Pharmacokinetics and biodistribution study of self-assembled Gd-micelles demonstrating blood-pool contrast enhancement for MRI.

Magnetic resonance angiography (MRA) requires the use of contrast agents (CAs) to enable accurate diagnosis. There are currently no CAs on the market with appropriate pharmacokinetic (PK) parameters, namely long persistence in the blood, that can be easily used for MRA. We have recently synthesized amphiphilic building blocks loaded with gadolinium (Gd), which self-assemble into Gd-micelles in aqueous media, and have evaluated their potential as a blood-pool contrast agent (BPCA) in vivo. To assess the short and long term PK of Gd-micelles, the blood and organs of the mice were analyzed at t = 30 min, 1, 2, 3 h, 7, 14 and 21 days. Gd-DOTA was used as a control because it is the gold-standard CA for MRA despite its rapid clearance from the blood compartment. Gd-micelles circulated in the blood for more than 3 h postinjection whereas Gd-DOTA was eliminated less than half an hour postinjection. No side effects were observed in the mice up to the end of the study at 21 days and no accumulation of Gd was observed in the brain or bones. The Magnetic Resonance Imaging (MRI) parameters and the results of this in vivo study indicate the true BCPA properties of Gd-micelles and warrant further development.

Anti-apoA-1 auto-antibodies increase mouse atherosclerotic plaque vulnerability, myocardial necrosis and mortality triggering TLR2 and TLR4.

Auto-antibodies to apolipoprotein A-1 (anti-apoA-1 IgG) were shown to promote inflammation and atherogenesis, possibly through innate immune receptors signalling. Here, we aimed at investigating the role of Toll-like receptors (TLR) 2 and 4 on anti-apoA-1 IgG-induced atherosclerotic plaque vulnerability, myocardial necrosis and mortality in mice. Adult male apolipoprotein E knockout (ApoE)-/- (n=72), TLR2-/-ApoE-/- (n=36) and TLR4-/-Apo-/- (n=28) mice were intravenously injected with 50 µg/mouse of endotoxin-free polyclonal anti-apoA-1 IgG or control isotype IgG (CTL IgG) every two weeks for 16 weeks. Atherosclerotic plaque size and vulnerability were assessed by histology. Myocardial ischaemia and necrosis, respectively, were determined by electrocardiographic (ECG) changes assessed by telemetry and serum troponin I (cTnI) measurements. Impact on survival was assessed by Kaplan-Meier analyses. In ApoE-/- mice, anti-apoA-1 IgG passive immunisation enhanced histological features of atherosclerotic plaque vulnerability (increase in neutrophil and MMP-9 and reduction in collagen content), induced a substantial cTnI elevation (p=0.001), and increased mortality rate by 23 % (LogRank, p=0.04) when compared to CTL IgG. On a subgroup of ApoE-/- mice equipped with telemetry (n=4), a significant ST-segment depression was noted in anti-apoA-1 IgG-treated mice when compared to CTL IgG recipients (p< 0.001), and an acute ST-segment elevation myocardial infarction preceding mouse death was observed in one case. The deleterious effects of anti-apoA-1 IgG on atherosclerotic plaque vulnerability, myocardial necrosis and death were partially reversed in TLR2-/-ApoE-/- and TLR4-/-ApoE-/- backgrounds. In conclusion, anti-apoA-1 auto-antibodies seem to be active mediators of atherosclerotic plaque vulnerability, myocardial necrosis, and mortality in mice through TLR2- and TLR4-mediated pathways.

Analysis of the expression of nine secreted matrix metalloproteinases and their endogenous inhibitors in the brain of mice subjected to ischaemic stroke.

Matrix metalloproteinases (MMPs) are a family of more than twenty secreted and cell-surface endopeptidases. Among them, MMP2, MMP3 and MMP9 are involved in blood-brain barrier injury and neuronal death after cerebral ischaemia. On the other hand, very little is known about the expression of the other secreted MMPs. Herein, we compared the global changes in MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12 and MMP13, and their endogenous inhibitors TIMP1 and TIMP2, both at the mRNA and protein levels, during the hyperacute (6 h), acute (24 h) and subacute (72 h) stages following transient focal cerebral ischaemia and treatment with recombinant tissue plasminogen activator (rtPA). We observed a significant increase in MMP1, MMP2, MMP9, MMP10, MMP13 and TIMP1 levels during the acute stage of reperfusion, which was further amplified during the subacute stage for MMP1, MMP2, MMP10 and TIMP1. In general, no change of MMP3, MMP7, MMP8, MMP12 and TIMP2 was observed. However, rtPA treatment induced a rapid increase in MMP1/TIMP2, MMP2/TIMP2, MMP8/TIMP2 and MMP9/TIMP2 ratios during the hyperacute stage of reperfusion compared to saline treatment, which may have potential implications in the early disruption of the blood-brain barrier after rtPA treatment.

Involvement of T-type calcium channels in the mechanism of action of 5-HT in rat glomerulosa cells: a novel signaling pathway for the 5-HT7 receptor.

We have previously demonstrated that, in rat, the stimulatory effect of 5-HT on aldosterone secretion is mediated through a 5-HT7 receptor subtype. The aim of the present study was to characterize the transduction mechanisms associated with activation of native 5-HT7 receptors. 5-HT induced a dose-dependent increase in cAMP production in rat glomerulosa cells. Pretreatment of cells with the adenylyl cyclase (AC) inhibitor SQ 22536 or the protein kinase A (PKA) inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Administration of 5-HT in the vicinity of glomerulosa cells induced a robust increase in cytosolic calcium concentration ([Ca2+]i) and this effect was abrogated by the T-type calcium channel blocker mibefradil. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. H-89 attenuated both the [Ca2+]i response and the activation of T-type calcium current induced by 5-HT. Reduction of extracellular calcium concentration in the medium or administration of mibefradil caused a marked reduction of the maximum effect (Emax) of 5-HT on aldosterone secretion. These data demonstrate that activation of native 5-HT7 receptors stimulates cAMP formation, which in turn provokes calcium influx through T-type calcium channels. Both the activation of the AC/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.

Rat glomerulosa cells express functional 5-HT7 receptors.

It has been previously demonstrated that serotonin (5-HT) is a potent stimulator of aldosterone secretion in amphibians and mammals. The aim of the present study was to characterize the type of serotonergic receptor involved in the action of 5-HT on rat glomerulosa cells. The effects of 10 serotonergic receptor agonists and 12 receptor antagonists on aldosterone secretion from perifused rat adrenocortical slices were investigated. Correlation analysis between the potencies of the different compounds in our model and those previously reported for transfected 5-HT receptors showed that the rat adrenal 5-HT receptor exhibits the pharmacological profile of the 5-HT7 receptor. RT-PCR amplification with specific primers for the 5-HT7 receptor confirmed the presence of 5-HT7 receptor mRNA in the adrenal cortex. Western blot analysis using antibodies against the 5-HT7 receptor revealed the occurrence of a protein with an apparent molecular mass of 66 kDa in the zona glomerulosa. In glomerulosa cells, 5-HT induced a concentration-dependent increase of cAMP formation. These data demonstrate that rat adrenal glomerulosa cells express functional 5-HT7 receptors positively coupled to adenylyl cyclase.

Role of 5-HT in the regulation of the brain-pituitary-adrenal axis: effects of 5-HT on adrenocortical cells.

Serotonin (5-HT) plays a pivotal role in the regulation of the brain-pituitary-adrenal axis. In particular, 5-HT has been shown to control the activity of hypothalamic CRF neurons and pituitary corticotrope cells through activation of 5-HT1A and (or) 5-HT(2A/2C) receptor subtypes. 5-HT, acting through 5-HT2 receptors, can also trigger the renin-angiotensin system by stimulating renin secretion and consequently can enhance aldosterone production. At the adrenal level, 5-HT produced locally stimulates the secretory activity of adrenocortical cells through a paracrine mode of communication. The presence of 5-HT in the adrenal gland has been demonstrated immunohistochemically and biochemically in various species. In the frog, rat, and pig adrenal gland, 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex, 5-HT is contained in nerve fibers. In man, 5-HT is present in perivascular mast cells. In vivo and in vitro studies have shown that 5-HT stimulates corticosteroid secretion in various species (including human). The type of receptor involved in the mechanism of action of 5-HT differs between the various species. In frogs and humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl cyclase and calcium influx. In the rat, the effect of 5-HT on aldosterone secretion is mediated via activation of 5-HT7 receptors. Clinical studies indicate that 5-HT4 receptor agonists stimulate aldosterone secretion in healthy volunteers and in patients with corticotropic insufficiency and primary hyperaldosteronism. Local serotonergic control of corticosteroid production may be involved in the physiological control of the activity of the adrenal cortex as well as in the pathophysiology of cortisol and aldosterone disorders.

Pharmacological and molecular characterization of 5-hydroxytryptamine(7) receptors in the rat adrenal gland.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent stimulator of aldosterone secretion in the rat adrenal gland but the type of receptor involved in the mechanism of action of 5-HT remains unknown. The aim of the present study was to determine the pharmacological profile and to clone the receptor responsible for the corticotropic effect of 5-HT in rat glomerulosa cells. A series of 10 serotonergic receptor agonists and 12 receptor antagonists was used to characterize the receptor mediating the effect of 5-HT on aldosterone secretion from perifused rat adrenocortical slices. Correlation analysis between the potencies of the different compounds in our model and those previously reported for various recombinant 5-HT receptors showed that the rat adrenal 5-HT receptor exhibits the same pharmacological profile as the 5-HT(7) receptor transiently expressed in COS-7 cells (r = 0.82 for agonists, p <.05; r = 0.83 for antagonists, p <.01). Polymerase chain reaction with specific primers revealed the expression of 5-HT(7) receptor mRNA in the rat adrenal gland. Cloning of the polymerase chain reaction product confirmed that the amplified DNA corresponded to the 5-HT(7) receptor cDNA sequence. Western blot analysis showed the presence of a protein with an apparent molecular mass of 66 kDa in the adrenal cortex but not in the medulla. Taken together, these data demonstrate that the rat adrenal glomerulosa expresses functional 5-HT(7) receptors. Rat glomerulosa cells will thus provide a robust and sensitive bioassay for future studies on native 5-HT(7) receptors.

Pharmacological and functional characterization of muscarinic receptors in the frog pars intermedia.

The secretion of alphaMSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates alphaMSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated alphaMSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced alphaMSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and alphaMSH release. In contrast, omega-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and alphaMSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked alphaMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on alphaMSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10(-4) M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of alphaMSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.

Identification of human p53 mutations with differential effects on the bax and p21 promoters using functional assays in yeast.

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect affecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 different mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower affinity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.

Pharmacological profile of serotonergic receptors in the adrenal gland.

The secretory activity of the adrenal gland is mainly regulated by peptidergic hormones (ACTH, angiotensin II) and ions. However, there is now increasing evidence that local factors, including neuropeptides and neurotransmitters, can also participate in the control of adrenocortical cells. In particular, serotonin (5-HT), produced by adrenochromaffin cells in frog and rat as well as by mast cells in the adrenal gland of rat and human, stimulates corticosteroid secretion. In both frog and human adrenal gland, the benzamide derivative (R,S)-zacopride induces a robust increase in corticosteroid release suggesting that the effect of 5-HT on steroidogenesis is mediated through activation of 5-HT4 receptors. In contrast, in rat, the stimulatory effect of 5-HT on aldosterone secretion is clearly not mediated by 5-HT4 receptors. In all three species, incubation of adrenocortical fragments with 5-HT induces a significant increase in cAMP formation. Our data suggest that 5-HT, released within the adrenal cortex, may act as a paracrine factor to stimulate steroid secretion. Although the corticotropic effect of 5-HT has been conserved from amphibians to primates, the type of receptors involved in the action of 5-HT markedly differs across species.