Transendothelial migration of two metastatic breast carcinoma cells depend on the SDF-lalpha-CXCR4 complexes.

The role of the SDF-1alpha chemokine-CXCR4 receptor system on tumor cell transendothelial migration was studied by culturing metastatic breast tumor cell lines, MDA-MB-231 and MDA-MB-435, either alone or on a HUVEC monolayer pre-established on a "Transwell" filter. After a 24-hour culture in the presence or absence of SDF-1alpha, tumor cell transmigration rates were compared. We showed that: CXCR4 is present on both cell lines; MDA-MB-231 but not MDA-MB-435 cell migration is stimulated by increasing concentrations of SDF-1alpha; neutralizing anti-CXCR4 antibody inhibits the SDF-1alpha chemoattractant effect; CXCR4 expression, measured by cytofluorometry, was enhanced after treatment with SDF-1alpha on MDA-MB-231 cells but remained unchanged on MDA-MB-435 cells; Scatchard analysis evidences 8.10(5) and 2.10(5) high affinity sites (KD range: 20 to 30 nM) on, respectively, MDA-MB-231 and MDA-MB-435 cells. These significant differences could explain the distinctive transendothelial migration responses of these two cell lines in the presence of SDF-1alpha.

Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice.

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.

Inhibition of epidermoid carcinoma A431 cell growth and angiogenesis in nude mice by early and late treatment with a novel dextran derivative.

We investigated the effect of a new dextran derivative, phenylacetate carboxymethyl benzylamide dextran (NaPaC), on epidermoid carcinoma A431 cells secreting a large quantity of angiogenic factor, vascular endothelial growth factor (VEGF). In vitro, NaPaC inhibited the proliferation of A431 cells (IC(50)=5 micro M). Also, NaPaC decreased the binding of radiolabelled VEGF(165) to endothelial cells (IC(50)=0.2 micro M). In vivo, we explored the effects of NaPaC (15 mg kg(-1)) on A431 xenograft growth starting the drug administration at the time of tumour cell inoculation (early treatment) and 1 week later, when tumours were well established (late treatment). Early treatment was more efficient on tumour inhibition (70% vs control) than late treatment (50% vs control). Early and late NaPaC-treatment increased the aponecrosis in tumour by 70 and 30%, respectively. Whatever treatment, NaPaC inhibited the intratumour endothelial cell density in the same manner. In contrast, vessel area was decreased only when NaPaC was injected early (35%). These results show that NaPaC has a potent inhibitory effect, dependent on treatment outset, on epidermoid carcinoma growth associated with an intratumour microvascular network diminution and an aponecrosis increase. As this drug is nontoxic at efficient dose, it offers interesting perspectives for the therapy of malignant lesions.

Sodium phenylacetate enhances the inhibitory effect of dextran derivative on breast cancer cell growth in vitro and in nude mice.

Sodium phenylacetate (NaPa) and carboxymethyl benzylamide dextran derivative (CMDB(LS4)) are able to inhibit growth of breast tumour cells. In this study, we explored whether the combination of NaPa and CMDB(LS4)may enhance their respective inhibitory effects on the MCF-7ras cell growth in vitro and in vivo. NaPa inhibited MCF-7ras cell proliferation by reducing the DNA replication concomitantly with a recruitment of cells in G0/G1 phase and by inducing apoptosis in a dose- and time-dependent manner. The addition of CMDB(LS4)potentiated the NaPa antiproliferative effect in the manner dependent on the ratio of CMDB(LS4)and NaPa concentrations. In nude mice, CMDB(LS4)(150 mg kg(-1)) or NaPa (40 mg kg(-1)) administrated twice a week, for 7 weeks inhibited MCF-7ras xenograft growth by 40% and 60%, respectively. The treatment by both, CMDB(LS4)and NaPa, decreased tumour growth by 83% without any toxicity. To better understand the mechanism of NaPa and CMDB(LS4)action we assessed their effect on mitogenic activity of MCF-7ras conditioned medium (CM) on BALBC/3T3 fibroblasts. CMDB(LS4)added to the CM, inhibited its mitogenic activity whereas NaPa had an anti-mitogenic effect when CM was prepared from MCF-7ras cells pretreated with NaPa. Thus, the antiproliferative effects of NaPa and CMDB(LS4)involve 2 different mechanisms explaining, at least in part, the possible synergism between them. Overall, this study points to the potential use of a combination of dextran derivatives with NaPa to inhibit the breast tumour growth.

Vascular endothelial growth factor 165 (VEGF(165)) activities are inhibited by carboxymethyl benzylamide dextran that competes for heparin binding to VEGF(165) and VEGF(165).KDR Complexes.

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.

Human alpha1-acid glycoprotein binds to CCR5 expressed on the plasma membrane of human primary macrophages.

We have reported previously that human alpha(1)-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229-1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with K(d) values of 16 nM and 4.9 microM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein-protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect ( approximately 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1beta (MIP-1beta) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33-36 kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1beta. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.

Amiodarone has exclusively non-genomic action on cardiac beta-adrenoceptor regulation.

The antiarrhythmic drug amiodarone down-regulates the density of cardiac beta-adrenoceptors behaving as a triiodothyronine (T(3)) antagonist. It is still unclear if amiodarone acts at the nuclear (genomic) and/or the non-genomic levels. Using Northern blot analysis, we showed that the amiodarone had no effect on the increase of beta(1)-adrenoceptor mRNA level induced by the T(3)-administration in the heart of thyroidectomised rats. Thus, our results suggest that amiodarone has no genomic effect. Consequently, we investigated whether amiodarone down-regulation of beta-adrenoceptor number in T(3)-stimulated cardiomyocytes could be explained by changes in the rate of cell surface receptor protein turnover. Indeed, the binding studies of cyclohexidemide-treated cells showed that amiodarone suppressed the T(3)-induced decrease in the rate of the cell surface receptor disappearance. In conclusion, our findings indicate that the modulation of cardiac beta-adrenoceptor density by amiodarone involves only non-genomic targets required in T(3)-dependent regulation of the cell surface beta-adrenoceptor turnover.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Carboxymethyl benzylamide dextran blocks angiogenesis of MDA-MB435 breast carcinoma xenografted in fat pad and its lung metastases in nude mice.

We previously showed that carboxymethyl benzylamide dextran (CMDB7) prevents tumor growth and tumor angiogenesis by binding to angiogenic growth factors, thereby preventing them from reaching their receptors on tumor or stromal cells (Bagheri-Yarmand et al. Br. J. Cancer, 78: 111-118, 1998; Bagheri-Yarmand et al. Cell Growth Differ., 9: 497-504, 1998). In this study, CMDB7 inhibited neovessel formation within the fibroblast growth factor 2-enriched matrigel in mice, and its anticancer effect was then tested in a metastatic breast cancer model. Human MDA-MB435 cells were injected into the mammary fat pad of nude mice, and breast tumors developed within 1 week; all of the mice had lung metastases at 12 weeks. CMDB7 treatment (50, 150, or 300 s.c. or 300 i.v. mg/kg/week for 10 weeks) reduced the incidence of lung metastases to 12%. Histological analysis showed markedly less tumor neovascularization in the CMDB7-treated mice. Pulmonary metastasis incidence was strongly dependent on the intratumoral neoangiogenesis in primary tumors.

Effects of triiodothyronine administration on the adenylyl cyclase system in brown adipose tissue of rat.

This study was undertaken to investigate the effect of triiodothyronine (T3) administration to euthyroid rats on beta 3-adrenoceptor (beta 3-AR) expression and on the different components of the adenylyl cyclase (AC) system in brown adipose tissue (BAT). In rats treated with T3, the beta 3-AR density (assessed by the binding of [3H]CGP-12177) showed a decrease of 50%, as did their mRNA, as analyzed by reverse transcriptase-polymerase chain reaction. In hyperthyroid rats, compared with control rats, there was a 40% increase in G alpha s activity (stimulated by NaF or GTP gamma S) and a fourfold increase in the protein concentration (Western blotting). In contrast, the level of the pertussis toxin substrate Gi declined by 35% in response to T3. Analysis of dose-response curves for isoproterenol and CGP-12177 revealed that neither basal nor stimulated AC activities nor 50% stimulatory concentration for these agonists was changed by T3 administration. In conclusion, these results suggest that downregulation of the beta 3-AR by T3 was counter-balanced by changes in other components of the AC cascade (i.e., Gs and Gi), so no change occurred in the capacity of BAT to generate adenosine 3',5'-cyclic monophosphate.

Nongenomic effect of triiodothyronine on cell surface beta-adrenoceptors in cultured embryonic cardiac myocytes.

We studied the time course of cell surface beta-adrenoceptors (BAR) in cardiomyocytes in response to a single triiodothyronine (T3) (10(-8) M) stimulation. An early first increase of BAR density was observed within 2 hr (+ 10% versus control cells, P < 0.05), and a plateau was maintained for 17-20 hr. This effect was followed by a much greater, late increase of BAR density, starting around 22 hr and lasting until 48 hr post T3 addition (+40% versus control cells; p < 0.05). Since reverse T3 studied in the same conditions had no effect in this system, we concluded that this T3 effect was specific. We hypothesized that the early response might be nongenomic because the early effect of T3 was still observed in the presence of cycloheximide (2 x 10(-5) M) whereas the late increase was totally suppressed by the drug. The early response to T3 required intact microtubules, since colchicine (2 x 10(-5) M) was able to block the increase in the cell surface BAR number, but it did not involve a change in BAR distribution between external and internal sites, as the external to total BAR ratio remained stable. The measurement of the rate of BAR disappearance from the cell surface allowed us to hypothesize that T3 induced a modification of the turnover in cell surface BAR.

Beta-adrenoceptor subtype expression and function in rat white adipocytes.

1. The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [3H]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta 1-antagonist) and ICI 118551 (selective beta 2-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22 +/- 0.07 nM) and the other with low affinity (23 +/- 7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [3H]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta 3-agonist, BRL 37344 (BRL), were clearly biphasic (P < 0.001). The rank orders of agonist potency (pKi) in competing for [3H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28 +/- 0.24) > NA (8.90 +/- 0.12) > Ad (8.65 +/- 0.12) > > BRL (4.53 +/- 0.17) and BRL (7.38 +/- 0.19) > > Iso (2.96 +/- 0.26) > or = NA (2.80 +/- 0.17) > Ad (2.10 +/- 0.11) indicating the expression of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta 1-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81 +/- 9 fmol mg-1) and high pKi value (7.23 +/- 0.26) confirming the presence of beta 1-adrenoceptors. 4. To assess a possible contribution of the beta 2-subtype, procaterol (Proc), a selective beta 2-agonist, was used to compete with 2 nM [3H]-CGP 12177. A single low affinity (4.61 +/- 0.07) population of binding sites was identified. The density of these sites (71 +/- 12 fmol mg-1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]-CGP 12177 from the beta 1-subtype. 5. The functional potency (pD2) order with BRL (9.07 +/- 0.20) and catecholamines (Iso: 7.26 +/- 0.06, NA: 6.89 +/- 0.02 and Ad: 6.32 +/- 0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31 +/- 0.06 and 5.66 +/- 0.10, respectively. Proc stimulated lipolysis with a low potency (5.59 +/- 0.21). 6. The lipolytic response to 0.001 microM BRL was inhibited by both, selective beta 1- and beta 2-antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: < 4.5 and ICI 118551 pKi: 5.57 +/- 0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 microM Iso or 0.13 microM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41 +/- 0.09 and 8.58 +/- 0.17, respectively, and for the second population of sites 4.73 +/- 0.22 and 4.27 +/- 0.27, respectively. The proportion of the first sites was low: 19 +/- 4 and 22 +/- 5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 microM Proc (CGP 20712A: pKi1: 8.17 +/- 0.08, site1: 23 +/- 6%, pKi2: 4.77 +/- 0.14; ICI 118551: pKi1: 7.78 +/- 0.03, site1: 37 +/- 2%, pKi2: 5.35 +/- 0.25). 7. Our results show that the radioligand [3H]-CGP 12177 allows the characterization of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta 1- and beta 3-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta 3-subtype.

Triiodothyronine and amiodarone effects on beta 3-adrenoceptor density and lipolytic response to the beta 3-adrenergic agonist BRL 37344 in rat white adipocytes.

The beta-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T3) effect on beta-adrenergic receptors (beta-ARs) in adipose tissue. AM decreases the expression of some T3 sensitive genes in various tissues and antagonizes the effect of T3 on its nuclear receptors. In this study, the T3, AM and AM + T3 effects on the beta 1- and beta 3-AR density were assessed on rat white adipocytes by radioligand binding using [3H]CGP 12177 after characterization of these subtypes by displacement of [3H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study beta 3-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (Emax x 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T3 effect on lipolysis. The beta 1-binding characteristics (receptor density [Bmax]: 45 +/- 4 fmol/mg of proteins; dissociation factor [Kd]: 0.96 +/- 0.10 nM) were not modified by either compound. Finally, T3 significantly increased beta 3-AR density (587 +/- 69 versus 363 +/- 25 fmol/mg of proteins) and Kd (38 +/- 2 versus 23 +/- 3 nM), while AM alone had no effect and did not antagonize the T3 effect on beta 3-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific beta 3-agonist and increased the beta 3-AR density without changing in beta 1-AR number and affinity. Furthermore, the lack of antagonism between AM and T3 on beta 3-AR expression suggests that T3 does not work directly on the beta 3-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.

Antagonism between T3 and amiodarone on the contractility and the density of beta-adrenoceptors of chicken cardiac myocytes.

3,3',5-Triiodothyronine (T3), at 10(-8) M, potentiated by 26.4-30.9% the isoproterenol-mediated inotropic effect in chick embryo cardiac myocytes in culture. Amiodarone (10(-6) M) decreased this response by 44.6% only in cells cultured with serum, where the T3 concentration was 10(-13) M. Amiodarone inhibited the potentiating effect of T3. Amiodarone alone had no influence on the beta-adrenoceptor density in cells cultured in serum-free medium. This confirms that the effects of amiodarone on cardiac beta-adrenoceptors are T3 dependent. T3 increased the density of beta-adrenoceptors through two concentration ranges, with an initial 30% increase between 10(-14) and 10(-11) M, followed by a second increase until 10(-7) M. Amiodarone not only inhibited the first positive effect of T3 but also decreased beta-adrenoceptor density far below the control value. The second positive T3 effect was also inhibited by 50% by amiodarone. This study suggests that T3 might increase the number of cell-surface beta-adrenoceptors and modify their cellular traffic through at least two mechanisms, one assumed to be non-genomic, the other being genomic, and that amiodarone could affect the two mechanisms differently.

Amiodarone decreases cardiac beta-adrenoceptors through an antagonistic effect on 3,5,3' triiodothyronine.

Because surprising similarities exist between the cardiac effects of amiodarone and those observed during hypothyroidism, we tested three different mechanisms of action in rats to determine whether amiodarone might act by inducing a tissular hypothyroidism: (a) a decrease in thyroid secretion, (b) an inhibiting effect on the conversion of thyroxine (T4) to 3,5,3' triiodothyronine (T3), and (c) a direct antagonistic effect on the cellular action of T3. Five groups of rats, treated orally for 7 or 13 days, were studied: I, control (0.5 ml saline for 7 or 13 days, n = 14); II, amiodarone (50 mg/kg for 7 days, n = 5); III, iopanoic acid (100 mg/kg for 13 days, n = 7); IV, control + T3 (0.5 ml saline for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5); V, amiodarone + T3 (amiodarone 50 mg/kg for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5). Cardiac beta-adrenoceptor density (CBARD) and heart rate (HR) were the two endpoint parameters investigated. Thyroid status was evaluated by serum thyrotropin (TSH), T4, T3, rT3 concentrations and liver type I 5'-deiodinase (5'D-I) activity. Amiodarone (group II) decreased CBARD (-22%, p less than 0.05) without altering thyroid secretion and T3 serum level, whereas 5'D-I was strongly inhibited (-90%, p less than 0.01). Iopanoic acid had no effect on CBARD and HR, but deeply inhibited 5'D-I.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo effects of amiodarone on cardiac beta-adrenoceptor density and heart rate require thyroid hormones.

To assess if the anti-beta-adrenergic effect and the bradycardia induced by amiodarone were mediated by thyroid hormone, we investigated these effects of amiodarone in euthyroid and hypothyroid rats. We studied control rats, thyroidectomized rats, control rats treated with amiodarone (50 mg/kg for 8 days), and thyroidectomized rats treated with amiodarone. At the end of the treatment, free thyroid hormone levels (FT4 and FT3) were determined, and cardiac beta-receptor density (Bmax) and affinity (Kd) were assayed by using (-)-[125I]iodocyanopindolol as radioligand. Resting heart rate (rHR) was also assessed every day in control and thyroidectomized rats, before and after amiodarone. In hypothyroid rats, in which free thyroxine (FT4) was not detectable and free 3,5,3'-triiodothyronine (FT3) was only 16% that of euthyroid rats, Bmax (14.1 +/- 2.5 fmol/mg, n = 7) and rHR (259 +/- 9.7 beats/min, n = 6) were significantly lowered compared with euthyroid rats (Bmax:18.4 +/- 3.4 fmol/mg, n = 7; rHR:277 +/- 4.1 beats/min, n = 5). Amiodarone treatment decreased Bmax (13.6 +/- 2.9 fmol/mg, n = 8) and rHR (252 +/- 5.5 beats/min, n = 5) only in euthyroid rats and did not produce significant cardiac effects in hypothyroid rats. (Values are given as mean +/- SD.) We conclude that a minimum serum thyroid hormone concentration is a necessary condition for amiodarone to produce some of its cardiac effects. An antagonistic reaction to thyroid hormones at the cellular level can be postulated as a mechanism of the cardiac anti-beta-adrenergic action of amiodarone.

Effect of nifedipine and nitrendipine on insulin release in non-diabetic obese patients: a double-blind, placebo-controlled study.

Calcium antagonists are known to decrease insulin release in vitro. In vivo, their effects on insulin secretion and glucose tolerance are more controversial. We carried out a double-blind, double-dummy, controlled trial to study the effects of nifedipine (40 mg/day; n = 9), nitrendipine (20 mg/day; n = 9) and placebo (n = 10) on insulin release in 3 parallel groups of obese patients with mild or transient hypertension and a normal oral glucose tolerance test. The treatment lasted one week. Patients were asked not to modify their diet throughout the trial, and their body weight did not vary significantly. A 2-hour i.v. glucose tolerance test was performed twice in every patient, just before the first drug intake and one hour after the last one. The following parameters were measured or calculated during each test: basal, peak and early phase levels of blood glucose, insulin and C-peptide; glucose disappearance rate; and glucose, insulin and C-peptide incremental areas under the time-curves. On day 1, the 3 groups of patients were comparable for age, sex, body weight and every biological parameter. One-way analysis of variance did not show any significantly different evolution of those parameters between the 3 treatment groups, but calcium antagonists tended to slightly reduce the early phase of insulin release compared with placebo.