Cardiotoxic Effects of the Antineoplastic Doxorubicin in a Model of Metabolic Syndrome: Oxidative Stress and Transporter Expression in the Heart.

ABSTRACT: The aim of the present work was to examine whether metabolic syndrome-like conditions in rats with fructose (F) overload modify the cardiotoxic effects induced by doxorubicin (DOX) and whether the treatment altered the expression of P-gp, breast cancer resistance protein, and organic cation/carnitine transporters in the heart. Male Sprague-Dawley rats received either tap water (control group [C]; n = 16) or water with F 10% wt/vol (n = 16) during 8 weeks. Three days before being killed, the animals received a single dose of DOX (6 mg/kg, ip, md) (C-DOX and F-DOX groups) or vehicle (VEH; ISS 1 mL/kg BW; ip) (C-VEH and F-VEH groups) (n = 8 per group). F overload enhanced thiobarbituric acid-reactive substance levels in the left ventricle, and DOX injection further increased those values. DOX did not alter thiobarbituric acid-reactive substance production in C animals. DOX caused a decrease of 30% in the ejection fraction and a nearly 40% reduction in the fractional shortening in F animals, but not in C rats. Cardiac tissue levels of P-gp decreased by about 30% in F rats compared with the C groups. DOX did not modify cardiac P-gp expression. Breast cancer resistance protein and organic cation/carnitine transporter (OCTN 1/2/3) protein levels did not change with either F or DOX. It is suggested that DOX could cause greater cardiotoxicity in rats receiving F, probably due to enhanced cardiac lipid peroxidation and lower expression of cardiac P-gp. These results support the hypothesis that the cardiotoxicity of DOX could be increased under metabolic syndrome-like conditions or in other health disorders that involve cardiovascular risk factors.

Role of CGRP and GABA in the hypotensive effect of intrathecally administered anandamide to anesthetized rats.

In urethane-anesthetized rats the intrathecal (i.t.) injection of 100 nmol anandamide produced a hypotensive effect (-19.3+/-1.6 mm Hg; n=6) that was mimicked by i.t. administration of 0.25 nmol calcitonin gene-related peptide (CGRP; -26.2+/-1.8 mm Hg, n=4). Both effects were antagonized either by the CGRP receptor antagonist CGRP(8-37) (5 nmol; i.t.) or by the gamma-aminobutyric acid (GABA)(A) receptor antagonist bicuculline (8.8 nmol, i.t) or by the GABA(B) receptor antagonist 2-hydroxy saclofen (110 nmol; i.t.). On the contrary, blockade of spinal CGRP receptors by CGRP(8-37) did not modify the hypotensive response to either the GABA(A)-receptor agonist muscimol (8.8 nmol; i.t.) or the GABA(B)-receptor agonist baclofen (100 nmol; i.t). This result suggests a unidirectional effect of CGRP on the GABAergic system. The response to anandamide remained unaltered after acute inhibition of nitric oxide (NO) synthase activity by either i.t. (1 micromol) or i.v. (10 mg/kg) injection of N(G)-nitro-L-arginine methyl ester (L-NAME), but increased significantly after long-term L-NAME administration (70 mg/kg/day; four weeks; p.o.), thus suggesting compensatory changes in cardiovascular homeostasis. It is proposed that the hypotensive effect of anandamide in urethane-anesthetized rats could involve the release of CGRP followed by the release of GABA in the spinal cord. NO does not appear to have a direct participation in the spinal mechanisms involved in the decrease of the blood pressure caused by anandamide.

Fructose-induced metabolic syndrome decreases protein expression and activity of intestinal P-glycoprotein.

OBJECTVES: Metabolic syndrome (MetS) is a health disorder that increases the risk for cardiovascular complications such as heart disease and type 2 diabetes. Some drugs used in patients with MetS are substrates of intestinal P-glycoprotein (P-gp), one of the most important efflux pumps that limit the absorption of xenobiotics. Thus, their bioavailability could be affected by changes in this transporter. Because one of the major causes of MetS in humans is excessive sugar intake, the aim of this study was to evaluate the effect of a fructose-rich diet on intestinal P-gp activity and protein expression in male Sprague-Dawley rats. METHODS: Fructose-drinking animals received standard chow and 15% (w/v) fructose in the drinking water over 8 wk; control rats were fed on standard chow and tap water. RESULTS: Ileal protein expression of P-gp was 50% lower in fructose-drinking rats than in control animals. This reduction was confirmed by immunofluorescence microscopy. These results correlated well with the decrease of about 50% in the transport rate of the substrate rhodamine 123 in everted intestinal sacs. Finally, an increase of 62% in the intestinal absorption of digoxin, a P-gp substrate used as therapeutic drug, was observed in vivo, in fructose-drinking animals. CONCLUSION: The present study demonstrated that MetS-like conditions generated by enhanced fructose intake in rats decreased the protein expression and activity of ileal P-gp, thus increasing the bioavailability of P-gp substrates.

Participation of nitric oxide and N-methyl-D-aspartic acid receptors in the pressor response to intrathecal injected noradrenaline at the spinal cord of the rat.

In pentobarbital-anesthetized rats, intrathecal injection of noradrenaline (NA; 6, 18 and 60 nmol) induced a dose-dependent increase in the mean blood pressure. The pressor response to NA (18 nmol) was blocked by pretreatment with the selective antagonist for N-methyl-D-aspartic acid (NMDA) receptors, 2-amino-5-phosphonovaleric acid (30 nmol), but not by pretreatment with the selective antagonist for (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors, 6,7-dinitroquinoxaline-2,3-dione (50 nmol). The pressor effect of NA was reduced after pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 micromol). The effect of L-NAME on the pressor response to NA was reverted by the precursor of nitric oxide (NO), L-arginine (5 micromol). The hypertension induced by NA was also reduced by the guanylate cyclase inhibitor methylene blue (0.3 micromol). These results suggest that spinal NMDA receptors and spinal NO are involved in the pressor response to NA.

Activation of spinal metabotropic glutamate receptors elicits cardiovascular responses in pentobarbital anesthetized rats.

The aim of this study was to examine whether intrathecal (i.t.) injection of metabotropic glutamate (mGlu) receptor agonists at the thoracolumbar level of the spinal cord causes changes either in the blood pressure or in the heart rate of pentobarbital anesthetized rats. The broad spectrum mGlu receptor agonist (+/-)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD) and the Group III mGluR agonist L-(+)-2-amino-4-phosphonobutyric acid ( L-AP4) induced pressor effects at doses of 300 nmol and 600 nmol (i.t.) but did not induce changes at a lower dose (150 nmol, i.t.). The specific Group I mGlu receptor agonist ( RS)-3,5-dihydroxyphenylglycine (3,5-DHPG), as well as the highly selective Group II mGlu receptor agonist 2 R,4 R-4-aminopyrrolidine-2,4-dicarboxilate (2 R,4 R-APDC), induced pressor effects at a dose of 300 nmol only. The compounds (150-600 nmol) did not modify the heart rate in these experiments. On the other hand, low doses of Group II mGlu receptor agonists (75 nmol 2 R,4 R-APDC; 1.5 nmol 2 S,2' R,3' R)-2-(2',3'-dicarboxychloropropyl)glycine; DCG IV) induced hypotension and bradycardia when spinal N-methyl- D-aspartate (NMDA) receptors were previously blocked by 2-amino-5-phosphonovaleric acid (APV; 30 nmol; i.t.). The pressor response to trans-ACPD was probably mediated by activation of both Group I and Group II mGluRs because i.t. injection of either the selective Group I mGlu receptor antagonist ( S)-4-carboxyphenylglycine (4CPG) or the selective Group II mGlu receptor antagonist (2 S,3 S,4 S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG) antagonized the increases in the blood pressure produced by the agonist. Moreover, 4CPG and MCCG antagonized the pressor effects of 3,5-DHPG and 2 R,4 R-APDC, respectively. Blockade of spinal Group II mGlu receptors by MCCG also prevented the hypotensive and bradycardic effects of 2 R,4 R-APDC and DCG IV in rats pretreated with APV. On the other hand, the pressor response to L-AP4 (300 nmol) was prevented by the selective antagonist ( S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4).These results suggest that activation of spinal Group I, II and III mGlu receptors increases the mean blood pressure in pentobarbital anesthetized rats and that, after blockade of NMDA receptors, low doses of Group II mGlu receptor agonists induce hypotension and bradycardia.

Hypotensive effect of anandamide through the activation of CB1 and VR1 spinal receptors in urethane-anesthetized rats.

This study examined the effect of intrathecal (i.t.) injection of the endocannabinoid anandamide in urethane-anesthetized rats. The tip of the i.t. cannula was positioned at the T(12)-L(1) level of the spinal cord. Either anandamide or its metabolically stable analogue methanandamide (25 to 100 nmol) produced dose-dependent decreases in the blood pressure that persisted at least for up to 30 min. The hypotensive responses to 100 nmol anandamide and to 100 nmol methanandamide were -17.7+/-1.6 mmHg ( n=5) and -17.9+/-2.0 mmHg ( n=4), respectively. Hypotensive effects were also obtained with the CB(1) cannabinoid receptor agonist WIN 55212-2 (20 nmol; i.t.) as well as with the vanilloid VR(1) receptor agonist capsaicin (3 nmol; i.t.). Nicotinic ganglionic blockade with hexamethonium bromide [10 mg/kg; intravenous(i.v.)] abolished the responses to both anandamide and capsaicin. The i.t. administration of the CB(1) receptor antagonist, 20 nmol SR 141716A, as well as the VR(1) receptor antagonist, 20 nmol capsazepine, prevented almost completely the hypotensive responses to both anandamide and methanandamide. SR 141716A prevented the hypotension caused by WIN 55212-2 but did not modify the response to the vanilloid receptor agonist capsaicin. On the contrary, capsazepine antagonized the hypotension caused by capsaicin but failed to affect the decrease in blood pressure caused by the CB1 cannabinoid receptor agonist WIN 55212-2. These results suggest that anandamide could modulate the blood pressure through the activation of cannabinoid CB(1) receptors and vanilloid VR(1) receptors localized at the spinal cord.

Enhancement of the hypotensive effects of intrathecally injected endocannabinoids by the entourage compound palmitoylethanolamide.

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (-10.6+/-1.6 mmHg and -15.0+/-1.7 mmHg, respectively; n=6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (-12.3+1.3 mmHg for anandamide; -12.1+/-0.8 mmHg for methanandamide; -12.1+/-0.8 mmHg for N-arachidonoyldopamine; n=4-6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB(1) receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB(1) receptor-mediated effects of endocannabinoids on the blood pressure control.

Efectos cardiovasculares del óxido nítrico a nivel espinal en ratas anestesiadas / Cardiovascular effects of spinal nitric oxide in anesthetized rats

En ratas anestesiadas con pentobarbital, la inyección intratecal del precursor de la síntesis de óxido nítrico, la L-arginina (10 y 20 µmoles), produjo una disminución en la presión arterial media, mientras que el inhibidor de la síntesis de óxido nítrico, el NG-nitro-L-arginina metil éster (L-NAME: 0,1-10 µmoles) produjo un efecto presor dosis-dependiente. La respuesta presora al L-NAME fue prevenida por el pretratamiento con L-arginina. Los isómeros inactivos (D-NAME y D-arginina) no modificaron la presión arterial media. El dador de óxido nítrico, nitroprusiato de sodio (0,125-0,250 µmoles) indujo una respuesta hipotensora seguida por un efecto presor. La respuesta dual al nitroprusiato de sodio, como así también el efecto hipotensor de L-arginina fueron abolidos por un inhibidor de la guanilato ciclasa, el azul de metileno (0,30 µmoles, inyección intratecal). El bloqueo de la transmisión nicotínica ganglionar con hexametonio (10 mg/kg, i.v.) redujo los efectos hipotensores de L-arginina y nitroprusiato de sodio y previno casi totalmente los efectos presores de L-NAME y nitroprusiato de sodio. El efecto presor del L-NAME fue abolido por el antagonista de receptores de glutamato de tipo NMDA, el ácido 2-amino-5-fosfonovalérico (AVP: 30 nmoles, inyección intratecal). Estos resultados sugieren que en la médula espinal de ratas anestesiadas con pentobarbital, el óxido nítrico produce efectos tanto inhibitorios como excitatorios sobre la actividad preganglionar simpática relacionada con el control de la presión arterial. La síntesis de óxido nítrico estaría tónicamente activada a través de la estimulación de receptores a glutamato de tipo NMDA

Participación de receptores NMDA en la respuesta presora inducida por noradrenalina a nivel espinal en ratas anestesiadas / Participation of spinal NMDA receptors in the pressor response induced by intrathecal injection of noradrenaline in anesthetized rats

En ratas anestesiadas con pentobarbital, la administración intratecal de noradrenalina (NA: 6, 18 y 60 nmoles) y de glutamato (1, 2,5 y 5 µmol) produjo un aumento dosis-dependiente de la presión arterial media, sin modificar la frecuencia cardíaca. El efecto presor inducido por glutamato (1 µmol, inyección intratecal) fue bloqueado por un antagonista no selectivo de receptores de glutamato, el ácido kinurénico (Kin: 125 nmoles, inyección intratecal), por un antagonista selectivo de receptores del subtipo NMDA, el ácido 2-amino-5-fosfonovalérico (AVP: 30 nmoles, inyección intratecal) y por un antagonista selectivo de receptores del subtipo no-NMDA, el DNQX (50 nmoles, inyección intratecal). El ácido kinurénico y el ácido 2-amino-5-fosfonovalérico, pero no el DNQX, bloquearon la respuesta presora inducida por noradrenalina (18 nmoles, inyección intratecal). El antagonista alfa1 adrenérgico prazosín (3,1 nmoles, inyección intratecal) bloqueó totalmente la respuesta presora a noradrenalina (19 nmoles, inyección intratecal) pero no modificó la respuesta presora inducida por glutamato (1 µmol, inyección intratecal). El ácido kinurénico, el ácido 2-amino-5-fosfonovalérico y el DNQX produjeron per se una disminución de la presión arterial media basal mientras que el prazosín no modificó dicho parámetro. Estos resultados sugieren que, a nivel espinal, la respuesta presora inducida por glutamato involucra la activación de receptores tanto NMDA como no-NMDA. La activación de receptores NMDA por el glutamato endógeno participaría en el efecto presor inducido por las catecolaminas a través de la estimulación de receptores alfa1 espinales