Receptors for angiotensin I1 on platelets from man

1984 ◽  
Vol 66 (6) ◽  
pp. 725-731 ◽  
Author(s):  
Yuan Ding ◽  
Christopher J. Kenyon ◽  
Peter F. Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Capacity ◽  
Specific Binding ◽  
Venous Blood ◽  
Kinetic Studies ◽  
Free Fluid ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Temperature Dependent ◽  
Single Class

1. Platelets were prepared from peripheral venous blood on iso-osmotic density gradients of Percoll, resulting in a good recovery of cells (50–80%) which were relatively free of contaminating blood cells (erythrocyte <0.1%, leucocyte <0.1%). 2. At 22°C, specific binding of 125labelled angiotensin II (300 pmol/l) was time and temperature dependent, saturable, reversible and linear with cell concentration. 3. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 ± 0.4 × 10−10 mol/l and total binding capacity 6.3 ± 1.2 receptorslplatelet. Similar values (Kd 2.4 ± 0.7 × 10−10 mol/l and binding capacity 6.5 ± 1.0 receptors/cell) were obtained by displacement analysis. From kinetic studies the forward and reverse rate constants were 3.1 × 108 mol min−1 1−1 and 3.6 × 10−2/min giving a Kd of 1.2 × 10−10mol/l. 4. The relative binding potencies for angiotensin I1 and analogues were: [Sar1, Thr8]ANC II > ANG II > ANG III > [Sar1, Ala8]ANG II > ANG I. 5. Incubation with an extracellular marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was not due to free fluid endocytosis.

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

1989 ◽  
Vol 121 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Tohru Yashiro ◽  
Yoshito Ohba ◽  
Hitomi Murakami ◽  
Takao Obara ◽  
Toshio Tsushima ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Type I ◽  
Single Class ◽  
Normal Tissues ◽  
Igf I ◽  
Cancer Tissues

Abstract. The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

Interaction of endothelin-1 with porcine thyroid cells in culture: a possible autocrine factor regulating iodine metabolism

1994 ◽  
Vol 142 (3) ◽  
pp. 463-470 ◽  
Author(s):  
T Tsushima ◽  
M Arai ◽  
O Isozaki ◽  
Y Nozoe ◽  
K Shizume ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Thyroid Cell ◽  
Scatchard Analysis ◽  
Camp Production ◽  
Iodide Uptake ◽  
Single Class ◽  
Thyroid Cells ◽  
Cells In Culture ◽  
Iodine Metabolism

Abstract Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a Kd of 0·45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake. These results are compatible with the notion that ET-1 negatively regulates TSH-induced iodide uptake in an autocrine manner. Journal of Endocrinology (1994) 142, 463–470

scholarly journals Human somatotropin binding to rabbit kidney microsomal fraction

1981 ◽  
Vol 200 (2) ◽  
pp. 257-264 ◽  
Author(s):  
L P Roguin ◽  
S H Sánchez ◽  
J S Bonifacino ◽  
A C Paladini
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Temperature Dependent ◽  
Binding Reaction ◽  
Dissociation Equilibrium ◽  
Microsomal Membranes ◽  
Kidney Microsomes

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.

Angiotensin II receptor isoforms in the rat adrenal gland: studies with the selective subtype antagonists DuP 753 and CGP42112A

1993 ◽  
Vol 11 (1) ◽  
pp. 69-75 ◽  
Author(s):  
M Montiel ◽  
S Barker ◽  
G P Vinson ◽  
E Jiménez
Keyword(s):  
Angiotensin Ii ◽  
Adrenal Gland ◽  
Binding Sites ◽  
Specific Binding ◽  
Zona Glomerulosa ◽  
Scatchard Analysis ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Receptor Isoforms

ABSTRACT The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using 125I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6·8, 6·7, 6·5 and 6·3. In binding assays in the presence of an excess of the Ang II receptor AT1 subtype antagonist DuP 753, a monophasic dose-dependent displacement of 125I-labelled Ang II binding by the Ang II receptor AT2 subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity. Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6·7 substantially, while CGP42112A significantly inhibited the specific 125I-labelled Ang II binding to the pI 6·3 isoform. When DuP 753 and CGP42112A were used together, specific binding of 125I-labelled Ang II to the isoforms of pI values 6·8, 6·7 and 6·3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT1 and AT2 receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6·7 and pI 6·3 are predominantly composed of AT1 and AT2 receptor subtypes respectively. Interestingly, in the presence of both antagonists, 8·7 ± 0·9% of the specific binding migrating at pI 6·5 remained unaffected. This finding suggests the presence of an additional subtype, which is neither AT1 nor AT2, in the rat adrenal zona glomerulosa. In further studies, pretreatment with DTT was found to increase the specific 125I-labelled Ang II binding of all four isoforms. Moreover, DTT also produced a further specific binding component between pI 6·5 and pI 6·7 which exhibited AT2 subtype pharmacology in DTT-treated preparations. Since DTT has been reported to enhance only AT2 subtype binding this also suggests that the different isoforms may contain components related to both AT1 and AT2 receptor subtypes.

Angiotensin II receptors in rabbit renal preglomerular vessels

1988 ◽  
Vol 255 (1) ◽  
pp. E16-E22 ◽  
Author(s):  
G. P. Brown ◽  
R. C. Venuto
Keyword(s):  
Angiotensin Ii ◽  
Vascular Tissue ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Ang Ii ◽  
Single Class ◽  
Binding Inhibition ◽  
Afferent Arterioles ◽  
Inhibition Potencies

Controversy exists regarding the specific sites within the renal microcirculation affected by angiotensin II (ANG II). Under some conditions, ANG II can elicit direct vasoconstrictor responses in the preglomerular vessels and efferent arterioles. These experiments were designed to evaluate the binding of 125I-ANG II in preglomerular vessels. Arcuate and interlobular arteries, with attached proximal segments of afferent arterioles, were microdissected from rabbit renal cortexes. A membrane preparation was obtained from the pooled freshly dissected vessels and utilized in an ANG II radioreceptor assay on the same day. Binding site concentrations [N] and dissociation constants [KD] were obtained by Scatchard analyses of binding inhibition data. Specific binding was saturable and reversible. The dissociation of bound ANG II was enhanced in the presence of a nonhydrolyzable analogue of GTP. Linear Scatchard plots were obtained, indicating the presence of a single class of high-affinity binding sites. The KD and N are similar to those for ANG II receptors in extrarenal vascular tissue. The order of binding inhibition potencies of ANG analogues was [Sar1,Ile8]-ANG II much greater than [Sar1,Ala8]ANG II = ANG II = ANG III much greater than ANG I, which is consistent with in vivo observations of the effects of these analogues on renal blood flow. The binding inhibition potencies of ANG III and [Sar1,Ile8]ANG II were greater in renal compared with reported values for extrarenal vasculature and rabbit glomeruli. Furthermore, there were no differences in ANG II receptor parameters in preglomerular vessels obtained from pregnant and nonpregnant rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased angiotensin II receptors in neuronal cultures from hypertensive rat brain

1984 ◽  
Vol 247 (5) ◽  
pp. C364-C372 ◽  
Author(s):  
M. K. Raizada ◽  
T. F. Muther ◽  
C. Sumners
Keyword(s):  
Angiotensin Ii ◽  
Rat Brain ◽  
Binding Sites ◽  
Specific Binding ◽  
Neuronal Cell ◽  
Neuronal Cultures ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Spontaneously Hypertensive ◽  
Wistar Kyoto

Binding of 125I-angiotensin II (ANG II) to neuronal cultures made from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats was time dependent, saturable, reversible, and 90-95% specific. Neuronal cultures from SH rats bound 50-70% more 125I-ANG II compared with their WKY controls. Scatchard analysis revealed that the increase in the specific binding of ANG II to SH rat neuronal cultures was due to an increase in the number of binding sites per cell rather than change in the affinity of receptors for ANG II. Light-microscopic autoradiographic analysis showed that ANG II specific binding sites were located on neuronal cell bodies and neurites. Treatment of neuronal cultures from both strains of rats with alpha-methyl-p-tyrosine caused a 50-60% decrease in the endogenous levels of norepinephrine (NE) and dopamine (DA). This decrease was associated with increases in the specific binding of 125I-ANG II in neuronal cultures from WKY rat brain. In contrast, ANG II binding in neuronal cultures from SH rat brain failed to respond to changes in NE and DA levels. These observations suggest that ANG II specific receptors are increased and that they are not under a negative-feedback control by catecholamines in SH rat brain neuronal cultures.

Charge heterogeneity of the AT1 angiotensin II receptor subtype in the rat lung

1995 ◽  
Vol 147 (1) ◽  
pp. 153-159 ◽  
Author(s):  
M Montiel ◽  
J Quesada ◽  
E Jiménez
Keyword(s):  
Angiotensin Ii ◽  
Specific Binding ◽  
Receptor Subtype ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Rat Lung ◽  
Angiotensin Ii Receptor ◽  
Charge Heterogeneity ◽  
Major Band ◽  
Receptor Sites

Abstract In order to obtain more information on the molecular structure of the angiotensin II (Ang II) binding sites from whole rat lung membranes these were characterized by isoelectric focusing (IEF) and SDS-PAGE. Whereas a single population of Ang II receptor sites was identified (Kd=2·2± 0·3 nmol/l; Bmax=203·9± 15·8 fmol/mg protein) by Scatchard analysis, using IEF three Ang II binding isoforms were observed; a major band which migrated to isoelectric point (pI) 6·7, and two minor bands with pI values of 6·5 and 6·3 Specific binding of 125I-Ang II to rat lung membrane preparations was sensitive to Losartan, a non-peptide AT1, receptor subtype antagonist, but was unaffected by the AT2 receptor subtype antagonist CGP42112A. Immunoblotting analyses on SDS gels, using a monoclonal antibody specific to the AT1, receptor, showed two immunoreactive protein species of 45 and 48 kDa. Enzymic deglycosylation using recombinant N-glycanase did not alter the molecular weight patterns of the AT1, receptor subtype. The results of the present study demonstrated that the Ang II receptor population in the whole rat lung consists solely of the AT1, receptor subtype and that the AT2 receptor subtype is absent. In addition, the data showed the existence of charge heterogeneity of the AT1, receptor subtype, and suggest that glycosylation probably does not contribute to its charge heterogeneity. Journal of Endocrinology (1995) 147, 153–159

Direct effect of endothelin-1 on the granulosa cells of the porcine ovary

1992 ◽  
Vol 134 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S. Kamada ◽  
T. Kubota ◽  
Y. Hirata ◽  
M. Taguchi ◽  
S. Eguchi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Endothelin 1 ◽  
Porcine Granulosa Cells

ABSTRACT Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells. Journal of Endocrinology (1992) 134, 59–66

BINDING OF HUMAN CHORIONIC GONADOTROPHIN BY RAT TESTIS: EFFECT OF SEXUAL MATURATION, CRYPTORCHIDISM AND HYPOPHYSECTOMY

1975 ◽  
Vol 64 (1) ◽  
pp. 59-66 ◽  
Author(s):  
JOACHIM FROWEIN ◽  
WOLFGANG ENGEL
Keyword(s):  
Dissociation Constant ◽  
Sexual Maturation ◽  
Leydig Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Scatchard Analysis ◽  
Rat Testis ◽  
Relative Binding

SUMMARY The specific binding of 125I-labelled human chorionic gonadotrophin (HCG) by rat testicular homogenate as compared with isolated Leydig cells differs with respect to total binding capacity but not to the dissociation constant (KD) as revealed by Scatchard analysis. The maximal binding capacity for [125I]HCG of crude testicular homogenate was 95 ng/g rat testis. Hypophysectomy causes a decline in binding capacity within the first three days but on the 20th and 30th day after hypophysectomy the relative binding capacity no longer differs from that of controls. Binding capacity is enhanced in cryptorchid testes relative to normal, and increases during sexual maturation to a peak shortly before puberty.

Characterization of [3H]nifedipine binding to intact vascular smooth muscle cells

1988 ◽  
Vol 254 (1) ◽  
pp. C45-C52 ◽  
Author(s):  
K. Sumimoto ◽  
M. Hirata ◽  
H. Kuriyama
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Porcine Coronary Artery ◽  
Control Value

Specific binding of the dihydropyridine Ca2+ antagonist [3H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of [3H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [3H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd value remained unchanged. MnCl2 and CdCl2 had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K+]o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [3H]nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.

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