Thermodynamic analysis of β-adrenergic partial agonists (befunolol and carteolol) interaction with low and high affinity binding sites of β-adrenoceptors in guinea-pig taenia caecum

The stereoselectivities of β-adrenergic partial agonists for the high affinity binding site of β-adrenoceptors in the rabbit ciliary body and the guinea-pig taenia caeci were studied. The pA2 values of the S-(−)-isomers of befunolol and carteolol against S-(−)-isoprenaline, which were calculated from the shift of each concentration–response curve in increasing cyclic AMP levels, were significantly larger than those of the R-(+)-isomers in the guinea-pig taenia caeci, while the pA2 values of the S-(−)-isomers were not significantly larger than those of the R-(+)-isomers in the rabbit ciliary body. The pKi values determined from the binding experiments were in good agreement with the pA2 values from the increases in cyclic AMP levels. These results suggest that the high affinity binding site of β-adrenoceptors in the guinea-pig taenia caeci may be able to discriminate stereoselectively between the R-(+)- and S-(−)-isomers, while in the rabbit ciliary body there is no stereoselectivity between the two enantiomers.Key words: stereoselectivity, β-adrenoceptor, partial agonist, rabbit ciliary body, guinea-pig taenia caeci.

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Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes

Biochemical Journal ◽

10.1042/bj2080083 ◽

1982 ◽

Vol 208 (1) ◽

pp. 83-88 ◽

Cited By ~ 81

Author(s):

S M Jarvis ◽

J R Hammond ◽

A R P Paterson ◽

A S Clanachan

Keyword(s):

Guinea Pig ◽

Binding Sites ◽

Species Differences ◽

Cell Types ◽

Nucleoside Transport ◽

Kinetic Properties ◽

Affinity Binding ◽

Transport Capacity ◽

High Affinity Binding ◽

High Affinity

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

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Interactions of some partial agonists with high and low affinity binding sites in β-adrenoceptors

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-004 ◽

1987 ◽

Vol 65 (1) ◽

pp. 18-22 ◽

Cited By ~ 12

Author(s):

I. Takayanagi ◽

K. Koike ◽

A. Nakagoshi

Keyword(s):

Guinea Pig ◽

Binding Sites ◽

Specific Binding ◽

The Other ◽

Affinity Binding ◽

High Affinity ◽

Partial Agonists ◽

Significant Difference ◽

High Affinity Site ◽

Derivatives Of

Interactions of derivatives of befunolol (BFE-37, BFE-55, and BFE-61), carteolol, and pindolol with β-adrenoceptors were tested in guinea pig isolated taenia caecum. All the drugs used acted as partial agonists on the β-adrenoceptors when compared with isoprenaline, a full agonist. The pA2 values of BFE-61, carteolol, and pindolol were significantly larger than their pD2 values, while there was no significant difference between the pA2 and pD2 values for BFE-37 and BFE-55. The specific binding of [3H]befunolol to microsomal fractions from the guinea pig taenia caecum distinguished two binding sites, high affinity and low affinity sites. Both sites are considered to be bound by 50 nM of [3H]befunolol. Specific 3H binding was displaced by BFE-61, carteolol, and pindolol in a biphasic manner but in a monophasic manner by BFE-37 and BFE-55. Furthermore, [3H]befunolol binding was only partially displaced by BFE-55 but completely displaced by the other drugs used. These results, together with our previous findings, suggest that BFE-61, carteolol, and pindolol discriminate between the two affinity binding sites in the β-adrenoceptors, which are not discriminated between by BFE-37, and further that BFE-55 may bind with only the high affinity site.

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Characterization of high-affinity binding sites for the antitussive [3H]noscapine in guinea pig brain tissue

European Journal of Pharmacology ◽

10.1016/0014-2999(88)90230-0 ◽

1988 ◽

Vol 145 (2) ◽

pp. 195-203 ◽

Cited By ~ 20

Author(s):

Mats O. Karlsson ◽

Bengt Dahlström ◽

Anders Neil

Keyword(s):

Guinea Pig ◽

Brain Tissue ◽

Binding Sites ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Pig Brain ◽

Guinea Pig Brain

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High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648890 ◽

1994 ◽

Vol 72 (03) ◽

pp. 465-474 ◽

Cited By ~ 20

Author(s):

Neelesh Bangalore ◽

William N Drohan ◽

Carolyn L Orthner

Keyword(s):

Binding Sites ◽

Protein C ◽

Umbilical Vein ◽

Activated Protein C ◽

Affinity Binding ◽

Cell Binding ◽

High Affinity Binding ◽

High Affinity ◽

Gla Domain ◽

Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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