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Inhibitory effect of gaseous neuromodulators in vasopressin and oxytocin release induced by endotoxin in rats
Neuroscience Letters 381 (2005) 320–324
Inhibitory effect of gaseous neuromodulators in vasopressin and oxytocin release induced by endotoxin in rats Alexandre Giusti-Paiva, Lucila Leico Kagohara Elias, Jos´e Antunes-Rodrigues ∗ Departamento de Fisiologia, Faculdade de Medicina de Ribeir˜ao Preto, Universidade de S˜ao Paulo, 14049-900 Ribeir˜ao Preto, S˜ao Paulo, Brazil Received 16 December 2004; received in revised form 14 February 2005; accepted 16 February 2005
Abstract Nitric oxide (NO) and carbon monoxide (CO) are endogenously synthesized gaseous molecules that act as neurotransmitters in central nervous system. In this study we investigated the modulatory role of NO and CO in lipopolysaccharide (LPS)-induced vasopressin and oxytocin secretion. Intracerebroventricular (i.c.v.) injection of N-l-nitro-arginine methyl ester (l-NAME), 3-morpholino-sydnonimine (SIN-1), zinc deuteroporphyrin 2,4-bis glicol (ZnDPBG) or hemin did not change the basal vasopressin and oxytocin plasma levels. After endovenous LPS administration, plasma vasopressin and oxytocin increased, reaching a peak at 60 min, and returning to basal levels afterwards. LPS administration induced a higher vasopressin and oxytocin plasma levels in rats previously treated with l-NAME and ZnDPBG (P < 0.05) compared to rats pre-treated with vehicle. On the other hand, in rats previously treated with SIN-1 or hemin, there was a significant reduction in the vasopressin and oxytocin secretion. These findings confirm the inhibitory role of NO and CO in the LPS-induced vasopressin and oxytocin secretion. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Vasopressin; Oxytocin; Nitric oxide; Carbon monoxide; Sepsis; Endotoxic shock; Stress
The neuroendocrine system plays an important role in maintaining homeostasis under a variety of stress conditions, including endotoxemia. The response of the organism to disturbance of its homeostasis caused by endotoxin includes release of hormones such as corticotropin-release hormone (CRH), vasopressin and oxytocin involved in the hypothalamus–pituitary–adrenal axis activation during stress [17]. Administration of endotoxin from Gram-negative bacteria, lipopolysaccharide (LPS), induces an intense nuclear Fos expression in vasopressin- and oxytocin-containing magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, suggesting that LPS activates magnocellular neurons in these brain areas [16]. Indeed, we have previously shown that there is a remarkable increase of vasopressin plasma leles in response to LPS intravenous stimulus [4,6]. However, the mechanism involved in this ∗
Corresponding author. Tel.: +55 16 602 3018; fax: +55 16 633 0017. E-mail address: [email protected] (J. Antunes-Rodrigues).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.02.040
control of vasopressin and oxytocin secretion during endotoxemia has not yet been well elucidated. Nitric oxide (NO) and carbon monoxide (CO) have been recently considered as potential neurotransmitters that influence physiological and pathological processes in both the central and the peripheral nervous systems [1]. Nitric oxide, which is generated by nitric oxide synthase (NOS), and CO, which is generated by heme oxygenase (HO), have been shown to modulate both vasopressin and oxytocin release, and both enzymes are located in the PVN and SON [2,28]. NO and CO have been shown to be essential for several physiological and pathophysiological manifestations, including hormonal responses to stress [2,19,22] and thermoregulation [24,25] during endotoxemia. It has been demonstrated that NO may act as an inhibitory regulator of vasopressin and oxytocin [9]. In addition, it has been recently reported that CO may inhibit the release of vasopressin and oxytocin from the rat hypothalamus in vitro [10,11,14]. Thus, the purpose of the present study was to investigate the interaction between the generation of NO and
A. Giusti-Paiva et al. / Neuroscience Letters 381 (2005) 320–324
CO in vasopressin and oxytocin release induced by LPS in rats. For this purpose we used NOS or HO inhibitors, and a NO donor or hemin, a substrate of HO, to evaluate the effects of the NO and CO system on plasma vasopressin and oxytocin release induced by LPS. Male Wistar rats (230–280 g), from the Central Animal Facility of the Faculdade de Medicina de Ribeir˜ao Preto—Universidade de S˜ao Paulo, were housed in individual cages, in a room with controlled temperature (23 ± 2 ◦ C) and lighting (lights on from 7.00 a.m. to 7.00 p.m.), and allowed free access to standard rat pellet food and tap water. One week after acclimatization, the rats were anesthetized with 2,2,2,-tribromoethanol (Aldrich, Milwaukee, WI, USA; 250 mg/kg, i.p.) and a stainless steel guide cannula was introduced into the right lateral cerebral ventricle with the use of a stereotaxic apparatus (Kopf Instruments, Kent, UK), following the stereotaxic coordinates from the atlas of Paxinos and Watson [21]; 8 mm posterior to the bregma, 1.4 mm lateral to midline, and 3.2–3.7 mm ventral to the skull surface; as previously described [5]. After surgery, the animals received an intramuscular prophylactic dose of 100,000 U of benzylpenicillin, and were handled daily during 7 days before the experiments. One day before the experiment, under anesthesia, a silastic catheter was introduced into the right external jugular vein and advanced to the right atrium for intravenous (i.v.) drug administration. The effects of LPS on vasopressin and oxytocin secretion was observed in rats treated with an i.v. bolus injection of 1.5 mg/kg of LPS or 0.15 M NaCl (Saline, 1 ml/kg, control group), and decapitated 0, 0.5, 1, 2, 3, 4, 5 and 6 h after. The NO participation in vasopressin and oxytocin release induced by LPS administration was determined in animals pre-treated with i.c.v. injection of isotonic saline (0.15 M NaCl, 5 l) or 10 g of 3-morpholino-sydnonimine (SIN-1, an NO donor), or 250 g of N-l-nitro-arginine methyl ester (l-NAME, an NOS inhibitor) 30 min before the i.v. injection of LPS or saline, being decapitated 1 h afterwards. To determine the CO participation in vasopressin and oxytocin release induced by LPS administration, in another set of experiments, the animals were pre-treated with i.c.v. injection of vehicle (50 mM Na2 CO3 , 5 l) or 200 nmol hemin, a substrate of HO, or 200 nmol zinc deuteroporphyrin 2,4bis glicol (ZnDPBG, inhibitor of HO) 30 min before the iv injection of LPS or saline and they were decapitated 1 h afterwards. Blood was collected into chilled heparinized plastic tubes, centrifuged (20 min, 3000 rpm, 4 ◦ C) and the plasma separated for vasopressin and oxytocin determination, after extraction from 1 ml of plasma using acetone and petroleum ether. Dried sample extracts were stored at −20 ◦ C until hormonal measurement by radioimmunoassay as previously described [3,18]. The heme oxygenase inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) and hemin were obtained from Porphyrin Products (UT, USA), dissolved in basic aqueous solution (50 mM Na2 CO3 ) and stored in the dark.
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Lipopolysaccharide (LPS, from Escherichia coli, serotype 026:B6), l-NAME and SIN-1 were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in isotonic saline (0.15 M NaCl). Doses and method of administration were chosen on the basis of previous studies [6,9,25]. Results are expressed as the mean ± S.E. and statistical significance was assessed by analysis of variance (ANOVA) followed by Newman–Keuls post hoc test. The level of significance was set at p < 0.05. Fig. 1 shows the time-course increase in plasma vasopressin and oxytocin levels after LPS treatment, with a peak at 1 h post-injection, followed by a gradual return to baseline. In control animals, vasopressin and oxytocin levels did not change from baseline during all experimental period. Fig. 2 shows the vasopressin and oxytocin secretion induced by LPS and the modulatory effect of NO on this response. Intracerebroventricular pre-treatment with saline, l-NAME or SIN-1 did not significantly change plasma vasopressin and oxytocin concentrations 1 h after i.v. saline injection. On the other hand, LPS induced a significant increase in plasma vasopressin and oxytocin concentration in the i.c.v. saline (p < 0.05) and l-NAME (p < 0.05) pre-treated groups. Vasopressin and oxytocin secretion were even higher in rats pre-treated with l-NAME (p < 0.05). Conversely, the increase in plasma vasopressin and oxytocin levels induced by LPS was significantly reduced by i.c.v. pre-treatment with SIN-1 (p < 0.05). Fig. 3 shows similar modulatory effects of CO on vasopressin and oxytocin secretion induced by LPS. The pre-treatment with vehicle, ZnDBPG and hemin did not
Fig. 1. Temporal effect of saline (open symbol) or lipopolysaccharide (LPS; closed symbol) injections on plasma vasopressin (top) and oxytocin (bottom) concentration. Value are the mean ± S.E., n = 8 per group. * P < 0.05 vs. saline group.
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Fig. 2. Effect of intracerebroventricular (i.c.v.) injection of saline, SIN-1 and l-NAME on vasopressin (top) and oxytocin (bottom) release 1 h after endovenous (i.v.) saline or LPS injection. Bars represent the mean ± S.E., n = 8 per group. * P < 0.05 vs. saline (i.c.v.) + saline (i.v.) group; # P < 0.05 vs. saline (i.c.v.) + LPS (i.v.) group.
Fig. 3. Effect of intracerebroventricular (i.c.v.) injection of vehicle, hemin and ZnDPBG on vasopressin (top) and oxytocin (bottom) release 1 h after endovenous (i.v.) saline or LPS injection. Bars represent the mean ± S.E., n = 8 per group. * P < 0.05 vs. vehicle (i.c.v.) + saline (i.v.) group; # P < 0.05 vs. vehicle i.c.v. + LPS (i.v.) group.
significantly change vasopressin and oxytocin plasma levels in control animals. LPS induced a significant increase in plasma vasopressin and oxytocin concentration in the i.c.v. vehicle pre-treated group. The administration of LPS in rats previously treated with ZnDPBG (i.c.v.) caused a significantly higher increment in vasopressin and oxytocin levels (p < 0.05), in comparison with controls. However, plasma vasopressin and oxytocin levels after LPS in rats i.c.v. pre-treated with hemin was reduced compared with vehicle pre-treated rats (p < 0.05). The present study suggests that the generation of NO and CO, within the central nervous system, may influence the neuroendocrine adaptive responses to immunological stress. The prevailing action seems to be inhibition of stress responses. In the present work we have shown that NO and CO are involved in the vasopressin and oxytocin release in response to the endotoxic shock. The administration of LPS in rats previously treated with ZnDPBG or l-NAME potentiates the vasopressin- and oxytocin-increase observed in response to LPS. On the other hand, rats pretreated with the NO donor (SIN-1) or hemin showed a decrease of vasopressin and oxytocin response to LPS. The neuroendocrine actions of NO and CO may have clear pathophysiological relevance, since bacterial endotoxins can concomitantly activate stress responses and induce the production of these gases [26]. The observation that NOS is present in the hypothalamus has suggested that NO might play a physiological role in regulating neuroendocrine functions [2]. This concept has been further supported by the detection of NOS staining in hypothalamic cells that contains vasopressin and oxytocin [8]. It has been demonstrated in rats that NO inhibits oxytocin secretion [9], however, the effect of NO on vasopressin secretion remains controversial. The intracerebroventricular injection of NO precursor, as well as NO donor has been shown to increase vasopressin plasma level [20]. In addition, NO donor has been shown to mediate the basal and stimulated release of vasopressin from rat hypothalamic explants [29]; and the inhibition of NOS decreases vasopressin release induced by hypertonic blood volume expansion [27]. Conversely, it has been demonstrated that NO tonically inhibits the basal vasopressin release in control rats [9], and the inhibition of NOS by l-NAME (non-selective inhibitor of NOS) or aminoguanidine (a selective inhibitor of inducible NOS) increases LPS-induced vasopressin plasma levels [5,6]. Most reports suggest that NO is inhibitory of both vasopressinergic and oxytocinergic hypothalamic neurons. In electrophysiological studies in vitro, the NO donor sodium nitroprusside (SNP) inhibited the supraoptic neurons, whereas the NOS inhibitor and the NO scavenger hemoglobin enhanced neuronal activity [12]. In the present study, we observed a significant higher increment of vasopressin and oxytocin plasma levels in response to the LPS administration in animals pre-treated with l-NAME and ZnDPBG. This result is in accordance with an inhibitory role of both NO and CO system on the vasopressin and oxytocin secretion during endotoxemia.
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Equimolar amounts of CO, iron, and biliverdin are generated from the catabolism of the tetrapyrrole ring of hemin (Fe-protoporphyrin-9) by heme oxygenase. Two isoforms of this enzyme have been characterized to date: an inducible isoform, heme oxygenase-1 (HO-1), and a constitutive enzyme, heme oxygenase-2 (HO-2). HO-1 can be detected in macrophages under resting conditions, but it is induced in virtually all cell types of our organism by a variety of stimuli including hemin itself, oxidative stress, heat shock, various disease states, and LPS administration [13]. The central nervous system is endowed with very high HO activity under basal conditions, mostly accounted for HO-2. In terms of influence of CO on neuroendocrine regulation, there are few published data. The formation of CO within the hypothalamus has been associated with inhibition of release of hormones such as CRH, vasopressin and oxytocin [11,14]. The present data are in accordance with previous reports showing that the HO–CO pathway acts centrally to attenuate LPS-stimulated vasopressin release [15]. It should be borne in mind that vasopressin has antipyretic effects [23] and that fever is one of the most essential responses to infection [7]. After systemic administration of high doses of LPS, there is an initial hypothermia followed by febrile response, and V1 antagonist blocked this drop in temperature [4]. In addition, i.c.v. injection of l-NAME potentiates the fall in body temperature and abolished the febrile response induced by LPS [4]. Steiner et al. [25] showed that HO inhibition reduces the febrile response to LPS and suggested that CO is required for the production of fever. Thus, increase of gaseous neuromodulator production induced by LPS could be an important adaptive mechanism to refrain vasopressin release and facilitate fever development. In conclusion, our data support the hypothesis that NO and CO participate as an inhibitory modulator of vasopressin and oxytocin release induced by LPS.
[4]
[5]
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[7] [8]
[9]
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Acknowledgements We thank Maria Valci A. Silva and Marina Holanda for their excellent technical assistance, and also to Fundac¸ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), PRONEX and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) for the support of our work.
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Inhibitory effect of gaseous neuromodulators in vasopressin and oxytocin release induced by endotoxin in rats Alexandre Giusti-Paiva, Lucila Leico Kagohara Elias, Jos´e Antunes-Rodrigues ∗ Departamento de Fisiologia, Faculdade de Medicina de Ribeir˜ao Preto, Universidade de S˜ao Paulo, 14049-900 Ribeir˜ao Preto, S˜ao Paulo, Brazil Received 16 December 2004; received in revised form 14 February 2005; accepted 16 February 2005
Abstract Nitric oxide (NO) and carbon monoxide (CO) are endogenously synthesized gaseous molecules that act as neurotransmitters in central nervous system. In this study we investigated the modulatory role of NO and CO in lipopolysaccharide (LPS)-induced vasopressin and oxytocin secretion. Intracerebroventricular (i.c.v.) injection of N-l-nitro-arginine methyl ester (l-NAME), 3-morpholino-sydnonimine (SIN-1), zinc deuteroporphyrin 2,4-bis glicol (ZnDPBG) or hemin did not change the basal vasopressin and oxytocin plasma levels. After endovenous LPS administration, plasma vasopressin and oxytocin increased, reaching a peak at 60 min, and returning to basal levels afterwards. LPS administration induced a higher vasopressin and oxytocin plasma levels in rats previously treated with l-NAME and ZnDPBG (P < 0.05) compared to rats pre-treated with vehicle. On the other hand, in rats previously treated with SIN-1 or hemin, there was a significant reduction in the vasopressin and oxytocin secretion. These findings confirm the inhibitory role of NO and CO in the LPS-induced vasopressin and oxytocin secretion. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Vasopressin; Oxytocin; Nitric oxide; Carbon monoxide; Sepsis; Endotoxic shock; Stress
The neuroendocrine system plays an important role in maintaining homeostasis under a variety of stress conditions, including endotoxemia. The response of the organism to disturbance of its homeostasis caused by endotoxin includes release of hormones such as corticotropin-release hormone (CRH), vasopressin and oxytocin involved in the hypothalamus–pituitary–adrenal axis activation during stress [17]. Administration of endotoxin from Gram-negative bacteria, lipopolysaccharide (LPS), induces an intense nuclear Fos expression in vasopressin- and oxytocin-containing magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, suggesting that LPS activates magnocellular neurons in these brain areas [16]. Indeed, we have previously shown that there is a remarkable increase of vasopressin plasma leles in response to LPS intravenous stimulus [4,6]. However, the mechanism involved in this ∗
Corresponding author. Tel.: +55 16 602 3018; fax: +55 16 633 0017. E-mail address: [email protected] (J. Antunes-Rodrigues).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.02.040
control of vasopressin and oxytocin secretion during endotoxemia has not yet been well elucidated. Nitric oxide (NO) and carbon monoxide (CO) have been recently considered as potential neurotransmitters that influence physiological and pathological processes in both the central and the peripheral nervous systems [1]. Nitric oxide, which is generated by nitric oxide synthase (NOS), and CO, which is generated by heme oxygenase (HO), have been shown to modulate both vasopressin and oxytocin release, and both enzymes are located in the PVN and SON [2,28]. NO and CO have been shown to be essential for several physiological and pathophysiological manifestations, including hormonal responses to stress [2,19,22] and thermoregulation [24,25] during endotoxemia. It has been demonstrated that NO may act as an inhibitory regulator of vasopressin and oxytocin [9]. In addition, it has been recently reported that CO may inhibit the release of vasopressin and oxytocin from the rat hypothalamus in vitro [10,11,14]. Thus, the purpose of the present study was to investigate the interaction between the generation of NO and
A. Giusti-Paiva et al. / Neuroscience Letters 381 (2005) 320–324
CO in vasopressin and oxytocin release induced by LPS in rats. For this purpose we used NOS or HO inhibitors, and a NO donor or hemin, a substrate of HO, to evaluate the effects of the NO and CO system on plasma vasopressin and oxytocin release induced by LPS. Male Wistar rats (230–280 g), from the Central Animal Facility of the Faculdade de Medicina de Ribeir˜ao Preto—Universidade de S˜ao Paulo, were housed in individual cages, in a room with controlled temperature (23 ± 2 ◦ C) and lighting (lights on from 7.00 a.m. to 7.00 p.m.), and allowed free access to standard rat pellet food and tap water. One week after acclimatization, the rats were anesthetized with 2,2,2,-tribromoethanol (Aldrich, Milwaukee, WI, USA; 250 mg/kg, i.p.) and a stainless steel guide cannula was introduced into the right lateral cerebral ventricle with the use of a stereotaxic apparatus (Kopf Instruments, Kent, UK), following the stereotaxic coordinates from the atlas of Paxinos and Watson [21]; 8 mm posterior to the bregma, 1.4 mm lateral to midline, and 3.2–3.7 mm ventral to the skull surface; as previously described [5]. After surgery, the animals received an intramuscular prophylactic dose of 100,000 U of benzylpenicillin, and were handled daily during 7 days before the experiments. One day before the experiment, under anesthesia, a silastic catheter was introduced into the right external jugular vein and advanced to the right atrium for intravenous (i.v.) drug administration. The effects of LPS on vasopressin and oxytocin secretion was observed in rats treated with an i.v. bolus injection of 1.5 mg/kg of LPS or 0.15 M NaCl (Saline, 1 ml/kg, control group), and decapitated 0, 0.5, 1, 2, 3, 4, 5 and 6 h after. The NO participation in vasopressin and oxytocin release induced by LPS administration was determined in animals pre-treated with i.c.v. injection of isotonic saline (0.15 M NaCl, 5 l) or 10 g of 3-morpholino-sydnonimine (SIN-1, an NO donor), or 250 g of N-l-nitro-arginine methyl ester (l-NAME, an NOS inhibitor) 30 min before the i.v. injection of LPS or saline, being decapitated 1 h afterwards. To determine the CO participation in vasopressin and oxytocin release induced by LPS administration, in another set of experiments, the animals were pre-treated with i.c.v. injection of vehicle (50 mM Na2 CO3 , 5 l) or 200 nmol hemin, a substrate of HO, or 200 nmol zinc deuteroporphyrin 2,4bis glicol (ZnDPBG, inhibitor of HO) 30 min before the iv injection of LPS or saline and they were decapitated 1 h afterwards. Blood was collected into chilled heparinized plastic tubes, centrifuged (20 min, 3000 rpm, 4 ◦ C) and the plasma separated for vasopressin and oxytocin determination, after extraction from 1 ml of plasma using acetone and petroleum ether. Dried sample extracts were stored at −20 ◦ C until hormonal measurement by radioimmunoassay as previously described [3,18]. The heme oxygenase inhibitor zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) and hemin were obtained from Porphyrin Products (UT, USA), dissolved in basic aqueous solution (50 mM Na2 CO3 ) and stored in the dark.
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Lipopolysaccharide (LPS, from Escherichia coli, serotype 026:B6), l-NAME and SIN-1 were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in isotonic saline (0.15 M NaCl). Doses and method of administration were chosen on the basis of previous studies [6,9,25]. Results are expressed as the mean ± S.E. and statistical significance was assessed by analysis of variance (ANOVA) followed by Newman–Keuls post hoc test. The level of significance was set at p < 0.05. Fig. 1 shows the time-course increase in plasma vasopressin and oxytocin levels after LPS treatment, with a peak at 1 h post-injection, followed by a gradual return to baseline. In control animals, vasopressin and oxytocin levels did not change from baseline during all experimental period. Fig. 2 shows the vasopressin and oxytocin secretion induced by LPS and the modulatory effect of NO on this response. Intracerebroventricular pre-treatment with saline, l-NAME or SIN-1 did not significantly change plasma vasopressin and oxytocin concentrations 1 h after i.v. saline injection. On the other hand, LPS induced a significant increase in plasma vasopressin and oxytocin concentration in the i.c.v. saline (p < 0.05) and l-NAME (p < 0.05) pre-treated groups. Vasopressin and oxytocin secretion were even higher in rats pre-treated with l-NAME (p < 0.05). Conversely, the increase in plasma vasopressin and oxytocin levels induced by LPS was significantly reduced by i.c.v. pre-treatment with SIN-1 (p < 0.05). Fig. 3 shows similar modulatory effects of CO on vasopressin and oxytocin secretion induced by LPS. The pre-treatment with vehicle, ZnDBPG and hemin did not
Fig. 1. Temporal effect of saline (open symbol) or lipopolysaccharide (LPS; closed symbol) injections on plasma vasopressin (top) and oxytocin (bottom) concentration. Value are the mean ± S.E., n = 8 per group. * P < 0.05 vs. saline group.
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Fig. 2. Effect of intracerebroventricular (i.c.v.) injection of saline, SIN-1 and l-NAME on vasopressin (top) and oxytocin (bottom) release 1 h after endovenous (i.v.) saline or LPS injection. Bars represent the mean ± S.E., n = 8 per group. * P < 0.05 vs. saline (i.c.v.) + saline (i.v.) group; # P < 0.05 vs. saline (i.c.v.) + LPS (i.v.) group.
Fig. 3. Effect of intracerebroventricular (i.c.v.) injection of vehicle, hemin and ZnDPBG on vasopressin (top) and oxytocin (bottom) release 1 h after endovenous (i.v.) saline or LPS injection. Bars represent the mean ± S.E., n = 8 per group. * P < 0.05 vs. vehicle (i.c.v.) + saline (i.v.) group; # P < 0.05 vs. vehicle i.c.v. + LPS (i.v.) group.
significantly change vasopressin and oxytocin plasma levels in control animals. LPS induced a significant increase in plasma vasopressin and oxytocin concentration in the i.c.v. vehicle pre-treated group. The administration of LPS in rats previously treated with ZnDPBG (i.c.v.) caused a significantly higher increment in vasopressin and oxytocin levels (p < 0.05), in comparison with controls. However, plasma vasopressin and oxytocin levels after LPS in rats i.c.v. pre-treated with hemin was reduced compared with vehicle pre-treated rats (p < 0.05). The present study suggests that the generation of NO and CO, within the central nervous system, may influence the neuroendocrine adaptive responses to immunological stress. The prevailing action seems to be inhibition of stress responses. In the present work we have shown that NO and CO are involved in the vasopressin and oxytocin release in response to the endotoxic shock. The administration of LPS in rats previously treated with ZnDPBG or l-NAME potentiates the vasopressin- and oxytocin-increase observed in response to LPS. On the other hand, rats pretreated with the NO donor (SIN-1) or hemin showed a decrease of vasopressin and oxytocin response to LPS. The neuroendocrine actions of NO and CO may have clear pathophysiological relevance, since bacterial endotoxins can concomitantly activate stress responses and induce the production of these gases [26]. The observation that NOS is present in the hypothalamus has suggested that NO might play a physiological role in regulating neuroendocrine functions [2]. This concept has been further supported by the detection of NOS staining in hypothalamic cells that contains vasopressin and oxytocin [8]. It has been demonstrated in rats that NO inhibits oxytocin secretion [9], however, the effect of NO on vasopressin secretion remains controversial. The intracerebroventricular injection of NO precursor, as well as NO donor has been shown to increase vasopressin plasma level [20]. In addition, NO donor has been shown to mediate the basal and stimulated release of vasopressin from rat hypothalamic explants [29]; and the inhibition of NOS decreases vasopressin release induced by hypertonic blood volume expansion [27]. Conversely, it has been demonstrated that NO tonically inhibits the basal vasopressin release in control rats [9], and the inhibition of NOS by l-NAME (non-selective inhibitor of NOS) or aminoguanidine (a selective inhibitor of inducible NOS) increases LPS-induced vasopressin plasma levels [5,6]. Most reports suggest that NO is inhibitory of both vasopressinergic and oxytocinergic hypothalamic neurons. In electrophysiological studies in vitro, the NO donor sodium nitroprusside (SNP) inhibited the supraoptic neurons, whereas the NOS inhibitor and the NO scavenger hemoglobin enhanced neuronal activity [12]. In the present study, we observed a significant higher increment of vasopressin and oxytocin plasma levels in response to the LPS administration in animals pre-treated with l-NAME and ZnDPBG. This result is in accordance with an inhibitory role of both NO and CO system on the vasopressin and oxytocin secretion during endotoxemia.
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Equimolar amounts of CO, iron, and biliverdin are generated from the catabolism of the tetrapyrrole ring of hemin (Fe-protoporphyrin-9) by heme oxygenase. Two isoforms of this enzyme have been characterized to date: an inducible isoform, heme oxygenase-1 (HO-1), and a constitutive enzyme, heme oxygenase-2 (HO-2). HO-1 can be detected in macrophages under resting conditions, but it is induced in virtually all cell types of our organism by a variety of stimuli including hemin itself, oxidative stress, heat shock, various disease states, and LPS administration [13]. The central nervous system is endowed with very high HO activity under basal conditions, mostly accounted for HO-2. In terms of influence of CO on neuroendocrine regulation, there are few published data. The formation of CO within the hypothalamus has been associated with inhibition of release of hormones such as CRH, vasopressin and oxytocin [11,14]. The present data are in accordance with previous reports showing that the HO–CO pathway acts centrally to attenuate LPS-stimulated vasopressin release [15]. It should be borne in mind that vasopressin has antipyretic effects [23] and that fever is one of the most essential responses to infection [7]. After systemic administration of high doses of LPS, there is an initial hypothermia followed by febrile response, and V1 antagonist blocked this drop in temperature [4]. In addition, i.c.v. injection of l-NAME potentiates the fall in body temperature and abolished the febrile response induced by LPS [4]. Steiner et al. [25] showed that HO inhibition reduces the febrile response to LPS and suggested that CO is required for the production of fever. Thus, increase of gaseous neuromodulator production induced by LPS could be an important adaptive mechanism to refrain vasopressin release and facilitate fever development. In conclusion, our data support the hypothesis that NO and CO participate as an inhibitory modulator of vasopressin and oxytocin release induced by LPS.
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
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[13]
[14]
[15]
Acknowledgements We thank Maria Valci A. Silva and Marina Holanda for their excellent technical assistance, and also to Fundac¸ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), PRONEX and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) for the support of our work.
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