Maurizio Cavalli

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

An abnormal gene expression of the β‐adrenergic system contributes to the pathogenesis of cardiomyopathy in cirrhotic rats

Decreased cardiac contractility and β‐adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study β‐adrenergic–stimulated contractility and β‐adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10−10 to 10−6 M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half‐maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha–inhibiting subunit 2 (Gαi2), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G‐protein signaling 2 (RGS2), and down‐expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Gαi2, PDE2a, and RGS2 down‐regulates the β‐adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy. (HEPATOLOGY 2008;48:1913‐1923.)

Cardamonin Is a Bifunctional Vasodilator that Inhibits Cav1.2 Current and Stimulates KCa1.1 Current in Rat Tail Artery Myocytes

An in-depth analysis of the effects of cardamonin, 2′,4′-dihydroxy-6′-methoxychalcone, on rat tail artery preparations was performed by means of whole-cell patch-clamp recordings of Cav1.2 Ca2+ [ICa(L)] or Ba2+ [IBa(L)] current as well as KCa1.1 currents in single myocytes and by measuring contractile responses in endothelium-denuded isolated rings. At a holding potential (Vh) of −80 mV, cardamonin decreased both IBa(L) and ICa(L) in a concentration-dependent manner with similar pIC50 values. The maximum of the IBa(L)-voltage relationship was shifted by 10 mV in the hyperpolarizing direction, but threshold remained unaffected. Cardamonin modified both the activation and the inactivation kinetics of IBa(L) and shifted the voltage dependence of both inactivation and activation curves to more negative potentials by 19 and 7 mV, respectively, thus markedly decreasing the Ba2+ window current. Block of IBa(L) was frequency-dependent, and rate of recovery from inactivation was slowed. Cardamonin increased KCa1.1 currents in a concentration-dependent manner; this stimulation was iberiotoxin- and BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid]-sensitive. On the contrary, iberiotoxin did not modify cardamonin-induced relaxation of rings precontracted either with phenylephrine or with (S)-(−)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(S)-(−)-Bay K 8644]. The overall effects of cardamonin were incompletely reversed by washout. In conclusion, cardamonin is a naturally occurring, bifunctional vasodilator that, by simultaneously inhibiting ICa(L) and stimulating KCa1.1 current, may represent a scaffold for the design of novel drugs of potential interest for treatment of systemic hypertension.

Anthracene Based Compounds as New L-type Ca2+ Channel Blockers : Design, Synthesis, and Full Biological Profile

L-Type Ca(2+) channels (LTCCs) play a key role in the regulation of vascular smooth muscle contraction, and substances that interfere with their function (Ca(2+) channel blockers) are widely used in the therapy of hypertension. Here, we report anthracene-maleimide derivatives as new LTCC blockers. Among these, 3, lacking intracellular effects, was investigated in more detail. The results show that 3 binds preferentially to inactivated LTCCs, directly interacting with the pore-forming subunit of the channel.

RELAXANT AND CA2+ CHANNEL BLOCKING PROPERTIES OF NORBORMIDE ON RAT NON-VASCULAR SMOOTH MUSCLES

We have investigated the effects of the rat-specific vasoconstrictor agent norbormide on the mechanical and electrophysiological properties of rat non-vascular smooth muscles. Norbormide (50 microM) did not affect the resting tone of urinary bladder, tracheal, and duodenal rings. In all tissues, KCl-induced concentration-response curves were shifted downward by norbormide (5 and 50 microM). In urinary bladder and tracheal rings, norbormide inhibited contractile responses to carbachol only at the higher concentration (50 microM). In single gastric fundus myocytes, 50 microM norbormide inhibited L-type Ca(2+) current (I(Ca(L))) by about 60%, neither affecting both activation and inactivation rates of the current nor the current-voltage curve along the voltage axis. Our results indicate that rat non-vascular smooth muscles are relaxed by norbormide with a mechanism likely involving a reduction of Ca(2+) entry through L-type Ca(2+) channels.

Effects of commonly used protein kinase inhibitors on vascular contraction and L-type Ca2+ current

Regulation of smooth muscle contraction is driven by a number of protein kinases: the evidence for this often originates from studies that investigate the effects of extracellularly added specific protein kinase inhibitors. Six compounds, thought to be selective inhibitors of various kinases, were analysed for their effects on vascular L-type Ca(2+) channels because this potential subsidiary activity could strongly influence our understanding of the pathways involved in smooth muscle contraction. Whole-cell L-type Ba(2+) currents [I(Ba(L))] were recorded in single myocytes, and contractile responses were measured from endothelium-denuded rings taken from the rat tail artery. Although ML-7, ML-9, and wortmannin (MLCK inhibitors), HA-1077 and Y-27632 (Rho-associated kinase inhibitors), and GF-109203X (PKC inhibitor) relaxed rings pre-contracted with high KCl in a concentration-dependent manner, their effect on I(Ba(L)) intensity was surprisingly variable. Wortmannin showed negligible effects while HA-1077 and Y-27632 were ineffective. I(Ba(L)) was partly inhibited by GF-109203X and blocked by ML-7 and ML-9 in a concentration-dependent manner, with the blockade by ML-7 being voltage-dependent. Whilst ML-7, ML-9, and GF-109203X sped up the inactivation kinetics of I(Ba(L)), GF-109203X did not modify ML-7- or ML-9-induced effects, with both intensity and kinetics of the current remaining unchanged. In contrast, application of Bay K 8644 on myocytes pre-treated with ML-7 or ML-9 raised I(Ba(L)) beyond control values. In conclusion, ML-7 and ML-9 inhibit L-type Ca(2+) channels via a mechanism independent of MLCK, PKC or Rho kinase activities, and as such caution should be used in employing these agents to elucidate the role of kinases in smooth muscle contraction.

Fatty Acid-Derived Pro-Toxicants of the Rat Selective Toxicant Norbormide.

Norbormide [5‐(α‐hydroxy‐α‐2‐pyridylbenzyl)‐7‐(α‐2‐pyridylbenzylidene)‐5‐norbornene‐2,3‐dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB‐derived pro‐toxicants (3a – i, 4a – i, and 5a – i) were prepared in an effort to ‘mask’ this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague–Dawley, wild Norway, and wild ship rats is described. Most notably, pro‐toxicant 3d was revealed to be free of all pre‐cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two‐choice bait‐palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB‐derived toxicants with enhanced palatability and efficacy profiles.

AZT dilates rat cardiac intercalated discs, and the effect is prevented by vitamin C

We investigated whether chronic zidovudine (AZT) administration in rats could impair cardiac function by affecting intercellular junctions and whether vitamin C could prevent these possible effects. Rats were treated for 8 months with AZT, vitamin C, and AZT plus vitamin C. Cardiac fractional shortening (FS) was assessed by echocardiographic examination, intercellular junctions morphology was detected by electron microscopy (EM) and immunocytochemistry (ICC). AZT-treated rats showed a reduced FS that was not prevented by vitamin C. EM revealed that AZT treatment did not affect coronary endothelial intercellular junctions whereas it caused an enlargement of fascia adherens of the intercalated discs that was prevented by vitamin C. AZT treatment did not induce either alterations of gap junctions morphology or distribution of connexin-43, the major protein expressed in the gap junctions. We conclude that AZT treatment may be potentially deleterious to the heart by inducing a ROS-mediated damage of cardiac intercalated discs.