Brain Research EuJJeri~, Vol. 27, pp. 739-742. 0
PergamonPress plc, 1991.printed in the U.S.A.
0361-92x1191 $3.00 + .oo
RAPID COMMUNICATION
Further Evidence for a Dopaminergic Involvement in the Renal Action of Centrally Administered Atria1 Natriureti~ Peptide in Rats ANITA ISRAEL,*’ MARYSABEL TORRES,1_ MARIA CIERCOS AND YARISMA BARBELLA* *Faculty of Pharmacy, Department of Biological Sciences, Universidad Central de Venezuela Apartado Postal 50174, Sabana Grande 1050 A, Caracas, Venezuela tSchoo1 of Medicine, Universidad de Carabobo #School of Medicine, Universidad National Experimental Francisco de Miranda Received 15 May 1991 ISRAEL, A., M. TORRBS, M. CIERCO AND Y. BARBBLLA. Further evidence for a dopaminergic involvement in the renal action of centrally administered atrial natriuretic peptide in rats. BRAIN RES BULL 27(S) 739-742, 1991. -Intracerebroventricular (ICV) administration of atria1 natriuretic peptide (ANP) induces an increase of urinary volume and sodium excretion in conscious male hydrated rats. The involvement of brain dopaminergic neurones in the ICV-ANP renal action was investigated. Diuretic and natriuretic action of ANP was prevented by inhibition of tyrosine hydroxylase activity with alpha-methyl-p-tyrosine treatment and was absent following selective central dopaminergic denervation with ICV administration of 6-hydroxydopamine in combination with desmethylimipramine (IP). Our results suggest that ANP acts centrally, at least in part, via an interaction with endogenom dopamine neurones. Natriuresis
Atria1 natriuretic peptide
Dopamine
Diuresis
gic and/or dopaminergic system function for its action. Further-
ATRIAL natriuretic peptides (ANP) are a group of polypeptides, originally identified in mammalian atrium extracts, with potent natriuretic and vasorelaxant activities (4,22). Several lines of evidence suggest a significant role for ANP in the coordinated central and peripheral control of fluid and electrolytes homeostasis (2, 10, 20, 22). Indeed, in addition to the diuretic and natriuretic actions in the kidney (19) and adrenal inhibition of aldosterone secretion (33), central administration of this peptide inhibits dehydration and AII-induced water intake (20), salt preference in sodium-depleted rats (2) and vasopressin secretion (9,18). Our initial report in 1986 (10) demonstrated that intracerebroventricular (XV) a~~s~ation of ANP induces a dose-related increase in urine volume and sodium excretion. These observations are in agreement with those reported by other authors (5, 18, 23, 27). Subsequent studies in our laboratory revealed that the diuretic and natriuretic response to ANP persists after chemical sympathectomy but is prevented by central chemical denervation with 6-hydroxydopamine (60HDA) (12-14). This implies that the central nervous system mechanism of diuresis and natriuresis is independent of the neural tone that the adrenergic system exerts on sodium and water reabsorption; however, central ANP requires the integrity of brain noradrener1_
more, we demonstrated that in euvolemic rats, ICY-GNP or dopamine-induced diuresis and natriuresis could be prevented by pretreatment with dopamine receptor blockers such as haloperido1 or domperidone (13, 14, 3 l), suggesting the involvement of brain and/or median eminence-pituitary gland DA receptors in the centrally mediated action of ANP on urine and sodium excretion. Several findings point to a possible interaction of ANP with brain dopaminergic system. In effect, it has been demonstrated that ANP alters dopamine levels and its metabolites in the hypothalamus and septum (21). In addition, several actions of ANP are similar to those produced by dopamine system activation, including effects on blood pressure and prolactin and LH secretion (6, 15, 24, 25). In order to establish the direct mediation of brain dopaminergic neurones in the ICV-ANP renal action, in the present study we assessed the effects of tyrosine hydroxylase inhibition by alpha-methyl-p-tyrosine in order to cause a temporary inhibition of endogenous DA production at medio-basal-hypothalamic dopaminergic neurones. In addition, we studied the effect of the ICV administration of the catecholaminergic neurotoxin 60HDA after desmethylimipramine (DMI) blockade of neuronal catechol-
_
‘&quests tar reprmts should be addressed to Anita Israef, Apartado Postal 50176, Sabana Grande 1050 A, Caracas, Venezuela.
739
740
ISRAEL, TORRES, CIERCO AND BARBELLA
amine uptake pathway for noradrenaline (NA), to cause a selective central dopaminergic dene~ation. Both treatments would result in a diminished dopaminergic tone in the median eminence and/or pituitary gland (1).
METHOD
Adult male Sprague-Dawley rats (230-290 gj were housed under controlled conditions of temperature and photoing (lights on from 0600 to 1800 h) and provided with free access to laboratory chow and water. A cannula (26) was implanted in the left lateral cerebroventricle, I mm caudal to the coronal suture and 1.5 mm lateral to the midsagittal suture, with the aid of a stereotaxic instrument and under pentobarbital anesthesia (40 mgi kg, IP). The cannula was secured to the skull with acrylic cement. A min~um of 2 days was allowed for recovery. Single ICV injections were made with a Hamilton syringe fitted with a stop to prevent needle penetration past the cannula tip. Experimental Protocol One day after ven~cul~ cannulation, the animals were randomly distributed into the following groups: 1) Tyrosine hydroxylase activity inhibition: sixteen and one hour before ICV-ANP, a group of rats received 250 mg/kg of alpha-methyl-p-tyrosine or vehicle (IP) (V) (N= 28); or 2) Centrally selective dopaminergic denervation: a group of rats was treated with desmethylimipramine (DMI) (25 mgikg, IP) half an hour before ICV injection of 6-hy~oxydopamine (6OHDA) (250 kg15 ~1) or vehicle (V) (0.9 NaCl, 0.1% ascorbic, 5 pl), 72 and 48 h before ICV-ANP injection (N= 28). 60HDA treatment is a routine method for destroying catecholaminergic axon terminals in the central nervous system (32). This action is secondary to displacement of endogenous NA and appears to be due to intraaxonal oxidation of 60HDA, with consequent production of free radicals and ~t~hond~al damage (f&29,32). Entry of 60HDA into catecholaminergic axons relies on active transport by the membrane processes that are normally responsible for endogenous amine reuptake. Although we did not measure catecholamine content in the hypothalamus, selective dopaminergic denervation and the NA protection were confirmed by the observation (30), in anaesthetized dogs, that following p~~eatment with uptake-l inhibitor DMI, 6OHDA had no significant attenuating effect on vasoconstrictor or antinatriuretic response to renal nerve stimulation or on nerve-evoked renal production of NA. ICV-ANP Administration Protocol Animals were weighed and placed in metabolic cages. At 0900 h, half of the rats receiving each pretreatment were injected ICY, as a bolus in 10 s, with saline solution (5 pl) or with freshly prepared rANP in saline solution (5 kg/5 ~1) (rat atria1 peptide, 28 amino acids, Peninsula Laboratories, Inc., Belmont, CA) followed by 20 ml/kg water orally. Urine was collected at 1, 3. and 6 h; the bladder was emptied at 6 h by gentle suprapubic massage. Food and water were not available during the experiment. Ventricular cannula placement was confirmed postmortem by examining the distribution of an ICV injection of 5 p,l of fast green dye, given before animal sacrifice. Data were used only if the dye was distributed in the lateral, third, and fourth ventricles. Urine samples were assayed for sodium and stasis content by flame photome~. All data are presented as mean 2 SEM. Statistical differences between groups were analyzed using two-way analysis of vari-
ante (ANOVA) statistics.
and by the
Newman-Keul’s
student
range
RESULTS
Eflects of Alpha-Methyl-P-Tyrosine (AMPT) Pretreatment on ICV-ANP-Induced Diuresis and Natriuresis The effect of tyrosine hydroxylase activity inhibition is illustrated in Fig. I. Two-way analysis of variance and NewmanKeul’s test revealed that ANP clearly evoked diuresis at the l-. 3- and 6-h periods of collection (all p’s
PergamonPress plc, 1991.printed in the U.S.A.
0361-92x1191 $3.00 + .oo
RAPID COMMUNICATION
Further Evidence for a Dopaminergic Involvement in the Renal Action of Centrally Administered Atria1 Natriureti~ Peptide in Rats ANITA ISRAEL,*’ MARYSABEL TORRES,1_ MARIA CIERCOS AND YARISMA BARBELLA* *Faculty of Pharmacy, Department of Biological Sciences, Universidad Central de Venezuela Apartado Postal 50174, Sabana Grande 1050 A, Caracas, Venezuela tSchoo1 of Medicine, Universidad de Carabobo #School of Medicine, Universidad National Experimental Francisco de Miranda Received 15 May 1991 ISRAEL, A., M. TORRBS, M. CIERCO AND Y. BARBBLLA. Further evidence for a dopaminergic involvement in the renal action of centrally administered atrial natriuretic peptide in rats. BRAIN RES BULL 27(S) 739-742, 1991. -Intracerebroventricular (ICV) administration of atria1 natriuretic peptide (ANP) induces an increase of urinary volume and sodium excretion in conscious male hydrated rats. The involvement of brain dopaminergic neurones in the ICV-ANP renal action was investigated. Diuretic and natriuretic action of ANP was prevented by inhibition of tyrosine hydroxylase activity with alpha-methyl-p-tyrosine treatment and was absent following selective central dopaminergic denervation with ICV administration of 6-hydroxydopamine in combination with desmethylimipramine (IP). Our results suggest that ANP acts centrally, at least in part, via an interaction with endogenom dopamine neurones. Natriuresis
Atria1 natriuretic peptide
Dopamine
Diuresis
gic and/or dopaminergic system function for its action. Further-
ATRIAL natriuretic peptides (ANP) are a group of polypeptides, originally identified in mammalian atrium extracts, with potent natriuretic and vasorelaxant activities (4,22). Several lines of evidence suggest a significant role for ANP in the coordinated central and peripheral control of fluid and electrolytes homeostasis (2, 10, 20, 22). Indeed, in addition to the diuretic and natriuretic actions in the kidney (19) and adrenal inhibition of aldosterone secretion (33), central administration of this peptide inhibits dehydration and AII-induced water intake (20), salt preference in sodium-depleted rats (2) and vasopressin secretion (9,18). Our initial report in 1986 (10) demonstrated that intracerebroventricular (XV) a~~s~ation of ANP induces a dose-related increase in urine volume and sodium excretion. These observations are in agreement with those reported by other authors (5, 18, 23, 27). Subsequent studies in our laboratory revealed that the diuretic and natriuretic response to ANP persists after chemical sympathectomy but is prevented by central chemical denervation with 6-hydroxydopamine (60HDA) (12-14). This implies that the central nervous system mechanism of diuresis and natriuresis is independent of the neural tone that the adrenergic system exerts on sodium and water reabsorption; however, central ANP requires the integrity of brain noradrener1_
more, we demonstrated that in euvolemic rats, ICY-GNP or dopamine-induced diuresis and natriuresis could be prevented by pretreatment with dopamine receptor blockers such as haloperido1 or domperidone (13, 14, 3 l), suggesting the involvement of brain and/or median eminence-pituitary gland DA receptors in the centrally mediated action of ANP on urine and sodium excretion. Several findings point to a possible interaction of ANP with brain dopaminergic system. In effect, it has been demonstrated that ANP alters dopamine levels and its metabolites in the hypothalamus and septum (21). In addition, several actions of ANP are similar to those produced by dopamine system activation, including effects on blood pressure and prolactin and LH secretion (6, 15, 24, 25). In order to establish the direct mediation of brain dopaminergic neurones in the ICV-ANP renal action, in the present study we assessed the effects of tyrosine hydroxylase inhibition by alpha-methyl-p-tyrosine in order to cause a temporary inhibition of endogenous DA production at medio-basal-hypothalamic dopaminergic neurones. In addition, we studied the effect of the ICV administration of the catecholaminergic neurotoxin 60HDA after desmethylimipramine (DMI) blockade of neuronal catechol-
_
‘&quests tar reprmts should be addressed to Anita Israef, Apartado Postal 50176, Sabana Grande 1050 A, Caracas, Venezuela.
739
740
ISRAEL, TORRES, CIERCO AND BARBELLA
amine uptake pathway for noradrenaline (NA), to cause a selective central dopaminergic dene~ation. Both treatments would result in a diminished dopaminergic tone in the median eminence and/or pituitary gland (1).
METHOD
Adult male Sprague-Dawley rats (230-290 gj were housed under controlled conditions of temperature and photoing (lights on from 0600 to 1800 h) and provided with free access to laboratory chow and water. A cannula (26) was implanted in the left lateral cerebroventricle, I mm caudal to the coronal suture and 1.5 mm lateral to the midsagittal suture, with the aid of a stereotaxic instrument and under pentobarbital anesthesia (40 mgi kg, IP). The cannula was secured to the skull with acrylic cement. A min~um of 2 days was allowed for recovery. Single ICV injections were made with a Hamilton syringe fitted with a stop to prevent needle penetration past the cannula tip. Experimental Protocol One day after ven~cul~ cannulation, the animals were randomly distributed into the following groups: 1) Tyrosine hydroxylase activity inhibition: sixteen and one hour before ICV-ANP, a group of rats received 250 mg/kg of alpha-methyl-p-tyrosine or vehicle (IP) (V) (N= 28); or 2) Centrally selective dopaminergic denervation: a group of rats was treated with desmethylimipramine (DMI) (25 mgikg, IP) half an hour before ICV injection of 6-hy~oxydopamine (6OHDA) (250 kg15 ~1) or vehicle (V) (0.9 NaCl, 0.1% ascorbic, 5 pl), 72 and 48 h before ICV-ANP injection (N= 28). 60HDA treatment is a routine method for destroying catecholaminergic axon terminals in the central nervous system (32). This action is secondary to displacement of endogenous NA and appears to be due to intraaxonal oxidation of 60HDA, with consequent production of free radicals and ~t~hond~al damage (f&29,32). Entry of 60HDA into catecholaminergic axons relies on active transport by the membrane processes that are normally responsible for endogenous amine reuptake. Although we did not measure catecholamine content in the hypothalamus, selective dopaminergic denervation and the NA protection were confirmed by the observation (30), in anaesthetized dogs, that following p~~eatment with uptake-l inhibitor DMI, 6OHDA had no significant attenuating effect on vasoconstrictor or antinatriuretic response to renal nerve stimulation or on nerve-evoked renal production of NA. ICV-ANP Administration Protocol Animals were weighed and placed in metabolic cages. At 0900 h, half of the rats receiving each pretreatment were injected ICY, as a bolus in 10 s, with saline solution (5 pl) or with freshly prepared rANP in saline solution (5 kg/5 ~1) (rat atria1 peptide, 28 amino acids, Peninsula Laboratories, Inc., Belmont, CA) followed by 20 ml/kg water orally. Urine was collected at 1, 3. and 6 h; the bladder was emptied at 6 h by gentle suprapubic massage. Food and water were not available during the experiment. Ventricular cannula placement was confirmed postmortem by examining the distribution of an ICV injection of 5 p,l of fast green dye, given before animal sacrifice. Data were used only if the dye was distributed in the lateral, third, and fourth ventricles. Urine samples were assayed for sodium and stasis content by flame photome~. All data are presented as mean 2 SEM. Statistical differences between groups were analyzed using two-way analysis of vari-
ante (ANOVA) statistics.
and by the
Newman-Keul’s
student
range
RESULTS
Eflects of Alpha-Methyl-P-Tyrosine (AMPT) Pretreatment on ICV-ANP-Induced Diuresis and Natriuresis The effect of tyrosine hydroxylase activity inhibition is illustrated in Fig. I. Two-way analysis of variance and NewmanKeul’s test revealed that ANP clearly evoked diuresis at the l-. 3- and 6-h periods of collection (all p’s
