Antonio Capuzzo

Effects of environmental concentrations of the antiepilectic drug carbamazepine on biomarkers and cAMP-mediated cell signaling in the mussel Mytilus galloprovincialis

Pharmaceuticals represent a diverse collection of thousands of bioactive chemicals used in human and veterinary medicine. The increased consumption, together with the recent development of more sensitive analytical techniques, has identified these as emerging contaminants in the aquatic environment. According to many investigations pharmaceuticals do not cause acute toxic effects in organisms when released in the environment. However, many independent studies agree that chronic exposure and more specific endpoints should be used in risk assessment of these compounds. We thus investigated the effects of exposure to environmentally relevant concentrations of the antiepileptic drug carbamazepine (CBZ) on Mediterranean mussels (Mytilus galloprovincialis) by considering the existing knowledge about the therapeutic and side effects of this drug on humans. To do so we analysed: (a) six consolidated biomarkers related primarily to oxidative stress; (b) cAMP levels and protein kinase A (PKA) activities; (c) mRNA expression of MXR-related genes. MXR proteins are involved both in the cAMP pathway and in the protective response of organisms towards xenobiotics. Mussels exposed to 0.1 or 10microg CBZ per liter water for 7 days showed a 60% and 80% reduction in haemocyte lysosome membrane stability, respectively. Moreover, increased neutral lipid and lipofuscin accumulation in the digestive gland, and lipid peroxidation in gills and mantle/gonads were observed. Also glutathione S-tranferase and catalase activities were increased in digestive gland and mantle, while no increase in primary DNA damage was observed. In agreement with the mode of action of CBZ in humans, exposure resulted in a significant reduction in cAMP levels and PKA activities in digestive gland, gills and mantle/gonads of mussels, and lowered the mRNA expression of genes encoding three different MXR-related transporters in the same tissues. Our data indicate that CBZ, at concentrations found in the environment, affects the Mediterranean mussel by acting on specific biochemical pathways that are evolutionarily conserved.

The role of circulating catecholamines in the regulation of fish metabolism: An overview

The physiological role of the catecholamines (CA), adrenaline and noradrenaline in fish has been frequently reviewed, but the metabolic consequences of these hormones have received less attention. The purpose of this review is to examine the recent literature dealing with CA actions on whole fish and tissue metabolism. The CA increase glucose production both in vivo and in vitro, at least in isolated hepatocytes. Although the data are less clear, lipid mobilization is also a consequence of elevated circulating CA. The difficulty with using the whole fish for such studies is that CA may alter other circulating hormone levels, CA turnover in the circulation quickly, and it is difficult to define precisely the tissue being affected. Much of our understanding is derived, therefore, from the study of isolated tissues, and especially the hepatocyte. Catecholamines stimulate both glycogenolysis and gluconeogenesis in hepatocytes isolated from a large number of fish species. This review examines the steps involved in the signal transduction system, from the binding of CA to alpha- and beta-adrenoceptors to the ultimate effects of specific enzyme phosphorylation. Recent literature demonstrates that the complexity of the adrenoceptor system noted for mammals, also is expressed in fish. Adrenoceptor subtypes are specific to species, to tissues and to function of the tissues, and these issues are discussed especially as they are related to external and to internal stressors. Future research will pursue better definitions of the adrenoceptor systems, molecular biology of the components of these receptor systems and development of alternative cell models. There still remains a poor explanation of the reason for the diversity of adrenoceptor systems, and there are a number of fish systems that may provide unique opportunities to understand this question.

α-Adrenoceptor-mediated glucose release from perifused catfish hepatocytes

In fish liver catecholamines bind to beta-adrenoceptors (AR) and increase glucose release via cAMP augmentation. Alpha1-AR have recently been shown to mediate IP3 and Ca2+ elevation in catfish and eel hepatocytes, although their coupling to a physiological response has remained doubtful. We have perifused isolated catfish hepatocytes in Bio-Gel P4 columns with epinephrine in the presence of prazosin and/or propranolol, alpha- and beta-AR antagonists, respectively. Ten nM epinephrine stimulated glucose release approximately 3-fold, and this effect was completely antagonized by the simultaneous presence of both alpha- and beta-AR blockers. The two AR antagonists separately inhibited about one-third and two-third of the total stimulation, respectively. Through alpha-AR occupancy, epinephrine provoked a significant increase of glucose release whereas no stimulation was detected in Ca2+-depleted hepatocytes. Glucose release was strongly elevated by both ionomycin and dibutyryl cAMP. These results represent the first direct evidence that alpha-AR transduction pathway is involved in epinephrine-induced glucose release from fish hepatocytes.

The β-blocker propranolol affects cAMP-dependent signaling and induces the stress response in Mediterranean mussels, Mytilus galloprovincialis.

Widespread occurrence of pharmaceuticals is reported in aquatic systems, posing concerns for the health of aquatic wildlife and a theoretical risk to humans. A recent concept was developed for the identification of highly active compounds amongst the environmental pharmaceuticals, based on their mode of action, the homology between human targets and possible targets in the environment, and the importance of the affected pathway for the target species. In line with this approach, this study investigated whether propranolol (PROP) affects the cAMP-dependent pathway in Mediterranean mussels, Mytilus galloprovincialis. PROP is a prototypical β-adrenoceptor antagonist, and these receptors exist in bivalves and show gross pharmacological properties similar to their mammalian counterparts. PROP also acts as a 5-HT1 receptor antagonist, which is the sole 5-HT receptor reported in bivalves to date. Importantly, β-adrenoceptor and 5HT-1 receptor subtypes are positively and negatively coupled to cAMP-mediated signaling, respectively. PROP was administered as either l-PROP or dl-PROP. A wide range of concentrations was tested including low (0.3, 3 and 30ng/L) and high (300ng/L) environmental ranges, and a concentration 5-fold above the maximum reported environmental level (30,000ng/L). After a 7-day exposure, mussel cAMP levels and PKA activities were significantly reduced in digestive gland, increased in mantle/gonads and unaffected in gills. Similar patterns were observed for the mRNA expression of the ABCB1 gene encoding the membrane transporter P-glycoprotein, hypothesised to be under PKA modulation. The effects on the digestive gland are consistent with PROP blocking β-adrenoceptors. The observed increased cAMP levels in the mantle/gonad tissue support PROP blocking 5-HT1 receptors. Catalase and glutathione-S tranferase were differently affected by PROP in the two tissues. Mussel haemocyte lysosome membrane stability, a sensitive biomarker of animal health status, was concentration-dependently reduced following PROP exposure. Our observations provide evidence for PROP affecting cell signaling in M. galloprovincialis. Moreover, the chemical interacts with specific and evolutionally conserved biochemical pathways for which it was designed. The mode of action of PROP in mussels is related with its therapeutic properties in humans, based upon these conserved human targets. It also induced a stress response, and all these effects were displayed at the lowest concentrations tested.

Cyclic AMP signaling in bivalve molluscs: an overview.

The cyclic AMP (cAMP)-dependent signaling accounts for the control of cellular cascades involved in many physiological functions, and a wealth of information is available on the cAMP system that operates in mammalian cells. Nevertheless, cAMP has a central role also in nonmammalian vertebrates and invertebrates. The present review aims at examining the information available on bivalve molluscs, from the first studies carried out in the early 1980s to the last progresses made in the present days. The major focus is on the structural and operational characteristics of the main actors of the signaling pathway, i.e., adenylyl cyclase, G proteins, and protein kinase A, and on the role played by the cyclic nucleotide on smooth muscle, heart, gills, gonads, and metabolism regulation. Moreover, recent evidence regarding the cAMP system as a target of environmental stress factors are discussed. It will become clear that cAMP does play a wide and important role in bivalve physiology. Several issues have been sufficiently clarified, although investigated only in a few model species. However, further fundamental aspects remain unknown, mainly regarding molecular features and interactions with other signaling pathways, thus requiring further elucidation.

β-Adrenergic receptors in catfish liver membranes: Characterization and coupling to adenylate cyclase ☆

beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.

Electrophysiological evidence for a PGE-mediated presynaptic control of acetylcholine output in the guinea-pig superior cervical ganglion

Abstract Intracellular recordings from single ganglion neurons show that 10 −8 −10 −7 M PGE 1 reversibly blocks synaptic transmission in isolated preparations of the guinea-pig superior cervical ganglion (SCG), when added to the superfusing medium. Neither resting potential nor membrane resistance of the impaled neurons appear to be affected by PGE 1 . Quantal analyses of transmitter release demonstrate that the number of quanta liberated per volley is sharply reduced by PGE 1 treatment whereas the amplitude of the elementary event does not appear to be significantly changed.

Cd2+ and Hg2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes.

Isolated hepatocytes of the European eel (Anguilla anguilla) have been used as experimental model to characterize the effects of Cd(2+) and Hg(2+) on either basal or epinephrine-stimulated glucose release. Cd(2+) strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 microM (IC(50) 0.04 microM). The epinephrine-stimulated glucose output was also reduced by Hg(2+), although a significant inhibition of about 60% was achieved only at 10 microM (IC(50) 5 microM). The possible influence of Cd(2+) and Hg(2+) on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd(2+) and Hg(2+) also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg(2+) was shown by Cd(2+) and Hg(2+), which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg(2+) was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 microM Cd(2+) and Hg(2+), respectively. These results indicate that Cd(2+) and Hg(2+) may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Abstract Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α 1 -adrenoceptors in EPI action, at least in some fish species. The role of the α 1 - and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist 3 H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors ( K d 0.36 nM, B max 8.61 fmol · mg −1 protein). We provide the first demonstration of α 1 -adrenoceptors in these same membranes; analysis of binding data with the α 1 -adrenergic antagonist 3 H-prazosin demonstrated a single class of binding sites with a K d of 15.4 nM and a B max of 75.2 fmol · mg −1 protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP 3 ) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP 3 ( K d 6.03 nM, B max 90.2 fmol · mg −1 protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α 1 -adrenergic agonists are able to increase intracellular concentrations of IP 3 and Ca 2+ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α 1 -binding sites affinity and of α 1 -adrenoceptor/IP 3 /Ca 2+ transduction systems.

Nasal chitosan microparticles target a zidovudine prodrug to brain HIV sanctuaries

Zidovudine (AZT) is an antiretroviral drug that is a substrate of active efflux transporters (AETs) that extrude the drug from the central nervous system (CNS) and macrophages, which are considered to be sanctuaries of HIV. The conjugation of AZT to ursodeoxycholic acid is known to produce a prodrug (UDCA-AZT) that is able to elude the AET systems, indicating the potential ability of this prodrug to act as a carrier of AZT in the CNS and in macrophages. Here, we demonstrate that UDCA-AZT is able to permeate and remain in murine macrophages with an efficiency twenty times higher than that of AZT. Moreover, we propose the nasal administration of this prodrug in order to induce its uptake into the CNS. Chitosan chloride-based microparticles (CP) were prepared by spray-drying and were characterized with respect to size, morphology, density, water uptake and the dissolution profile of UDCA-AZT. The CP sample was then nasally administered to rats. All in vitro and in vivo measurements were also performed for a CP parent physical mixture. The CP sample was able to increase the dissolution rate of UDCA-AZT and to reduce water uptake with respect to its parent physical mixture, inducing better uptake of UDCA-AZT into the cerebrospinal fluid of rats, where the prodrug can act as an AZT carrier in macrophages.