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Only a limited number of studies have compared the therapeutic effects of repetitive cycles of radiolabeled somatostatin (SST) analogues or assessed potential synergy of these analogues with chemotherapy agents. Hence, a comparison was conducted in an in vivo AR42J tumor model with an SST2 antagonist, [177Lu]Lu-satoreotide tetraxetan ([177Lu]LuSSO110), and a SST2 agonist, [177Lu]Lu-DOTA-TATE. The efficacy of treatment combining [177Lu]Lu-satoreotide tetraxetan with capecitabine plus temozolomide was also assessed. Swiss Nude mice were xenografted with AR42J tumor cells. For the evaluation of the tumor response to radiation therapy, animals received one weekly dose of [177Lu]LuSSO110 or [177Lu]Lu-DOTA-TATE for four consecutive weeks. For combined treatments, animals received one weekly dose of [177Lu]LuSSO110 at 20 MBq for four consecutive weeks in association with daily doses of capecitabine and temozolomide (CAPTEM). Weekly treatment for four consecutive weeks with [177Lu]LuSSO110 at 15 MBq revealed a significantly reduced tumor growth, with 68 days to reach a tumor volume of 850 mm3, compared to [177Lu]Lu-DOTA-TATE at 15 MBq or 30 MBq, with, respectively, 43 days and 48 days. This was associated with a 3.5 fold higher tumor uptake of [177Lu]LuSSO110 (15 MBq) compared to [177Lu]Lu-DOTA-TATE, with no or mild effects on body weight, hematological toxicity, or renal toxicity. In AR42J tumor-bearing mice treated once a week for four consecutive weeks, [177Lu]LuSSO110 (15 MBq) associated with CAPTEM regimen did not significantly increase the median time to reach a tumor volume of 1000 mm3 as compared to treatment with [177Lu]Lu-satoreotide alone: 50.7 vs 42.2 days, respectively. The association of the two treatments only led to mild weight losses. Repeated administrations of [177Lu]LuSSO110 were able to potentiate peptide receptor radionuclide therapy with a higher tumor uptake and longer median survival compared to [177Lu]Lu-DOTA-TATE. The The combination of [177Lu]Lu-satoreotide with capecitabine and temozolomide did not show any synergy, but no antagonism was observed either. Citation Format: Olivier Raguin, Marcel Krüger, Peggy Provent, Funda Cay, Elodie Marie dit chatel, Marie Lux, Stéphane Lezmi, Florence Meyer-Losic, Pascale Plas, Ben Pais, Cyril Berthet. Antitumor activity comparison of two somatostatin receptor ligands radiolabeled with Lutetium-177 (SSO110 and DOTA-TATE) alone or combined with chemotherapy in mice bearing AR42J SST2-positive tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5044.

BACKGROUND Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment. METHODS Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro. RESULTS Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-β. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment. CONCLUSIONS The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions. Prostate 76:383–393, 2016. © 2015 Wiley Periodicals, Inc.

A novel series of benzimidazoles was identified and optimized, leading to the discovery of potent and selective antagonists of the human melanocortin-4 receptor. In addition, compound 5i was shown to cross the blood-brain barrier after intravenous dosing in rats.

Background Tasquinimod (a quinoline-3-carboxyamide) is a small molecule immunotherapy with demonstrated effects on the tumor microenvironment (TME) involving immunomodulation, anti-angiogenesis and inhibition of metastasis. A target molecule of tasquinimod is the inflammatory protein S100A9 which has been shown to affect the accumulation and function of suppressive myeloid cell subsets in tumors. Given the major impact of myeloid cells to the tumor microenvironment, manipulation of this cell compartment is a desirable goal in cancer therapeutics. Methods To understand the consequences of tasquinimod treatment on the TME, we evaluated early treatment effects in tumor infiltrating myeloid cells. Cellular phenotypes were studied by flow cytometry while gene expression both in tumor tissue and in isolated CD11b+ cells or tumor cells were measured by real time-PCR. Effects on angiogenesis were monitored by changes in CD31 levels and by gene expression in tumor tissue. Effects on cytokine levels in tumor tissue and serum were determined by multiplex analysis. Results The MC38-C215 colon carcinoma tumors showed a substantial infiltration of primarily myeloid cells that were dominated by Ly6ClowF4/80+CD206+ M2-polarized tumor associated macrophages (TAMs), an immuno-suppressive and pro-angiogenic cell population. Here, we show that tasquinimod treatment induces an anti-tumor effect which is subsequent to a reduction in tumor infiltrating CD206+ M2 macrophages and a simultaneous increase in M1 macrophages expressing MHC class II and CD86. The tasquinimod-induced changes in TAM polarization were evident within 24 h of exposure, emphasizing the ability of tasquinimod to rapidly reprogram the tumor microenvironment. This change in the tumor associated myeloid compartment preceded an increased IL12-production within the tumor and a decrease in tumor neovascularization. The switch in TAM polarization by tasquinimod was confirmed in the 4T1 breast cancer model where tasquinimod also reduce lung metastasis development. Conclusion Our data show that tasquinimod affects tumor infiltrating myeloid cells early after exposure, leading to a change in phenotype from pro-angiogenic and immunosuppressive M2-like TAMs to pro-inflammatory M1-like macrophages. These changes are consistent with the effects of tasquinimod seen on tumor vascularization, immune suppression and metastasis giving further insights to the anti-tumor mechanism of action of tasquinimod. Electronic supplementary material The online version of this article (doi:10.1186/s40425-015-0098-5) contains supplementary material, which is available to authorized users.

Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment.Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro.Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-β. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment.The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions.

Novel 3-thio-1,2,4-triazoles have been obtained via a solution-phase parallel synthesis strategy, affording potent non-peptidic human somatostatin receptor subtypes 2 and 5 agonists.

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.

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 (P0.01) and 633.2 +/- 56.4 fmol/mg protein (P0.001), 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, P0.05), 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.

The infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an “inflamed” milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa.

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.

Human melanocortin 4 receptor (hMC4R) mutations with in vitro functional effects are responsible for 0.5–2.5% of severe obesity. Designing ligands that are able to counteract this in vitro-associated molecular defect is crucial to develop specific anti-obesity drugs in these genetically associated cases. We analyzed the in vitro effect of two novel melanocortin agonists, IRC-022493 and IRC-022511, on typical hMC4R mutations chosen based on the nature of their functional alterations, i.e. intracytoplasmic retention and/or reduced basal activity and/or reduced α-MSH potency. We assessed the in vitro ability of IRC-022493 and IRC-022511 to bind and activate hMC4R mutants. These mutations were found earlier in 11 obese French patients (median age (range) was 17.6 years (5.7–48.0) and body mass index (BMI)-Z-score 4.2 s.d. (1.5–5.5). The MC4R agonists were responsible for a significant activation of mutated hMC4R depending on the functional characteristics of the mutations. Both agonists were able to activate mutated hMC4R with decreased α-MSH potency, associated with or without decreased basal activity, to the same extent than α-MSH in wild-type MC4R. This result suggests that those mutations would be the best targets for the MC4R agonists among MC4R mutation-bearing obese patients. No specific clinical phenotype was associated with the differential response to pharmacological agonists. We identified two novel melanocortin agonists that were able in vitro to efficiently activate mutated hMC4R with impaired endogenous agonist functional response. These results stimulate interest in the development of these drugs for hMC4R mutations-associated obesity.

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood-brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (approximately 50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (approximately 50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.

Purpose: Tasquinimod is in phase III development (10TASQ10) for treatment of metastatic castration-resistant prostate cancer. Tasquinimod has shown proof of concept by delaying disease progression and increasing overall survival in a randomized phase II study. Tasquinimod binds to the S100A9 protein and inhibits its interaction with the RAGE and TLR4 receptors, an interaction postulated to be important for the accumulation and function of tumor infiltrating myeloid cells. Treatment with tasquinimod in preclinical tumor models has been shown to result in modulation of immune responses, inhibition of angiogenesis and inhibition of metastasis. Since myeloid cells can contribute to all of the above mentioned areas, we analyzed the effect of tasquinimod on infiltrating cell populations in MC38 adenocarcinoma tumors. Experimental design: Murine colon carcinoma cells (MC38) were inoculated in matrigel in wt or nude C57/Bl6 mice and tumor growth was monitored in the presence or absence of tasquinimod. Effects on vascularization and cell populations were studied by immunohistochemistry on tumors or flow cytometry on isolated cell suspensions. Results: Treatment with tasquinimod inhibited growth of subcutaneously inoculated MC38 tumors. This effect was not restricted to a changed T cell response since tumor growth was also inhibited in nude mice. Treatment effects were associated with a decrease in tumor neovascularization as evident by CD31 immunostaining. A distinct unvascularized necrotic core can be observed in the treated tumors compared to vehicle treated mice. Untreated MC38 tumors showed a substantial infiltration of primarily myeloid cells where most were Ly6ClowCD206+ (M2) macrophages, a population that has been shown to have pro-angiogenic properties. Treatment with tasquinimod led to a reduction in this CD206+ cell population and a concomitant increase in CCR7+ (M1) macrophages. No change was detected in infiltration of CD4+ or CD8+ T cells, including Foxp3+ cells, in accordance with the lack of T cell dependency of tasquinimod effects in this tumor. Conclusions: S100A9 and RAGE has previously been shown to have roles in infiltration of myeloid cells in MC38 tumors. Treatment with tasquinimod, an S100A9-RAGE/TLR4 inhibitor, changes the distribution of myeloid cell populations in the tumor which includes a reduced number of M2 macrophages in the infiltrate. These data suggest that tasquinimod has an effect on tumor infiltrating myeloid cells which could contribute to T cell independent effects on angiogenesis and inhibition of tumor growth. Citation Format: David Liberg, Anders Olsson, Pascale Plas, Marie Torngren, Catherine George, Martin Stenstrom, Fabien Schmidlin, Helena Eriksson, Tomas Leanderson. Immunomodulation by tasquinimod: Skewing of tumor infiltrating myeloid cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B78.

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 (p0.001) and creatinine clearance was reduced from 1.04 +/- 0.15 ml/min to 0.23 +/- 0.06 ml/min (p0.001). Renal ET-1 receptor density was significantly increased in glycerol-treated rats to 255 +/- 14 fmol/mg protein in renal cortex (p0.01), and 576 +/- 55 fmol/mg protein in renal medulla (p0.01), 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.

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.

Two atrial natriuretic factor (ANF) receptor subtypes are present in vascular nooth muscle cells: the B receptors (or biologically active) coupled to a guanylate cyclase ad 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, All) 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 All reduced the total umber of ANF binding sites after 18 h, ANF induced a desensitization of the guanylate cyclase whereas All elicited a potentialization of this system. From these results, we have included that in vascular cells B receptors are sensitive to homologous regulation and C receptors are sensitive to heterologous regulation by All. This also highlights a specific interaction between ANF and All at the receptor level.

[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.

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.

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.

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.

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.