooperativity was detected when GBP/FLFQP-NH2/FLP-NH2 were incubated together. These novel binding sites may provide new approaches to modulate L-type Ca2+ channels.

Properties of single Drosophila Trpl channels expressed in Sf9 insect cells

1997 ◽  
Vol 272 (1) ◽  
pp. C27-C34 ◽  
Author(s):  
D. L. Kunze ◽  
W. G. Sinkins ◽  
L. Vaca ◽  
W. P. Schilling
Keyword(s):  
Transient Receptor Potential ◽  
Insect Cells ◽  
Single Channel ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Single Channels ◽  
Channel Activity ◽  
Whole Cell ◽  
Bradykinin Receptor ◽  
Sf9 Insect Cells

The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.

Vagus Nerve Stimulation Induced Synchrony Modulation of Local Field Potential in the Rat Cerebral Cortex

2013 ◽  
Vol 133 (8) ◽  
pp. 1493-1500 ◽  
Author(s):  
Ryuji Kano ◽  
Kenichi Usami ◽  
Takahiro Noda ◽  
Tomoyo I. Shiramatsu ◽  
Ryohei Kanzaki ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Vagus Nerve ◽  
Local Field ◽  
Nerve Stimulation ◽  
Local Field Potential ◽  
Vagus Nerve Stimulation ◽  
Field Potential ◽  
Rat Cerebral Cortex

Features of Histostructural Changes in Rat Cerebral Cortex in Hemorrhagic Stroke Modeling

2013 ◽  
Vol 4 (2) ◽  
pp. 113-121 ◽  
Author(s):  
Sergiy I. Savosko ◽  
Juriy B. Chaikovsky ◽  
Nelly Kh. Pogorela ◽  
Alexandr N. Makarenko
Keyword(s):  
Cerebral Cortex ◽  
Hemorrhagic Stroke ◽  
Rat Cerebral Cortex

scholarly journals Adenyl Cyclase of Rat Cerebral Cortex

1971 ◽  
Vol 246 (1) ◽  
pp. 62-68 ◽  
Author(s):  
John P. Perkins ◽  
Marilyn M. Moore
Keyword(s):  
Cerebral Cortex ◽  
Adenyl Cyclase ◽  
Rat Cerebral Cortex

scholarly journals Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex

1994 ◽  
Vol 269 (24) ◽  
pp. 16780-16784
Author(s):  
N. Brose ◽  
G.W. Huntley ◽  
Y. Stern-Bach ◽  
G. Sharma ◽  
J.H. Morrison ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Glutamate Receptor ◽  
Rat Cerebral Cortex
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