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

1988 ◽  
Vol 255 (6) ◽  
pp. C771-C780 ◽  
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.

Leukotriene D4 binding and signal transduction in rat glomerular mesangial cells

1989 ◽  
Vol 257 (2) ◽  
pp. F280-F287 ◽  
Author(s):  
K. F. Badr ◽  
S. Mong ◽  
R. L. Hoover ◽  
M. Schwartzberg ◽  
J. Ebert ◽  
...  
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Mesangial Cells ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Rank Order ◽  
Glomerular Mesangial Cells ◽  
Leukotriene D4 ◽  
Specific Binding Sites ◽  
Dose Dependent

We examined the characteristics of [3H]leukotriene D4 (LTD4) binding to mesangial cells in culture. Binding is stereoselective, specific, saturable, and rapidly reversible. Two binding sites are recognized with dissociation constants and binding site densities at equilibrium of 2.2 and 16.8 nM and 1.1 x 10(4) and 3 x 10(4) binding sites per cell. LTD4, LTE4, (5R,6S)LTD4, LTB4, and the LTD4-receptor antagonist, SKF 104353, competitively inhibit radioligand binding in the following rank order of potency: LTD4 greater than LTE4 = SKF 104353 greater than (5R,6S)LTD4 greater than LTB4. LTD4 also induces time- and concentration-dependent phosphoinositide hydrolysis in mesangial cells. Formation of inositol 1,4,5-trisphosphate (IP3) is maximal at 5 s, followed by a time-dependent increase in inositol monophosphate generation, and inhibited by 100-fold excess concentration of SKF 104353. Addition of LTD4 to mesangial cells is associated with an increase in intracellular pH and dose-dependent stimulation of [3H]thymidine incorporation and mitogenesis. Thus rat mesangial cells possess specific binding sites for LTD4, the activation of which stimulates IP3 formation and induces cellular alkalinization and mitogenic responses. These studies provide insight into the cellular basis for LTD4-mesangial cell interactions, which are of potential pathophysiological relevance during acute glomerular inflammatory injury.

Evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2

1997 ◽  
Vol 272 (4) ◽  
pp. F477-F483 ◽  
Author(s):  
M. Fukunaga ◽  
T. Yura ◽  
R. Grygorczyk ◽  
K. F. Badr
Keyword(s):  
Binding Sites ◽  
Cellular Localization ◽  
Radioligand Binding ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Biological Significance ◽  
Thromboxane A2 ◽  
Functional Responses ◽  
Binding Studies ◽  
Binding Characteristics

In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

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

2001 ◽  
Vol 281 (1) ◽  
pp. F172-F178 ◽  
Author(s):  
Max Salomonsson ◽  
Melinda Oker ◽  
Susan Kim ◽  
Hua Zhang ◽  
James E. Faber ◽  
...  
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Rt Pcr ◽  
Binding Studies ◽  
Single Class ◽  
Site Model ◽  
Adrenoceptor Antagonist ◽  
Resistance Vessels ◽  
Afferent Arterioles ◽  
Radioligand Binding Studies

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.

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

1990 ◽  
Vol 265 (2) ◽  
pp. 393-398 ◽  
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.

Oxytocin receptors in rat kidney during development

1990 ◽  
Vol 259 (6) ◽  
pp. F872-F881 ◽  
Author(s):  
A. Schmidt ◽  
S. Jard ◽  
J. J. Dreifuss ◽  
E. Tribollet
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Juxtaglomerular Apparatus ◽  
Binding Studies ◽  
Single Class ◽  
Ligand Selectivity ◽  
Oxytocin Receptors ◽  
Two Stages

The development and characteristics of oxytocin (OT) receptors in the rat kidney were studied by light-microscopic autoradiography and on membrane preparations using the iodinated OT antagonist 125I-d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]OT. Specific binding was first detected by autoradiography at embryonic day 17 (E17) in both the cortex and the medulla. Cortical labeling was found thereafter at all ages examined including in the adult (postnatal day 90, PN90). It was localized on the distal tubule at the level of the juxtaglomerular apparatus. Medullary binding was detected only transiently during two stages of development, first, before PN6, and second, at the approximate time of weaning (PN20-PN30). Binding studies on crude membranes prepared from whole kidneys of animals aged between PN1 and PN15 showed a single class of high-affinity binding sites, with a dissociation constant of 0.13 +/- 0.08 nM. Thus transient OT binding sites expressed in the medulla do not differ from cortical OT binding sites; moreover, the ligand selectivity of kidney OT receptors regardless of location and age appears similar to that of previously characterized OT receptors. Our results suggest that OT may play a role in both renal development and renal function.

Thromboxane binding and signal transduction in rat glomerular mesangial cells

1993 ◽  
Vol 264 (2) ◽  
pp. F292-F299 ◽  
Author(s):  
R. F. Spurney ◽  
J. J. Onorato ◽  
F. J. Albers ◽  
T. M. Coffman
Keyword(s):  
Signal Transduction ◽  
Dissociation Constant ◽  
Binding Sites ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Maximal Response ◽  
Scatchard Analysis ◽  
Glomerular Mesangial Cells ◽  
Single Class ◽  
Alpha 7

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for functional thromboxane A2-prostaglandin H2 receptors in human placenta

1989 ◽  
Vol 256 (2) ◽  
pp. E256-E263
Author(s):  
A. Hedberg ◽  
P. F. Mento ◽  
E. C. Liu ◽  
A. M. Hollander ◽  
B. M. Wilkes
Keyword(s):  
Human Placenta ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Single Class ◽  
Receptor Sites ◽  
H2 Receptors ◽  
Placental Membranes

The aim of this investigation was to study the role of thromboxane (TX) A2 in the modulation of human fetoplacental vascular resistance. By use of the isolated perfused fetoplacental cotyledon, TX generation (measured by direct radioimmunoassay of TXB2) was demonstrated on the fetal side of the placental circulation. The stable TX mimetic U-46619 caused a dose-dependent increase in perfusion pressure that was inhibited by the TX receptor antagonist SQ 29548. To further characterize the putative TXA2-prostaglandin H2 receptors, binding studies were performed in placental membranes using [3H]SQ 29548. Kinetic analysis revealed rapid and reversible specific binding of [3H]SQ 29548. Saturation binding and Scatchard analysis indicated radioligand binding to a single class of receptors (dissociation constant, 9.11 +/- 0.60 nM; receptor density, 103 +/- 8 fmol/mg protein, n = 4). Prostaglandins D2, E1, E2, F2a, and I2 did not inhibit the specific binding of [3H]SQ 29548 at concentrations less than or equal to 10 microM. This study demonstrates that the human placenta produces and releases TXA2, which can act locally via specific receptor sites to constrict the fetoplacental vasculature.

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney

1992 ◽  
Vol 135 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Y. Song ◽  
E. A. Ayre ◽  
S. F. Pang
Keyword(s):  
Acetic Acid ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Hill Coefficient ◽  
Scatchard Analysis ◽  
Equilibrium Studies ◽  
Single Class ◽  
Urine Production ◽  
Daily Urine

ABSTRACT The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44·6±4·4 pmol/l (n= 6) and a total number of binding sites (Bmax) of 6·43 ±0·60 fmol/mg protein (n= 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1·0, suggesting a single class of 125I-labelled iodomelatoninbinding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53·4% higher at mid-light than at mid-point of the dark period (P<0·05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22·5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin » 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, l-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function. The possibility of a direct effect of melatonin on renin secretion and daily urine production is hypothesized. Journal of Endocrinology (1992) 135, 353–359

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

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