Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells.

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca(2+) chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies.

Pharmacological strategies and micrometastasis: what is known? What must be done?

Metastasis accounts for over 90% of cancer patient deaths. In spite of this , the majority of the currently used drugs are aimed at eradicating or controlling the primary tumor. Hardly any cancer therapies used at the moment interfere only with metastasis and there is a need for drugs acting specifically on the metastatic processes, curing the minimal residual disease and being less toxic than the current drugs. Searching for drugs that do not affect the growth of the primary tumor but inhibit the colonization and/or the outgrowth in distant sites could lead to new drugs specifically aimed at treating the minimal residual disease. The aim of this work is to search at what conditions such drugs might be discovered and if some exist already. For that, mechanisms of actions of current experimental and clinical antimetastatic drugs are described and their utility for different clinical settings (primary prevention, adjuvant setting) is analyzed. In conclusion, clues exist that specific antimetastatic drugs could exist and will be developed in a near future.

New pharmacological strategies against metastatic spread.

Although metastatic spread is the most frequent cause of death in cancer patients, there are very few drugs specifically targeting this process. Bases for a new antimetastatic drug discovery strategy are weak because a great number of unknowns characterize the complete understanding of the metastatic cascade mechanisms. Moreover, the current experimental models are too simplistic and do not account for the complexity of the phenomenon. Some targets have been identified but too few are validated. Among them, the metastasis suppressor genes seem to be the most promising. In spite of this, during recent years, a dozen of molecules, which fulfil the definition of a specific metastatic drug that inhibits the metastases without altering the growth of the primary tumour (which can be eradicated by surgery), have been identified and assessed for the proof of the concept. The continuation of this effort would benefit in terms of efficiency, if the objectives were defined more precisely. It is particularly important to distinguish molecules that prevent spread of the metastatic cells of the early-stage primary tumour from the ones which induce a regression of the established metastases or to inhibit the transition from disseminated occult tumour cells to dormant micrometastasis. This second goal is a priori more relevant in the current clinical setting where the detection of early metastatic spread is very difficult, and therefore would call for greater effort on the part of the scientific community.

[Metastatic spread: mechanisms and therapies]. / Les mécanismes de la dissémination métastatique et ses thérapies.

Although metastatic spread is the most frequent cause of deaths in cancer patients, there are very few drugs specifically targeting this process. Bases for a new antimetastatic drug discovery strategy are weak because a great number of unknowns characterizes the whole understanding of the metastatic cascade mechanisms. Moreover, the current experimental models are too simplistic and do not account for the complexity of the phenomenon. Some targets have been identified but too few are validated. Among them, metastasis suppressor genes seem to be the most promising. In spite of this, during the last years, a dozen of molecules which fulfill the definition of a specific metastatic drug, namely that inhibit metastases without altering growth of the primary tumor (which can be eradicated by surgery), have been identified and tried out to assess the proof of the concept. The continuation of this effort would be more efficient if the objectives were defined more precisely. It is particularly important to distinguish molecules aimed at preventing metastic cell spreading at the primary tumour early stage and molecules which have to induce a regression of established metastases or to inhibit the transition from disseminated occult tumour cells to dormant micrometastasis. This second goal is a priori more relevant in the current clinical setting where detection of the early metastatic spread is very difficult, and therefore it should focus a greater effort of the scientific community.

Microsatellite instability and sensitivitiy to FOLFOX treatment in metastatic colorectal cancer.

BACKGROUND: Microsatelite instability (MSI) is the consequence of the inactivation of a mismatch repair gene and is observed in approximately 15% of colon cancer cases. Patients with MSI colon cancer do not benefit from 5-fluorouracil (5-FU)-based chemotherapy. A current treatment of reference for colon cancer is a combination of 5-FU and oxaliplatin (FOLFOX). The aim of this study was to determine the efficiency of the FOLFOX treatment in patients with metastatic MSI colon cancer. PATIENTS AND METHODS: Tumour specimens were collected from patients with metastatic colon cancer treated with FOLFOX 4 modified or FOLFOX 6; these two regimens are based on 85 mg/m2 and 100 mg/m2 oxaliplatin, respectively. The MSI status was assessed by measuring the length of five monomorphic mononucleotide markers. The FOLFOX regimen was evaluated as a first-line treatment according to WHO criteria. RESULTS: Forty patients (22 men, 18 women), median age 63.5 years (27-83 years) were treated with FOLFOX 4 or 6. Nine patients had tumours exhibiting high MSI (MSI group) and 31 patients had tumours exhibiting microsatellite stability (MSS group). In the MSS group, 11 partial responses (36%) were observed, while there were only two in the MSI group (22%) (no significant difference). The two patients who were responders in the MSI group were treated with FOLFOX 6. The overall survival was not significantly different for MSI and MSS patients. CONCLUSION: No significant differences in the overall response rate or overall survival between the two groups of patients were observed. However, these results suggest that patients with MSI colon cancer are more sensitive to a higher dose of FOLFOX.

Microvessel density and VEGF expression are prognostic factors in colorectal cancer. Meta-analysis of the literature.

We performed a meta-analysis of all published studies relating intratumoural microvessel density (MVD) (45 studies) or vascular endothelial growth factor (VEGF) expression (27 studies), both reflecting angiogenesis, to relapse free (RFS) and overall survival (OS) in colorectal cancer (CRC). For each study, MVD impact was measured by risk ratio between the two survival distributions with median MVD as cutoff. Eleven studies did not mention survival data or fit inclusion criteria, six were multiple publications of same series, leaving 32 independent studies for MVD (3496 patients) and 18 for VEGF (2050 patients). Microvessel density was assessed by immunohistochemistry, using antibodies against factor VIII (16 studies), CD31 (10 studies) or CD34 (seven studies). Vascular endothelial growth factor expression was mostly assessed by immunohistochemistry. Statistics were performed for MVD in 22 studies (the others lacking survival statistics) including nine studies (n = 957) for RFS and 18 for OS (n = 2383) and for VEGF in 17 studies, including nine studies for RFS (n = 1064) and 10 for OS (n = 1301). High MVD significantly predicted poor RFS (RR = 2.32 95% CI: 1.39-3.90; P < 0.001) and OS (RR = 1.44; 95% CI: 1.08-1.92; P = 0.01). Using CD31 or CD34, MVD was inversely related to survival, whereas it was not using factor VIII. Vascular endothelial growth factor expression significantly predicted poor RFS (RR = 2.84; 95% CI: 1.95-4.16) and OS (RR=1.65; 95% CI: 1.27-2.14). To strengthen our findings, future prospective studies should explore the relation between MVD or VEGF expression and survival or response to therapy (e.g. antiangiogenic therapy). Assessment of these angiogenic markers should be better standardised in future studies.

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.

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.

Regulation of beta 1- and beta 3-adrenergic agonist-stimulated lipolytic response in hyperthyroid and hypothyroid rat white adipocytes.

1. This study examined the effects of thyroid status on the lipolytic responses of rat white adipocytes to beta-adrenoceptor (beta-AR) stimulation. The beta 1- and beta 3-AR mRNAs and proteins were measured by Northern and saturation analyses, respectively. Glycerol production and adenyl cyclase (AC) activity induced by various non-selective and selective beta 1/beta 3-AR agonists and drugs which act distal to the receptor in the signalling cascade were measured in cells from untreated, triiodothyronine (T3)-treated and thyroidectomized rats. 2. The beta 3-AR density was enhanced (72%) by T3-treatment and reduced (50%) by introduction of a hypothyroid state while beta 1-AR number remained unaffected. The beta 1- and beta 3-AR density was correlated with the specific mRNA level in all thyroid status. 3. The lipolytic responses to isoprenaline, noradrenaline (beta 1/beta 3/beta 3-AR agonists) and BRL 37344 (beta 3-AR agonist) were potentiated by 48, 58 and 48%, respectively in hyperthyroidism and reduced by about 80% in hypothyroidism. 4. T3-treatment increased the maximal lipolytic response to the partial beta 3-AR (CGP 12177) and beta 1-AR (xamoterol) agonists by 234 and 260%, respectively, increasing their efficacy (intrinsic activity: 0.95 versus 0.43 and 1.02 versus 0.42). The maximal AC response to these agonists was increased by 84 and 58%, respectively, without changing their efficacy. 5. In the hypothyroid state, the maximal lipolytic and AC responses were decreased with CGP (0.17 +/- 0.03 versus 0.41 +/- 0.08 mumol glycerol/10(6) adipocytes; 0.048 +/- 0.005 versus 0.114 +/- 0.006 pmol cyclic AMP min-1 mg-1) but not changed with xamoterol. 6. The changes in lipolytic responses to postreceptor-acting agents (forskolin, enprofylline and dibutenyl cyclic AMP, (Bu)2cAMP) suggest the modifications on receptor coupling and phosphodiesterase levels in both thyroid states. 7. Thyroid status affects lipolysis by modifying beta 3-AR density and postreceptor events without changes in the beta 1-AR functionality.

Interaction of amiodarone and triiodothyronine on the expression of beta-adrenoceptors in brown adipose tissue of rat.

1. This study was undertaken to evaluate in vivo the influence of amiodarone on the effects of triiodothyronine (T3) in brown adipose tissue (BAT) which are independent of thyroid hormone synthesis and of the conversion of thyroxine (T4) to T3. Thyroidectomized rats were given a replacement dose of T3 (0.5 mg kg(-1) p.o. daily for 3 days) with or without amiodarone (50 mg kg(-1) p.o. daily for 1 week). 2. As assessed by RT-PCR, treatment of thyroidectomized rats with T3 caused a 2 fold decrease in beta3-adrenoceptor (beta3-AR) mRNA levels and a 2 fold increase in beta1-AR mRNA levels. 3. Binding studies using [3H]-CGP 12177 as a ligand showed that treatment of thyoidectomized rats with T3 resulted in a 70% decrease in beta3-AR number and in an 80% increase in beta1-AR in BAT membranes. 4. T3-treatment abolished the increase in BAT adenylyl cyclase (AC) activity induced by CGP12177 in thyroidectomized rats. It also decreased the amount of Gi protein (ADP-ribosylation) by 30%. 5. At variance with the literature on the heart, amiodarone administration did not inhibit the positive effect of T3 on beta1-AR expression in BAT in thyroidectomized rats. However, it antagonized the effect of T3 on beta3-AR number, but not on AC activity or on Gi expression. 6. These results indicate that the effects of thyroid hormones on the responsiveness of BAT to catecholamines involves both receptor and post-receptor mechanisms, they also suggest that interaction between amiodarone and thyroid hormones is highly tissue-specific and depends on the beta-AR subtype.

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.

Effects on bone mass of long term treatment with thyroid hormones: a meta-analysis.

Osteoporosis is the main cause of spine and hip fractures. Morbidity, mortality, and costs arising from hip fractures have been well documented. Thyroid hormones (TH) are widely prescribed, mainly in the elderly. Some studies (but not all) found a deleterious effect of suppressive TH therapy on bone mass. These conflicting data raised a controversy as to the safety of current prescribing and follow-up habits, which, in turn, raised major health-care issues. To look for a detrimental effect on bone of TH therapy, we performed a meta-analysis (by pooling standardized differences, using a fixed effect model) of all published controlled cross-sectional studies (41, including about 1250 patients) concerning the impact of TH therapy on bone mineral density (BMD). Studies with women receiving estrogen therapy were excluded a priori, as were studies with a high percentage of patients with postoperative hypoparathyroidism, when no separate data were available. We decided to stratify the data according to anatomical site, menopausal status, and suppressive or replacement TH therapy, resulting in 25 meta-analysis on 138 homogeneous subsets of data. The main sources of heterogensity between studies that we could identify were replacement or suppressive TH therapy, menopausal status, site (lumbar spine, femoral neck, Ward's triangle, greater trochanter, midshaft and distal radius, with various percentages of cortical bone), and history of hyperthyroidism, which has recently been found to impair bone mass in a large epidemiological survey. To improve homogeneity, we excluded a posteriori 102 patients from 3 studies, who had a past history of hyperthyroidism and separate BMD data, thus allowing assessment of the TH effect in almost all 25 subset meta-analyses. However, controls were usually not matched with cases for many factors influencing bone mass, such as body weight, age at menarche and at menopause, calcium dietary intake, smoking habits, alcohol intake, exercise, etc. For lumbar spine and hip (as for all other sites), suppressive TH therapy was associated with significant bone loss in postmenopausal women (but not in premenopausal women), whereas, conversely, replacement therapy was associated with bone loss in premenopausal women (spine and hip), but not in postmenopausal women. The detrimental effect of TH appeared more marked on cortical bone than on trabecular bone. Only a large long term prospective placebo-controlled trial of TH therapy (e.g. in benign nodules) evaluating BMD (and ideally fracture rate) would provide further insight into these issues.

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.

Effects of aerosolized pentamidine on glucose homeostasis and insulin secretion in HIV-positive patients: a controlled study.

OBJECTIVE: Intravenous pentamidine induces hypo- and hyperglycaemia (dose-dependent toxicity on islet beta cells), pancreatitis and nephrotoxicity. Conversely, aerosolized pentamidine (AP) is usually devoid of systemic side-effects: few reports of hypo- or hyperglycaemia have been published. Our study aimed to assess the influence on glucose homeostasis and insulin secretion of long-term exposure to AP used for prophylaxis of Pneumocystis carinii pneumonia in HIV-positive patients, and to compare the impact on insulin secretion of AP, whether administered for the first time or after prolonged monthly exposure. DESIGN: Retrospective cross-sectional controlled study (main objective) and non-randomized prospective controlled study. PATIENTS: We compared glucose homeostasis and C peptide response to 1 mg intravenous glucagon in patients who had previously inhaled > or = 10 prophylactic aerosols (group 1, n = 21) and in HIV-positive controls (groups 2 and 3, n = 28) who had received none. Both groups were comparable for age and body-mass index, but CD4 T-lymphocyte counts and Karnofsky scores were both significantly higher in the control group. RESULTS: Fasting (T0) blood glucose, fructosamine and response to the first glucagon test were similar in both groups, but postprandial glucose, glycated haemoglobin and fasting C peptide were significantly higher (P < 0.05) in the pentamidine group. A second glucagon test was performed on the same day, 3 h (T3) after AP inhalation in 35 patients (in 21 after > or = 10 aerosols, group 1; in 14 after the first, group 2) and in 14 HIV-positive controls (group 3). The only significant difference between the three groups in C peptide response to this second test was a lower peak T3/peak T0 ratio in group 1. Plasma amylase and creatinine were not altered by the aerosol. CONCLUSION: Long-term prophylactic exposure to AP had minor but significant effects on glucose homeostasis and insulin secretion but did not modify pancreatic and renal function. The detrimental effects induced by long-term exposure to AP found in our study are probably not clinically relevant, but a more prolonged exposure to AP might conceivably induce more severe alterations.

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.

Transient hypocalcemia after thyroidectomy.

The causes of transient hypocalcemia after thyroid surgery are not fully understood. In 95 consecutive patients undergoing total thyroidectomy (n = 30), subtotal thyroidectomy (n = 14), or hemithyroidectomy (n = 51), we serially measured total calcium, parathyroid hormone (PTH), and proteins before surgery and 6, 24, 48, 72, and 96 hours after surgery, and we calculated the corresponding ionized calcium levels. In the whole population, there was a statistically significant decrease of PTH, total calcium, and proteins at nearly every time of blood withdrawal, when compared with the preoperative levels. The PTH decreased earlier and total calcium levels were significantly lower after total thyroidectomy than after hemithyroidectomy (at 48, 72, and 96 hours). Ten patients had on 2 occasions serum calcium levels below or equal to 2 mmol/L and were defined as having severe hypocalcemia. Severe hypocalcemia was found in 8 patients after total thyroidectomy, compared with 2 after hemithyroidectomy (p < .05), and was present in 3 of the 5 patients with thyroid carcinoma, compared with 7 of the 90 patients with nonmalignant thyroid diseases (p < .01). Despite careful preservation of the parathyroid glands and their blood supply, thyroidectomy was often followed by transient hypocalcemia, the determinants of which are hypoparathyroidism and hemodilution. No patients had persistent symptoms of hypocalcemia from 2 to 3 months after surgery.

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.