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P Plas

Publications and source records attributed to P Plas.

18 recordsLinked to original sources

Identification of potent non-peptide somatostatin antagonists with sst(3) selectivity.

Using a solution-phase parallel synthesis strategy, a series of non-peptide somatostatin analogues were prepared, and their binding affinities to the five human somatostatin receptor subtypes (sst(1-5)) were determined. Imidazolyl derivatives 2 were found to bind with moderate affinity but with high selectivity to the sst(3) receptor subtype. Further modifications of these structures led to a more potent class of ligands, the tetrahydro-beta-carboline derivatives 4. Among these, compounds 4k (BN81644) and 4n (BN81674) bind selectively and with high affinity to the sst(3) receptor subtype (K(i) = 0.64 and 0.92 nM, respectively). Furthermore, 4k and 4n reverse the inhibition of cyclic AMP accumulation induced by 1 nM somatostatin via sst(3) receptors, with IC(50) = 2.7 and 0.84 nM, respectively. The most potent compound 4n was shown to be a competitive antagonist of human sst(3) receptors by increasing the EC(50) of SRIF-14-mediated inhibition of cAMP accumulation with a K(B) of 2.8 nM (where K(B) is the concentration of antagonist that shifts the agonist dose-response 2-fold). These new derivatives are, to our knowledge, the first potent and highly selective non-peptide human sst(3) antagonists known and, as such, are useful tools for investigating the physiological role of sst(3) receptors.

Animals↗

Decrease in endothelin-1 renal receptors during the 1st month of life in the rat.

Endothelin-1 (Et1), like angiotensin II, is implicated in postnatal maturation and development. The present study was designed to identify Et1 receptors and subtype Et1 receptors present in rat kidney between 1 and 30 days of postnatal life. On day 1, high-affinity and high-density Et1 binding sites were identified in rat kidney. The dissociation constant and maximum binding for ET1 to membranes from whole kidney were 0.073 +/- 0.05 nM and 1,345.9 +/- 73 fmol/mg protein, respectively. On day 30, affinity and receptor density were markedly decreased. The dissociation constant and maximum binding were 0.147 +/- 0.021 nM (P < 0.01) and 633.2 +/- 56.4 fmol/mg protein (P < 0.001), respectively. Using BQ 123 (EtA-selective antagonist) and sarafotoxin S6c (EtB-selective agonist), the two Et1 receptor subtypes EtA and EtB were identified in 1- and 30-day-old rat kidney. BQ 123 selectively recognized EtA receptors with high affinity (2.9 +/- 0.44 on day 1 and 4.0 +/- 0.5 nM on day 30) and sarafotoxin S6c bound with higher affinity EtB receptors (0.871 +/- 0.14 on day 1 and 0.717 +/- 0.12 nM on day 30). Between birth and day 30, the EtA binding capacity was decreased (304 +/- 27 vs. 752 +/- 202 fmol/mg protein, P < 0.05), whereas EtB binding was not affected (514 +/- 87 vs. 656 +/- 171 fmol/mg protein, NS). The decrease in the total number of Et1 receptors during the 1st month of life may be due to the concomitant decrease in the number of EtA receptors. Increased Et1 receptor density in early postnatal life suggests an influence of Et1 on immature kidney circulation and/or kidney growth.

Animals↗

Endothelin receptor subtypes A and B are up-regulated in an experimental model of acute renal failure.

The two endothelin (ET) receptor subtypes (ETA and ETB) have been characterized in rat kidney from normal rats and rats with acute renal failure induced by hypertonic glycerol administration. In control rats, the total number of ET receptors in kidney cortex and medulla was 155 and 386 fmol/mg of protein, respectively. The ratio of ETA to ETB receptors was 54:46 in renal cortex and 35:65 in renal medulla. Treatment of rats with 10 ml/kg glycerol (50%, w/v) intramuscularly resulted in severe renal dysfunction; the serum urea concentration increased from 0.46 to 2.65 g/liter and the creatinine clearance decreased from 1.06 to 0.30 ml/min. Ligand binding studies showed that glycerol-induced acute renal failure was associated with a marked up-regulation of ETA and ETB receptor subtypes in both cortex and medulla. In glycerol-treated rats, the total ET receptor density in kidney cortex and medulla was increased to 294 and 1172 fmol/mg of protein, with ETA/ETB ratios of 52:48 and 31:69, respectively. The upregulatory effect of glycerol treatment was significantly more pronounced in renal medulla than renal cortex and affected ETB receptors preferentially, compared with ETA receptors. Subsequently, ETA and ETB receptor mRNA levels were markedly increased by glycerol administration in both kidney cortex and medulla, as assessed by polymerase chain reaction coupled to reverse transcription. These results suggest that up-regulation of renal ET receptors, particularly ETB receptors in kidney medulla, may account for or contribute to renal function impairment induced by glycerol, and they support a pathophysiological role for ET in acute renal failure.

Acute Kidney Injury↗

Upregulation of renal endothelin receptors in glycerol-induced acute renal failure in the rat.

Acute renal failure was induced in rat with a hypertonic glycerol solution and endothelin-1 (ET-1) binding was measured in kidney membrane preparations. In control animals, [125I]-ET-1 bound to specific recognition sites in kidney cortex (Bmax = 134 +/- 11 fmol/mg protein) and medulla (Bmax = 300 +/- 9 fmol/mg protein) with an apparent dissociation constant (Kd) of 0.16 +/- 0.06 nM and 0.39 +/- 0.07 nM for cortex and medulla, respectively. A single i.m. dose of 10 ml/kg glycerol (50% w/v) resulted in alterations of renal function that were maximal 48 h after glycerol administration. After this 48-h period, serum urea was increased from 0.20 +/- 0.01 g/L to 1.16 +/- 0.20 g/L (p < 0.001) and creatinine clearance was reduced from 1.04 +/- 0.15 ml/min to 0.23 +/- 0.06 ml/min (p < 0.001). Renal ET-1 receptor density was significantly increased in glycerol-treated rats to 255 +/- 14 fmol/mg protein in renal cortex (p < 0.01), and 576 +/- 55 fmol/mg protein in renal medulla (p < 0.01), with no significant modification of the Kd values. These results suggest that upregulation of ET-1 receptors is involved in renal hemodynamic impairment induced by glycerol.

Acute Kidney Injury↗

Binding characteristics of endothelin isoforms (ET-1, ET-2, and ET-3) in vascular smooth muscle cells.

The existence of distinct endothelin (ET) receptor subtypes has been reported in several tissues. In the present study, we investigated the binding characteristics of the three endothelin isoforms to cultured rat aortic smooth muscle cells. [125I]ET-1, [125I]ET-2, and [125I]ET-3 bound to an apparent single class of binding sites with apparent dissociation constants (Kd) of 111, 123, and 1410 pM and binding capacities (Bmax) of 54.1, 46.0, and 7.9 fmol/10(6) cells, respectively. The binding of the three radiolabeled endothelin isoforms was equally inhibited by ET-1 and ET-2. ET-3 was more effective in competing with [125I]ET-3 than with [125I]ET-1 or [125I]ET-2. In contrast to ET-1 and ET-2, the binding of ET-3 was reversible. Furthermore, 18 h of pre-exposure of the cells to 1 nM ET-1 or ET-2 decreased the ET-1 binding capacity, whereas ET-3 (10 nM) was ineffective. ET-3 binding characteristics were not affected by pretreatment of the cells with any of the endothelin isoforms. These results suggest the presence of two distinct endothelin receptor subtypes in rat aortic smooth muscle cells. The ET-1 and ET-2 preferring receptor (80-85%), sensitive to downregulation or internalization, elicits an irreversible binding. The second subtype (15-20%) binds the three endothelin isoforms with the same affinity in a reversible manner, and is insensitive to downregulation or internalization.

Animals↗

[Multiplicity of endothelin receptors of aortic smooth muscle cells in rats].

Among the different endothelin (ET) isoforms, ET-3 has been reported to exhibit a less potent constrictor activity than ET-1 and ET-2. Furthermore, distinct endothelin receptor subtypes have been identified in several tissues or cell types. In this study, we investigated the binding characteristics of the three endothelin isoforms in cultured rat aortic smooth muscle cells. [125I]ET-3 exhibited an apparent affinity and a number of binding sites 10 and 6 times smaller, respectively, than [125I]ET-1 and [125I]ET-2. In contrast to ET-1 and ET-2, ET-3 appeared to elicit a reversible binding and did not modify ET-1 binding characteristics in receptor-regulation experiments. In competition experiments ET-1 and ET-2 equally inhibited the binding of the three endothelin isoforms, whereas ET-3 was less potent in competing with [125I]ET-1 and [125I]ET-2 than [125I]ET-3. These results suggest that rat aortic smooth muscle cells possess 2 subtypes of endothelin receptors (A and B) differing by their affinity for ET-3, their proportion, the reversibility of the binding and their sensitivity to down-regulation.

Animals↗

Down-regulation of endothelin binding sites in rat vascular smooth muscle cells.

In cultured rat aortic smooth muscle cells, [125I]endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted in an apparent dose-dependent reduction of the number of endothelin binding sites with no significant modification of its binding affinity. The time course of the down-regulation of ET-1 binding sites showed that this effect was present after 30 min incubation and persisted after 18 h. This indicates that down-regulation of ET-1 binding sites can modulate the activity of ET-1 and suggests a rapid internalization of ET-1 in vascular cells.

Animals↗

Receptor regulation of atrial natriuretic factor.

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular smooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase and the C receptors (clearance) representing 95% of the total number of ANF binding sites but noncoupled to a guanylate cyclase. Using binding experiments with 125I-ANF and measurement of cGMP production stimulated by ANF, we were able to demonstrate that ANF receptors are sensitive to homologous (induced by ANF) and heterologous regulation (induced by angiotensin II, AII) in rat cultured vascular smooth muscle cells. The effect of the two hormones showed marked differences, in their time course, their reversibility and their consequence on guanylate cyclase activity. Although both ANF and AII reduced the total number of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas AII elicited a potentialization of this system. From these results, we have concluded that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by AII. This also highlights a specific interaction between ANF and AII at the receptor level.

Animals↗

[Homologous and heterologous regulations of endothelin receptors on smooth muscle cells].

In vascular smooth muscle cells, the vasoconstrictor peptide, endothelin (ET-1) possesses specific binding sites sensitive to homologous and heterologous regulation. In this study, we have compared the regulation of ET-1 receptors induced by ET-1 and by angiotensin II. After 18 hours preincubation of cultured rat aortic smooth muscle cells at 37 degrees C in presence of vasoactive substances (1 microM) such as norepinephrine, Met- and Leu-enkephalins, bradykinin, serotonin, histamine or carbachol, the binding characteristics of [125I]ET-1 were not modified. On the same conditions, Arg-vasopressin (1 microM) was able to down-regulate ET-1 receptors by less than 30 p. 100 whereas both ET-1 (1 nM) and angiotensin II (10 nM) reduced the number of ET-1 binding sites (Bmax) by more than 50 p. 100 without modification of the affinity (Kd). The time course of the effect of the two peptides showed a rapid decrease of ET-1 binding sites induced by ET-1 and a comparatively slow regulation elicited by angiotensin II. Sar1-Ile8-angiotensin II blocked the effect of angiotensin II. These results show that ET-1 and angiotensin II can regulate ET-1 receptors and suggest a possible modulation of ET-1 activity by endogenous levels of the two peptides.

Angiotensin II↗

Angiotensin II and phorbol-esters potently down-regulate endothelin (ET-1) binding sites in vascular smooth muscle cells.

[125I]ET-1 binding to vascular smooth muscle cells showed an apparent single class of high affinity recognition sites with a Kd of 2.12 +/- 0.46 nM and a Bmax of 81.2 +/- 5.2 fmol/10(6) cells. The specific binding was equally and totally displaced by ET-1 and ET-2 whereas ET-3 presented a different pattern. We investigated heterologous regulation of ET-1 binding sites by preincubating the cells with angiotensin II (AII), Arg-vasopressin, bradykinin, enkephalins, serotonin, norepinephrine and carbachol, for 18 h at 37 degrees C. Only AII pretreatment resulted in an important and dose-dependent decrease of ET-1 binding capacity. Sar1-Ile8-AII inhibited the regulatory effect of AII. Furthermore, preexposure of the cells with phorbol-12,13 dibutyrate but not with phorbol-12,13 didecanoate also resulted in a concentration-dependent diminution of ET-1 binding sites. These findings suggest that AII may selectively down-regulate ET-1 binding sites in vascular smooth muscle cells by a mechanism involving protein kinase C.

Angiotensin II↗

Atrial natriuretic factor in chronic obstructive lung disease with pulmonary hypertension. Physiological correlates and response to peptide infusion.

To investigate the physiological role of atrial natriuretic factor (ANF) in patients with hypoxic pulmonary hypertension secondary to chronic obstructive lung disease (COLD), we infused synthetic alpha-human ANF in seven such patients, and investigated the physiological correlates to circulating peptide levels in 24 patients with COLD. ANF infusion, at incremental rates of 0.01, 0.03, and 0.1 micrograms/kg.min, increased basal plasma immunoreactive (ir) ANF (136 +/- 38 pg/ml) by 3-, 10-, and 26-fold, respectively, and reduced pulmonary artery pressure (from 33 +/- 3 to 25 +/- 2 mmHg, P less than 0.001) and systemic arterial pressure (from 88 +/- 4 to 79 +/- 4 mmHg, P less than 0.001) in a dose-related fashion. Cardiac index increased by 13.5% (P less than 0.01) while heart rate was unchanged. Cardiac filling pressures decreased at 0.1 micrograms/kg.min ANF. Pulmonary and systemic vascular resistance fell by 37% (P less than 0.001) and 19% (P less than 0.001), respectively. Arterial oxygenation was impaired during ANF infusion, suggesting partial reversal of hypoxic pulmonary vasoconstriction. Plasma renin activity remained unchanged but aldosterone fell by 44% (P less than 0.01). The levels of plasma irANF in 24 patients correlated directly with the degree of hemoconcentration (r = 0.67, P less than 0.001), respiratory acidosis (r = -0.65, P less than 0.001), and pulmonary hypertension (r = 0.52, P less than 0.01). The results suggest that ANF may serve as a potent pulmonary vasodilator involved in the circulatory homeostasis of patients with COLD.

Adult↗

[Presence of a specific binding site of endothelin on cultured smooth muscle cells].

The 21 amino-acids endothelium-derived peptide, endothelin, recently isolated by Yanagisawa et al. (Nature 1988; 33, 411-5) possesses potent vasoconstrictive properties in vivo and in vitro. In the present study, we investigated the binding of endothelin on cultured rat aortic smooth muscle cells using 125I-iodotyrosyl-endothelin labelled by the chloramine T method. 125I-endothelin bound to a single class of hight affinity binding sites in vascular smooth muscle cells. After 2 hours incubation at 37 degrees C, dissociation constant (Kd) was 1.2 +/- 0.3 nM and binding capacity (Bmax) was 59 +/- 11 fmol/10(6) cells (n = 5). 125I-endothelin was displaced by unlabelled endothelin with a inhibition constant (Ki) of 0.2 nM, whereas an absence of competition was observed with 1 microM of vasoactive substances such as angiotensin II, arg-vasopressin, atrial natriuretic factor, histamine, epinephrine and norepinephrine, and with the calcium entry blocks nifedipine, diltiazem and D 600. 125I-endothelin binding was not reversible by addition of unlabelled endothelin (1 microM) and not dissociable by acetic acid (10 mM) or trypsin (0.1 p. 100) treatment of the cells. Furthermore, preincubation of vascular smooth muscle cells with endothelin (1 nM) at 37 degrees C induced a rapid down-regulation of endothelin binding capacity by about 50 p. 100. These data indicate that specific endothelin bindind sites are present in smooth muscle cells, and suggest a tight binding or a rapid captation of endothelin into the cell membrane leading to contractile events.

Animals↗

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells.

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.

Angiotensin II↗

Interaction between atrial natriuretic factor and angiotensin II receptors in the regulation of blood pressure.

Atrial natriuretic factor (ANF) and angiotensin II (Ang II) appear to act as physiological antagonists in the regulation of blood pressure and fluid homeostasis. After 18 h incubation in cultured vascular smooth muscle cells, Ang II (10(-8) mol/l) induced down-regulation of ANF receptors (reduced by 60% of total binding capacity) that was inhibited by Sar1-Ile8-Ang II (10(-7) mol/l), whereas ANF (10(-8), 10(-7) mol/l) was not able to affect Ang II receptors. The down-regulation provoked by Ang II was associated with an enhancement of ANF-stimulated cyclic (c) GMP formation and was confined to the non-guanylate cyclase-coupled ANF receptor subtype. This suggests that the decrease in ANF receptors elicited by Ang II and the paradoxical increase in the biological activity of ANF may represent a mechanism that represses excessive or long-term pressor effects of Ang II.

Angiotensin II↗

Blood-brain barrier and atrial natriuretic factor.

In brain, binding sites for atrial natriuretic factor (ANF) have been characterized in areas such as circumventricular organs that lack the tight capillary endothelial junctions of the blood-brain barrier and therefore are exposed to circulating peptides. Since atrial natriuretic factor acts directly on vascular endothelium and has been proposed to be actively involved in blood pressure regulation and fluid homeostasis, it is interesting to know whether ANF receptors exist on brain capillaries that constitute the blood-brain barrier and participate in the constant fluid exchange between blood and brain. The present paper reports recent evidence of the presence of ANF receptors located on the structure. It assesses the specific binding of 125I-labelled ANF on bovine brain microvessel preparations and its coupling with a guanylate cyclase system. The potential physiological role of ANF on brain microcirculation and blood-brain barrier functions is discussed.

Animals↗

Down-regulation of atrial natriuretic factor receptors and correlation with cGMP stimulation in rat cultured vascular smooth muscle cells.

The relationship between the binding of 125I-labeled rat ANF and the responsiveness in cGMP production of ANF receptors were examined in cultured rat thoracic smooth muscle cells after preexposure with the peptide. Binding assay of 125I-labeled ANF showed a specific, reversible and saturable binding with a KD value of 3.1 +/- 0.3 10(-10) M and a maximum binding (Bmax) of 240 +/- 30 fmol/10(6) cells. Pretreatment of the cells with increasing concentrations of unlabeled ANF (10(-9) M to 10(-7) M) resulted in a dose-dependent decrease of the number of binding sites without a change in the affinity. This effect was clearly associated with a desensitization of ANF-induced cGMP production.

Animals↗

Endothelin receptor regulation by endothelin synthesis in vascular smooth muscle cells: effects of dexamethasone and phosphoramidon.

One of the major biological effects of the endothelium-derived peptide endothelin-1 (ET-1) is its receptor-mediated constrictive action on vascular smooth muscle. In this study, we have examined the effects on the ET-1 pathway of 18 h exposure at 37 degrees C of cultured rat aortic smooth muscle cells to dexamethasone (DEX) and phosphoramidon. ET-1 synthesis was evaluated by radioimmunoassay, ET-1 binding characteristics were determined with [125I]iodo-ET-1, and ET-1-induced intracellular calcium mobilization was measured using fura-2-loaded cells. DEX (100 nM) led to a 2- to 3-fold-increase of ET-1 production, it down-regulated ET-1 receptors and reduced ET-1-stimulated calcium mobilization by 70%. In contrast, phosphoramidon (100 microM) inhibited ET-1 production by 60%, up-regulated ET-1 receptors and potentiated ET-1-induced calcium mobilization by 75%. These results indicate that the regulatory effects of DEX and phosphoramidon on ET-1 receptors are mediated via ET-1 production by the cells. This suggests an autocrine control of ET-1 receptors by endogenous ET-1 synthesis in vascular smooth muscle cells.

Animals↗