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.

Prodrugs of N-dicarboximide derivatives 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, one major drawback of NRB as a viable rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due to either its unpleasant 'taste' or rapid onset of effects. A series of NRB-derived prodrugs were prepared in an effort to 'mask' this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Prodrug 2 displayed the most promising profile with respect to a delay in the onset of symptoms and was subsequently demonstrated to be significantly more palatable to rats. Moreover, prodrug 25 was found to be largely accepted by rats in a choice trial, resulting in high mortality.

Design and synthesis of prodrugs 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, one major drawback of NRB as a viable rodenticide relates to an evolutionary aversion developed by the rat leading to sub-lethal dosing due to either its unpleasant 'taste' or rapid onset of effects. A series of NRB prodrugs were prepared in an effort to 'mask' this acute response. Their synthesis and biological evaluation (in vitro vasoconstrictory activity, in vitro hydrolytic and enzymatic stability and lethality/palatability in vivo) is described. Compound 19 displayed the most promising profile with respect to a delay in the onset of symptoms and was subsequently demonstrated to be significantly more palatable to rats.

Cardamonin is a bifunctional vasodilator that inhibits Ca(v)1.2 current and stimulates K(Ca)1.1 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 Ca(v)1.2 Ca(2+) [I(Ca(L))] or Ba(2+) [I(Ba(L))] current as well as K(Ca)1.1 currents in single myocytes and by measuring contractile responses in endothelium-denuded isolated rings. At a holding potential (V(h)) of -80 mV, cardamonin decreased both I(Ba(L)) and I(Ca(L)) in a concentration-dependent manner with similar pIC(50) values. The maximum of the I(Ba(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 I(Ba(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 Ba(2+) window current. Block of I(Ba(L)) was frequency-dependent, and rate of recovery from inactivation was slowed. Cardamonin increased K(Ca)1.1 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 I(Ca(L)) and stimulating K(Ca)1.1 current, may represent a scaffold for the design of novel drugs of potential interest for treatment of systemic hypertension.

An abnormal gene expression of the beta-adrenergic system contributes to the pathogenesis of cardiomyopathy in cirrhotic rats.

Decreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study beta-adrenergic-stimulated contractility and beta-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 (Galpha(i2)), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G-protein signaling 2 (RGS2), and down-expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Galpha(i2), PDE2a, and RGS2 down-regulates the beta-adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy.

Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide.

It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity. Our results show that: (i) norbormide isomers affect PTP in a rat-selective fashion; however, no relevant differences between lethal and non-lethal forms are observed suggesting that drug regulation of PTP-activity and lethality in rats are unrelated phenomena; (ii) a (phenylvinyl)pyridine moiety represents the key element conferring the PTP-activating effect; (iii) cationic derivatives of rat-active compounds accumulate in the matrix via the membrane potential and activate the PTP also in mouse and guinea pig mitochondria. These findings suggest that the norbormide-sensitive PTP-target is present in all species examined, and is presumably located on the matrix side. The species-selectivity may depend on the unique properties of a transport system allowing drug internalisation in rat mitochondria.

Vitamin C prevents zidovudine-induced NAD(P)H oxidase activation and hypertension in the rat.

OBJECTIVE: Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. METHODS: Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. RESULTS: AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. CONCLUSION: Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.

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.

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.

Distribution of the vasoconstrictor and vasorelaxant effects of norbormide along the vascular tree of the rat.

UNLABELLED: Norbormide is a vasoconstrictor of rat peripheral arteries and a relaxant in rat aorta. To characterise norbormide actions within the rat vascular tree we have investigated its effects on the contractile function of rings from several arteries and veins. A maximal norbormide concentration (50 microM) failed to contract thoracic aorta and carotid artery, whereas in pulmonary artery, abdominal aorta, iliac, caudal, and femoral arteries it induced a contractile effect that was respectively 4.8 +/- 0.6, 18.4 +/- 1.5, 39 +/- 5, 144 +/- 7, and 260 +/- 22% of that induced by 90 mM KCl. In pulmonary, carotid, and iliac arteries, and in thoracic and abdominal aorta, 50 microM norbormide inhibited KCl-induced responses. Norbormide (50 microM) contracted all veins investigated. The effect, expressed as % of KCl-induced contraction, was 121 +/- 25, 154 +/- 14.5, 154 +/- 18.2, 203 +/- 19, and 267 +/- 33 for pulmonary vein, thoracic and abdominal vena cava, iliac and jugular veins, respectively. In jugular vein, as previously shown in rat caudal artery, norbormide contraction was abolished in Ca2+-free medium, was unaffected by the Ca2+ channel blocker nifedipine, and was relaxed by SK&F 96365, a blocker of store-operated Ca2+ channels. IN CONCLUSION: i) rat veins represent the main target for contractile norbormide action; ii) in both artery and veins norbormide contractions are generally inversely related to the calibre of the vessel; iii) norbormide-induced contraction is mediated by the same mechanism/s in arteries and veins; iiii) in norbormide-contracted arteries the drug activates both contractile and relaxing mechanisms.

Effects of commonly used protein kinase inhibitors on vascular contraction and L-type Ca(2+) 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.

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.

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.

Synthesis and activity studies of analogues of the rat selective toxicant norbormide.

Norbormide [5-(alpha-hydroxy-alpha-2-pyridylbenzyl)-7-(alpha-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. A series of NRB-related analogues were prepared to investigate the structural features responsible for, and the in vitro biological markers indicative of, in vivo lethality of the parent molecule in rats. Their synthesis and biological evaluation (vasoconstriction, vasodilation, mitochondrial dysfunction, cardiotoxicity and lethality) is described.

Sodium pump alpha2 subunits control myogenic tone and blood pressure in mice.

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

Characterization of intrinsic sympathomimetic activity of carteolol in rat cardiovascular preparations.

We evaluated in vitro, in myocardial and vascular preparations isolated from reserpine-treated rats, the intrinsic sympathomimetic activity (ISA) of carteolol, a beta(1)/beta(2)-adrenoceptor blocking agent used in cardiovascular and non-cardiovascular diseases. In spontaneously beating atria, carteolol, at low concentrations (0.01 and 0.1 microM), antagonized the positive inotropic effect of isoprenaline, whereas at higher concentrations (1 microM to 1 mM), it caused an increase in the force of contraction (EC(50): 4.6 +/- 0.1 microM, E(max): 17.1 +/- 1.1%, with respect to the maximum isoprenaline response) and a slight increase (7.8 +/- 1.9% over basal values) in the heart rate. The positive inotropic effect of carteolol was abolished by concentrations of propranolol or timolol (10 microM) much higher than those blocking isoprenaline effects in the same preparations. A similar positive inotropic effect was also observed in electrically driven left atrium and in Langendorff perfused hearts. Functional and biochemical evidences supported the involvement of cAMP in the cardiac action of carteolol. In peripheral arteries (femoral and tail) pre-contracted with phenylephrine, carteolol exerted ISA-related relaxing effects, independent of the presence of endothelium and sensitive to high concentrations (10 microM) of conventional beta-blockers. On the basis of these results, we propose to categorize carteolol as a non-conventional partial agonist of both cardiac and vascular beta-adrenoceptors.