α1-Adrenoceptor subtypes on rat afferent arterioles assessed by radioligand binding and RT-PCR

We utilized [3H]prazosin saturation and competition radioligand binding studies to characterize the expression of α1-adrenoceptors in preglomerular vessels. mRNA for adrenoceptor subtypes was assayed using RT-PCR. The vessels were isolated using an iron oxide-sieving method. [3H]prazosin bound to a single class of binding sites ( K d0.087 ± 0.012 nM, Bmax 326 ± 56 fmol/mg protein). Phentolamine displaced [3H]prazosin (0.2 nM) with a p K i of 8.37 ± 0.09. Competition with the selective α1A-adrenoceptor antagonist 5-methylurapidil fit a two-site model (p K i9.38 ± 0.21 and 7.04 ± 0.15); 59 ± 3% of the sites were high-affinity, and 41 ± 3% were low-affinity binding sites. Competition with the α1D-adrenoceptor antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (p K i 6.83 ± 0.03). The relative contents of α1A-, α1B-, and α1D-adrenoceptor mRNAs were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively. Thus there was a very good correlation between mRNA and receptor binding for the subtypes. These data indicate a predominance of the α1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of α1B- and α1D-adrenoceptors.

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Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum

Biochemical Journal ◽

10.1042/bj2650393 ◽

1990 ◽

Vol 265 (2) ◽

pp. 393-398 ◽

Cited By ~ 20

Author(s):

D L Nunn ◽

B V L Potter ◽

C W Taylor

Keyword(s):

Binding Sites ◽

Radioligand Binding ◽

Inositol Phosphate ◽

Rank Order ◽

Binding Protein ◽

Molecular Target ◽

Binding Studies ◽

Single Class ◽

Surface Receptors ◽

High Affinity

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.

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Identification and transmembrane signaling of leukotriene D4 receptors in human mesangial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1988.255.6.c771 ◽

1988 ◽

Vol 255 (6) ◽

pp. C771-C780 ◽

Cited By ~ 31

Author(s):

M. S. Simonson ◽

P. Mene ◽

G. R. Dubyak ◽

M. J. Dunn

Keyword(s):

Binding Sites ◽

Radioligand Binding ◽

Mesangial Cells ◽

Specific Binding ◽

Transmembrane Signaling ◽

Binding Studies ◽

Single Class ◽

Leukotriene D4 ◽

Human Mesangial Cells ◽

Leukotriene Receptors

Although peptidoleukotriene (LTC4, LTD4) receptors have been characterized by radioligand binding studies, pathways of transmembrane signaling by activated leukotriene receptors remain obscure. We employed [3H]LTD4 binding studies and fluorescent measurements of intracellular Ca2+ concentration ([Ca2+]) and pH to identify LTD4 receptors and mechanisms of transmembrane signaling in cultured human mesangial cells. Mesangial cells expressed a single class of saturable, specific binding sites for [3H]LTD4. Kinetic, competition, and saturation analyses gave an average KD of approximately 12.0 nM with a Bmax of 987 fmol/mg protein. LTC4 competed with high affinity for [3H]LTD4 binding sites, as did LTB4 but with much lower affinity. [3H]LTD4 binding was blocked by a specific LTD4 receptor antagonist, SKF 102922. LTD4 and LTC4 also evoked a rapid (2-3 s), transient increase in intracellular [Ca2+], followed by a second, sustained increase. The transient phase was independent of extracellular Ca2+, whereas the sustained phase was dependent on extracellular Ca2+. Intracellular [Ca2+] was unaffected by LTB4. The LTD4-stimulated Ca2+ transients were dose dependent (1 nM-1 microM) and, similar to [3H]LTD4 binding, Ca2+ transients were inhibited by LTD4 receptor antagonists. We also report evidence that LTD4 affects intracellular pH and activates Na+-H+ exchange. Specifically, LTD4 induced an initial acidification within 1-2 min, followed by net alkalinization at 5 min. Alkalinization was due to activation of an amiloride-inhibitable Na+-H+ exchanger. LTD4 receptors were apparently not coupled to adenylate cyclase or phospholipase A2 as we detected no changes of adenosine 3',5'-cyclic monophosphate (cAMP) or prostanoids. Thus we conclude that [3H]LTD4 binding sites on human mesangial cells are coupled to a Ca2+-signaling system and Na+-H+ exchange. Moreover LTD4, a potent inflammatory mediator, failed to stimulate cAMP or prostaglandin E2/prostaglandin I2, two counterregulatory autacoids that preserve normal mesangial function.

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Enhanced responsiveness of endothelium in the growing/motile state to tumor necrosis factor/cachectin.

Journal of Experimental Medicine ◽

10.1084/jem.170.3.913 ◽

1989 ◽

Vol 170 (3) ◽

pp. 913-931 ◽

Cited By ~ 40

Author(s):

H Gerlach ◽

H Lieberman ◽

R Bach ◽

G Godman ◽

J Brett ◽

...

Keyword(s):

Endothelial Cell ◽

Binding Sites ◽

Cell Shape ◽

Radioligand Binding ◽

Cell Binding ◽

Binding Studies ◽

Endothelial Cell Surface ◽

High Affinity ◽

Radioligand Binding Studies

Some in vivo observations have suggested that growing or perturbed endothelium, such as that which occurs during angiogenesis, is more sensitive to the action of cytokines (TNF/cachectin, TNF, or IL-1) than normal quiescent endothelial cells. This led us to examine the responsiveness of endothelium to TNF as a function of the growth/motile state of the cell. TNF-induced modulation of endothelial cell surface coagulant function was half-maximal at a concentration of approximately 0.1 nM in subconfluent cultures, whereas 1-2 nM was required for the same effect in postconfluent cultures. Perturbation of endothelial cell shape/cytoskeleton was similarly more sensitive to TNF in subconfluent cultures. Consistent with these results, radioligand binding studies demonstrated high affinity TNF binding sites, Kd approximately 0.1 nM on subconfluent cultures, whereas only lower affinity sites (Kd approximately 1.8 nM) were detected on postconfluent cultures. The mechanisms underlying this change in the affinity of endothelium for TNF were studied in four settings. Crosslinking experiments with 125I-TNF and endothelium showed additional bands corresponding to Mr approximately 66,000 and approximately 84,000 with subconfluent cultures that were not observed with postconfluent cultures. Experiments with X-irradiated endothelium, whose growth but not motility was blocked, indicated that proliferation was not required for induction of high affinity TNF sites. Postconfluent endothelium, triggered to enter the proliferative cycle by microbutuble poisons, expressed high affinity TNF binding sites together with changes in cell shape/cytoskeleton well before their entry into S phase. Using wounded postconfluent monolayers, cells that migrated into the wound and those close to the wound edge displayed enhanced TNF binding and modulation of coagulant properties. These results suggest a model for targetting TNF action within the vasculature; regulation of high affinity endothelial cell binding sites can direct TNF to activated cells in particular parts of the vascular tree.

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Thymosthenic Agents, A Novel Approach in the Treatment of Schizophrenia

The British Journal of Psychiatry ◽

10.1192/s0007125000295950 ◽

1989 ◽

Vol 155 (S5) ◽

pp. 33-36 ◽

Cited By ~ 78

Author(s):

Yves G. Gelders

Keyword(s):

Binding Sites ◽

Psychotic Disorders ◽

Radioligand Binding ◽

Receptor Subtypes ◽

Binding Studies ◽

Novel Approach ◽

Definition Of ◽

Radioligand Binding Studies ◽

Β Adrenergic Receptors ◽

The Brain

The pharmacotherapy of psychotic disorders which started in 1952, based on empirical results obtained with chlorpromazine by Delay & Deniker, has revolutionised the treatment of psychiatric diseases of non-organic origin. The discovery of haloperidol by Janssen in 1959 represented further progress in this field, since this was found to be more potent and to have generally less disturbing adverse effects than chlorpromazine. Nevertheless it took several years before dopamine-antagonism was put forward as possible mechanism of action for neuroleptic drugs (Carlsson & Lindqvist, 1963). From the early 1970s, however, the advent of radioligand binding studies provided new tools for studying neurotransmitter receptors in the brain and for investigating the interaction of drugs with various receptors. This technique allowed identification of receptor-binding sites related to pharmacologically defined receptors and receptor subtypes, e.g. α and β-adrenergic receptors. It also led to further subclassification and refined definition of receptors, e.g. dopamine-D1 and -D2, and different pharmacological effects mediated by the receptor subtypes have been identified.

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Evidence for a novel angiotensin II receptor involved in angiogenesis in chick embryo chorioallantoic membrane

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.264.2.r460 ◽

1993 ◽

Vol 264 (2) ◽

pp. R460-R465 ◽

Cited By ~ 15

Author(s):

F. A. Le Noble ◽

N. H. Schreurs ◽

H. W. van Straaten ◽

D. W. Slaaf ◽

J. F. Smits ◽

...

Keyword(s):

Angiotensin Ii ◽

Chick Embryo ◽

Binding Sites ◽

Radioligand Binding ◽

Chorioallantoic Membrane ◽

Affinity Constant ◽

Binding Studies ◽

At1 Antagonist ◽

Radioligand Binding Studies ◽

At2 Receptors

Angiotensin II acts as a growth factor in the cardiovascular system and has been implicated in angiogenesis. The existence of at least two types of angiotensin II receptors, the AT1 and the AT2 receptors, has been suggested by ligand binding studies. We used three different AT receptor antagonists to study the receptor mediating angiotensin II-induced angiogenesis in the chorioallantoic membrane (CAM) of the chick embryo. Angiotensin II caused pronounced angiogenesis of pre- and postcapillary vessels of 30-40%. This response could only be blocked by adding the peptidergic AT2 antagonist CGP-42112A. The nonpeptidergic AT2 antagonist PD123319 and AT1 antagonist losartan (DuP 753) were not effective. In addition, we used radioligand binding studies with a range of ligands to define the nature of the receptor. Our results show a high density of specific single class AT receptor with a total number of binding sites of 1,190 fmol/mg protein and an affinity constant for angiotensin II of 2.7 nM. The inhibitory concentrations (IC50) for CGP-42112A, PD 123319 and losartan were 724, > 100,000, and 59,000 nM, respectively. Our studies suggest that these binding sites act as receptors for angiotensin II-induced angiogenesis. Both functional and radioligand binding studies suggest that the receptor is different from the classical mammalian AT1 and AT2 receptors.

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A New Nonlinear Regression Approach That Allows Detection of Inter-Individual Differences in Single-Point Radioligand Binding Studies

10.21236/ada455975 ◽

2000 ◽

Author(s):

C.-M. Staschen ◽

M. J. Munazza ◽

P. B. Massell ◽

L. D. Homer ◽

S. T. Ahlers

Keyword(s):

Individual Differences ◽

Nonlinear Regression ◽

Radioligand Binding ◽

Single Point ◽

Binding Studies ◽

Regression Approach ◽

Radioligand Binding Studies

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Bronchospasmolytic and Adenosine Binding Activity of 8- (Proline / Pyrazole)-Substituted Xanthine Derivatives

Current Drug Discovery Technologies ◽

10.2174/1570163817666200922121005 ◽

2020 ◽

Vol 17 ◽

Author(s):

Sneha Singh ◽

Madhwi Ojha ◽

Divya Yadav ◽

Sonja Kachler ◽

Karl-Norbert Klotz ◽

...

Keyword(s):

Binding Affinity ◽

Radioligand Binding ◽

Binding Activity ◽

Receptor Subtypes ◽

Significant Protection ◽

Binding Studies ◽

Standard Drug ◽

Xanthine Derivatives ◽

Radioligand Binding Studies ◽

Micromolar Range

Background: ABSTRACT: Background: 8-Phenyltheophylline derivatives exhibit prophylactic effects at a specific dose but do not produce the cardiovascular or emetic side effects associated with xanthines, thereby exhibiting unique characteristics of potential therapeutic importance. Methods: Novel series of 8-(proline/pyrazole)-substituted xanthine analogs has been synthesized. The affinity and selectivity of compounds to adenosine receptors have been assessed by radioligand binding studies. The synthesized compounds also showed good bronchospasmolytic properties (increased onset of bronchospasm; decreased duration of jerks) with 100% survival of animals in comparison to the standard drug. Besides, compound 8f & 9f showed good binding affinity in comparison to other synthesized compounds in the micromolar range. Results: The maximum binding affinity of these compounds was observed for A2B receptors, which is ~ 7 or 10 times higher as compared to A1, A2A and A3 receptors. The newly synthesized derivatives 8f, 9a-f, 17g-m, and 18g-m displayed significant protection against histamine aerosol induced bronchospasm in guinea pigs. Conclusion: Newly synthesized proline/pyrazole based xanthines compounds showed a satisfactory binding affinity for adenosine receptor subtypes. Replacement or variation of substituted proline ring with substituted pyrazole scaffold at 8thposition of xanthine moiety resulted in the reduction of adenosine binding affinity and bronchospasmolytic effects.

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