scholarly journals Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Exceptionally high-affinity binding in A7r5 and A10 cells.

1989 ◽  
Vol 94 (4) ◽  
pp. 669-692 ◽  
Author(s):  
R T McCarthy ◽  
C J Cohen
Keyword(s):  
Dissociation Constant ◽  
Cell Lines ◽  
Open Channel ◽  
Affinity Binding ◽  
Channel Block ◽  
Ca Channels ◽  
Open Channel Block ◽  
High Affinity Binding ◽  
High Affinity ◽  
A7r5 Cells

Calcium channel currents were studied in the A10 and A7r5 cell lines derived from rat thoracic aorta muscle cells. The whole-cell variation of the patch voltage clamp technique was used. Results with each cell line were nearly identical. Two types of Ca channels were found in each cell line that are similar to the L-type and T-type Ca channels found in excitable cells. Nimodipine block of the L-type Ca channels in both cell lines is more potent than in previously studied tissues. The kinetics of nimodipine block are accounted for by a model that postulates 1:1 drug binding to open Ca channels with an apparent dissociation constant (KO) of 16-45 pM. In A7r5 cells, the rate of onset of nimodipine block increases with the test potential, in quantitative agreement with the model of open channel block. The apparent association rate (f) is 1.4 x 10(9) M-1 s-1; the dissociation rate (b) is about 0.024 s-1. In anterior pituitary cells (GH4C1 cells), KO is 30 times larger; b is only twice as fast, but f is 15 times slower. The comparative kinetic analysis indicates that the high-affinity binding site for nimodipine is similar in both GH4C1 and A7r5 cells, but nimodipine diffuses much faster or has a larger partition coefficient into the plasmalemma of A7r5 cells than for GH4C1 cells. Unusually high-affinity binding was not observed in earlier 45Ca flux studies with A10 and A7r5 cells. The model of open channel block accounts for the discrepancy; only a small fraction of the Ca channels are in the high affinity open state under the conditions used in 45Ca flux studies, so an effective binding constant is measured that is much greater than the dissociation constant for high-affinity binding.

Interleukin-15 (IL-15) Can Replace the IL-2 Signal in IL-2–Dependent Adult T-Cell Leukemia (ATL) Cell Lines: Expression of IL-15 Receptor α on ATL Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4265-4272 ◽  
Author(s):  
Yasuaki Yamada ◽  
Kazuyuki Sugawara ◽  
Tomoko Hata ◽  
Kazuto Tsuruta ◽  
Ryozo Moriuchi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Affinity Binding ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
High Affinity Binding ◽  
Adult T Cell Leukemia ◽  
High Affinity ◽  
Interleukin 15 ◽  
Α Chain

Abstract Interleukin-15 receptor (IL-15R) and IL-2R have the same β and γ chains, but IL-15R has a specific α chain distinct from that of IL-2Rα, which is indispensable for the high affinity binding of IL-15. In the present study, we examined four IL-2-dependent adult T-cell leukemia (ATL) cell lines for their IL-15R expression. All cell lines bound IL-15, which was not inhibited by a 100-fold excess amount of IL-2, proliferated in response to IL-15 to the same degree as to the stimulation with IL-2, and were maintained without IL-2. The responses to 1L-15 were inhibited by the antibodies against IL-2R β or γ chains but was not by the IL-2R α chain antibody. [125I]–IL-15 exhibited a single high-affinity binding with an apparent kd of 0.17 nmol/L. Reverse transcription–coupled polymerase chain reaction (RT-PCR) showed that the cell lines had the mRNA of IL-15R α. The cell lines also had IL-15 mRNA. Despite the presence of IL-15 mRNA, the cell lines did not secrete IL-15, and the culture supernatants of fresh ATL cells and plasma from the patients did not contain a detectable amount of IL-15 with a few exceptional cases, although fresh ATL cells also responded to IL-15. These results suggest that ATL cells have the complete form of IL-15R and respond to IL-15. Such an IL-15–dependent cell proliferation mechanism might be used in the development of ATL and for the invasion and proliferation of ATL cells in the visceral organs.

scholarly journals Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity

2000 ◽  
Vol 352 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Michael R. HOLBROOK ◽  
Linda L. SLAKEY ◽  
David J. GROSS
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dissociation Constant ◽  
Growth Factor Receptor ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Epidermal Growth

The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988–992) of the EGFr. Equilibrium 125I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions.

scholarly journals Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

2004 ◽  
Vol 124 (6) ◽  
pp. 691-701 ◽  
Author(s):  
Sho-Ya Wang ◽  
Jane Mitchell ◽  
Edward Moczydlowski ◽  
Ging Kuo Wang
Keyword(s):  
Local Anesthetics ◽  
Open Channel ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Alternative View ◽  
Channel Block ◽  
Na Channels ◽  
Open Channel Block ◽  
Channel Activation ◽  
High Affinity

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.

Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex

1991 ◽  
Vol 127 (2) ◽  
pp. 227-230 ◽  
Author(s):  
Dario Acuña Castroviejo ◽  
Carmen María del Aguila ◽  
Begoña Fernández ◽  
María Dolores Gomar ◽  
JoséLuis Castillo
Keyword(s):  
Cerebral Cortex ◽  
Dissociation Constant ◽  
Binding Sites ◽  
Rat Cerebral Cortex ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

Interleukin-15 (IL-15) Can Replace the IL-2 Signal in IL-2–Dependent Adult T-Cell Leukemia (ATL) Cell Lines: Expression of IL-15 Receptor α on ATL Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4265-4272 ◽  
Author(s):  
Yasuaki Yamada ◽  
Kazuyuki Sugawara ◽  
Tomoko Hata ◽  
Kazuto Tsuruta ◽  
Ryozo Moriuchi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Affinity Binding ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
High Affinity Binding ◽  
Adult T Cell Leukemia ◽  
High Affinity ◽  
Interleukin 15 ◽  
Α Chain

Interleukin-15 receptor (IL-15R) and IL-2R have the same β and γ chains, but IL-15R has a specific α chain distinct from that of IL-2Rα, which is indispensable for the high affinity binding of IL-15. In the present study, we examined four IL-2-dependent adult T-cell leukemia (ATL) cell lines for their IL-15R expression. All cell lines bound IL-15, which was not inhibited by a 100-fold excess amount of IL-2, proliferated in response to IL-15 to the same degree as to the stimulation with IL-2, and were maintained without IL-2. The responses to 1L-15 were inhibited by the antibodies against IL-2R β or γ chains but was not by the IL-2R α chain antibody. [125I]–IL-15 exhibited a single high-affinity binding with an apparent kd of 0.17 nmol/L. Reverse transcription–coupled polymerase chain reaction (RT-PCR) showed that the cell lines had the mRNA of IL-15R α. The cell lines also had IL-15 mRNA. Despite the presence of IL-15 mRNA, the cell lines did not secrete IL-15, and the culture supernatants of fresh ATL cells and plasma from the patients did not contain a detectable amount of IL-15 with a few exceptional cases, although fresh ATL cells also responded to IL-15. These results suggest that ATL cells have the complete form of IL-15R and respond to IL-15. Such an IL-15–dependent cell proliferation mechanism might be used in the development of ATL and for the invasion and proliferation of ATL cells in the visceral organs.

High affinity binding of human interleukin 4 to cell lines

1987 ◽  
Vol 149 (3) ◽  
pp. 995-1001 ◽  
Author(s):  
Hélène Cabrillat ◽  
Jean-Pierre Galizzi ◽  
Odile Djossou ◽  
Naoko Arai ◽  
Takashi Yokota ◽  
...  
Keyword(s):  
Cell Lines ◽  
Interleukin 4 ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity
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