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.

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.

Alpha1-adrenoceptors in testosterone-induced prostatic hypertrophy.

Modifications of rat prostatic alpha1-adrenoceptors were investigated in testosterone-induced prostatic hypertrophy. [3H]prazosin bound to a single class of binding sites with a dissociation constant of 57.9+/-5.02 pM. The greater part of the binding capacity (24.6+/-1.02 fmol/mg protein) was made up of chloroethylclonidine-resistant binding sites that showed high-affinity for oxymetazoline and 5-methyl-urapidil, and was identified as alpha1A-adrenoceptors. The remaining chloroethylclonidine-sensitive binding sites that showed low-affinity for oxymetazoline and 5-methyl-urapidil were preferentially identified as alpha1B-adrenoceptors. mRNA for the three alpha1-adrenoceptors (alpha1a, alpha1b and alpha1d) was detected. Testosterone administration produced a 23% decrease of alpha1-adrenoceptor density, likely by an increase of prostatic glandular epithelium and a decrease in the relative proportion of smooth muscle, thus of alpha1-adrenoceptor density. The steady state level of mRNAs for alpha1-adrenoceptors was not modified by testosterone treatment. These results indicate that prostate alpha1-adrenoceptors are not affected in the prostatic hypertrophy induced by testosterone.

[Homologous and heterologous regulation of atrial natriuretic factor receptors in smooth muscle cells]. / Régulation homologue et hétérologue des récepteurs du facteur natriurétique atrial sur cellules musculaires lisses.

Two subtypes of atrial natriuretic factor (ANF) receptors are present in vascular smooth muscle cells: B(biologically active) receptors coupled to a guanylate cyclase and C (clearance) receptors (95 per cent of the total number of ANF binding sites) non coupled to any identified second messenger system. We compared the homologous receptor regulation induced by ANF to the heterologous one elicited by angiotensin II (Ag II). Binding of (3-[125I]iodotyrosyl) rat ANF and cGMP production stimulated by ANF were measured after 18 hours preincubation of rat cultured vascular smooth muscle cells (10(6) cells/dish) at 37 degrees C with ANF or Ag II. The hormones (10 nM) decreased to the same extent the total apparent number of ANF binding sites (control: 208 +/- 25 fmol/10(6); ANF : 82 +/- 20 fmol/10(6) cells; Ag II : 90 +/- 9 fmol/10(6) cells) The diminution of the number of ANF binding sites induced by ANF exposure was reversed by 85 per cent following 10 minutes treatment of the cells with 10 mM AcOH. Moreover, treatment with ANF (10 nM) led to a diminution of cGMP stimulation induced by ANF, this effect being still present after washing the cells with 10 mM AcOH. In contrast, diminution of ANF building sites consecutive to Ag II exposure was not affected by AcOH treatment and a potentiation of cGMP production elicited by ANF was observed. These results suggest that, in rat vascular smooth muscle cells, B receptors are sensitive to homologous down regulation and C receptors are sensitive to heterologous regulation by Ag II.

[Homologous and heterologous regulations of endothelin receptors on smooth muscle cells]. / Régulations homologue et hétérologue des récepteurs de l'endothéline sur cellules musculaires lisses.

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.

[Presence of a specific binding site of endothelin on cultured smooth muscle cells]. / Présence d'un site de liaison spéfique de l'endothéline sur cellules musculaires lisses en culture.

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.

[Multiplicity of endothelin receptors of aortic smooth muscle cells in rats]. / Multiplicité des récepteurs de l'endothéline sur les cellules musculaires lisses d'aorte de rat en culture.

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.

[Endothelin receptors and endothelin-1 immunoreactivity in the human lung]. / Récepteurs de l'endothéline et immunoréactivité de l'endothéline-1 au niveau des poumons humains.

We investigated in human lung preparations the characteristics of endothelin-1 (ET-1) binding and the amount of ET-1-like immunoreactivity. Saturation experiments revealed the presence of a large number of high affinity specific ET-1 binding sites with a dissociation constant (Kd) of 1.35 nM and a binding capacity (Bmax) of 9.74 pmol/mg of protein. The binding was time- and temperature-dependent and dissociated by only 10% by the addition of 1 microM unlabeled ET-1. In competition experiments, [125I]ET-1 binding was totally inhibited by unlabeled ET-1 and ET-2 with inhibition constant (Ki) values of 0.20 and 0.21 nM respectively, and 80% inhibited by ET-3 with Ki value of 0.50 nM. The binding was not affected by 1 microM structurally unrelated compounds. Moreover a high level of ET-1-like immunoreactivity (2.3 pg/mg wet weight) was found in human lung by using a specific radioimmunoassay of ET-1 after extraction. HPLC analysis revealed the presence of both ET-1 and Big-ET. These results suggest that the lung may be an important target organ for ET-1 action and/or metabolism in human.

[Effect of Ginkgo biloba extract on the hemato-encephalic barrier]. / Effet de l'extrait de Ginkgo biloba sur la barrière hémo-encéphalique.

The different methods used to explore the blood-brain barrier (made up of cerebral capillary vessels), and notably, at molecular level, isolated microvessel preparations, have greatly improved our knowledge in this particular field. Some of these methods could be used to evaluate the protective effects of therapeutic substances, such as Ginkgo biloba extract, on the blood-brain barrier.

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.

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.

Specific binding of atrial natriuretic factor in brain microvessels.

Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. We examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity (dissociation constant, approximately 10(-10) M) and with a binding capacity of 58 fmol/mg of protein. The binding of 125I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.

Endothelin receptors in testosterone-induced prostatic hypertrophy in rats.

Endothelin receptors were characterized in rat prostate and potential modification of these receptors was investigated in prostatic hypertrophy induced by testosterone. Both ET(A) and ET(B) endothelin receptor mRNA were detected in rat prostate, whereas binding experiments show the presence of only ET(A) receptors. Testosterone administration produced a 75% increase in prostate weight. Although the density of prostatic endothelin receptors was decreased from 348 +/- 75.0 fmol/mg protein in control rats to 252 +/- 39.9 fmol/mg protein in testosterone-treated animals, the total amount of receptors per prostate was unchanged. The steady-state level of ET(A)- and ET(B)-receptor mRNA was not altered by testosterone treatment. These results suggest that endothelin receptors are not affected in prostatic hypertrophy induced by testosterone.

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.

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.

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.

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.

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.

Cross-desensitization between angiotensin II and endothelin-1 in the pithed rat.

It has been shown that in vitro angiotensin II (Ang II) potently downregulates endothelin-1 (ET-1) binding sites. In this study, we investigated in vivo the interactions between the renin-angiotensin system and ET-1. Sprague-Dawley rats were pithed, ventilated, and diastolic blood pressure (DBP) was recorded. ET-1 (1 nmol/kg) induced a biphasic response: a transient hypotension followed by a sustained increase of DBP. Captopril (5 mg/kg, i.v.) or Sar1-Ile8-Ang II (10 ng/kg/min) did not affect ET-1 responses. In other experiments, DBP was increased by infusion of methoxamine (MTX: 10, 20, 25, 32.5 micrograms/kg/min) or Ang II (50, 100, 150, 200 ng/kg/min). ET-1-induced hypotension correlated with the level of DBP (r = 0.94) for both agonists. The elevation of DBP in response to ET-1 was also related to the vascular tone but was dose-dependently attenuated by Ang II infusion when compared with MTX. Conversely, infusion of ET-1 (0.1 nmol/kg/min) blunted the pressor response to Ang II (0.1 micrograms/kg) but not to MTX (50 micrograms/kg). These doses induced the same increase of DBP in pithed control rats. Similarly, increased plasma renin activity induced by chronic salt depletion (0% NaCl) in pithed rats provoked a shift to the right of the dose-response curves to Ang II and ET-1 but not to MTX. These results suggest an in vivo cross-desensitization between ET-1 and Ang II.