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An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats
Regulatory Peptides 179 (2012) 6–9
Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats☆ Josilene Fioravanti dos Santos a, Gabriela Ravanelli de Oliveira-Pelegrin a, Letícia Antunes Athayde a, b, Maria José Alves da Rocha a,⁎ a b
Departamento de Morfologia, Estomatologia e Fisiologia da Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Faculdade de Saúde Ibituruna, Montes Claros, MG, Brazil
a r t i c l e
i n f o
Article history: Received 18 April 2012 Received in revised form 1 July 2012 Accepted 27 August 2012 Available online 4 September 2012 Keywords: Antidiuretic hormone Neuroendocrine system Hyperosmolality FLAP inhibitor
a b s t r a c t Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2 μl of the LT synthesis inhibitor MK-886 (1, 2, or 4 μg/kg), or vehicle (DMSO 5%), 1 h before an intraperitoneal injection of hypertonic saline (NaCl 2 M) or isotonic saline (NaCl 0.01 M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P b 0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P b 0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Vasopressin (AVP), also known as antidiuretic hormone, is a nonapeptide synthetized by magnocellular neurons, primarily located in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, but also found in the circularis nucleus situated between the SON and PVN. The axons of these neurons project to the neurohyophysis, where the hormone is stored and, following adequate stimuli, secreted into the circulation [1,2]. The most important stimulus for AVP secretion is hyperosmolality [3], but other non-osmotic stimuli, viz. hypovolemia, hypotension, hypoxia, hypercapnia, pain, nausea, and fever can also lead to an increase in plasma hormone levels [2,4–6]. AVP release is also augmented by endotoxins, prostaglandins and interleukin-1 [7–9]. Recently, we reported an increase in AVP secretion occurring during the early phase of sepsis that correlated with the production of leukotrienes in the central nervous system [10].
☆ Conflict of Interest: All authors declare that there are no conflicts of interest. ⁎ Corresponding author at: Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n, 14049‐900 Ribeirão Preto, SP, Brazil. Tel.: +55 16 3602 3974; fax: +55 16 3633 0999. E-mail address: [email protected] (M.J.A. Rocha). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.08.012
Leukotrienes (LTs) are produced in several organs, including the central nervous system [11,12], being derived from arachidonic acid via the 5-lipoxygenase (5-LO) pathway. The enzyme 5-LO, in conjunction with its helper protein 5-LO-activating protein (FLAP), catalyzes the reaction that transforms arachidonic acid into the unstable intermediate 5-hydroxyperoxy-6,8,11,14-eicosatetranoic acid (5-HPETE), which is then rapidly converted to LTA4. This is also an unstable intermediate that can be either hydrolyzed to form LTB4, or conjugated with glutathione by LTC4 synthase to produce the cysteinylleukotrienes (cys-LTs) LTC4, LTD4 and LTE4 [13,14]. 5-LO and FLAP are found in neurons of several brain regions, including the hippocampus, cerebellum, thalamus, hypothalamus, and brain stem [15]. LTB4 is produced rather uniformly throughout the entire rat brain, with the highest concentrations of LTC4 being detected in the hypothalamus and the median eminence [16,17]. LTC4 synthase was shown to be selectively localized in neurons of the PVN and SON of the hypothalamus, as well as in the suprachiasmatic and retrochiasmatic nuclei in mice. Such LTC4 synthase localization seems to be specific for vasopressinergic neurons, since oxytocinergic neurons did not show LTC4 synthase immunoreactivity, suggesting that the enzyme is particularly involved in AVP functions within the neuroendocrine system [18]. While an osmotic stimulus is a typical physiological condition for AVP secretion, sepsis conditions also lead to a major increase in AVP secretion [7–9], but in a pathological background, not driven by an osmotic stimulus. Hence, we asked whether the relationship between LTs and
J.F. Santos et al. / Regulatory Peptides 179 (2012) 6–9
AVP, observed under this pathophysiological condition, would also hold during hyperosmolality, and hitherto we investigated the effects of a centrally administered LT synthesis blocker on AVP release during hyperosmolality in rats. 2. Material and methods 2.1. Animals Wistar male rats (200–300 g) obtained from the Animal Care Facility of Universidade de São Paulo (USP), Campus Ribeirão Preto were used in the present experiments. The rats were housed in a controlled temperature (25 ± 1°C) and photoperiod (12:12 h night:day cycle) conditions, with food (Nuvilab CR-1, NUVITAL) and water available ad libitum. All experimental protocols were performed according to the guidelines of the Ethics Committee of USP (CEUA)Campus Ribeirão Preto. 2.2. Drug The 5-lipoxygenase-activating protein (FLAP) and LTC4 synthase inhibitor MK-886, chemically known as 3-[1-(p-chlorobenzyl)-5(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (Merck), was dissolved in 5% dimethylsulfoxide (DMSO). 2.3. Cannulation procedure The animals were anesthetized with 2, 2, 2-tribromoethanol (Across Organics, 250 mg/kg i.p.) and fixed in a stereotaxic frame. A stainless guide cannula (0.7 mm outer diameter) was introduced into the right lateral ventricle (coordinates: A: − 0.8 mm, L: 1.4 mm, D: 3.2–3.7 mm). The cannula was attached to the bone via stainless steel screws and acrylic cement. A tight-fitting style was kept inside the guide cannula to prevent occlusion. After surgery, the animals were treated with 100,000 units of benzyl–penicillin and allowed to recover for 5–7 days. 2.4. Experimental protocol Intracerebroventricular (i.c.v.) injections were performed using a 10 μl Hamilton syringe and a dental injection needle (200 μm outer diameter; Mizzy, Brazil). The volume of each injection was 2 μl for all protocols. Injections were given over a period of 1 min, and an additional minute was allowed to elapse before the injection needle was withdrawn from the guide cannula, so as to avoid reflux. The animals received an i.c.v. injection of MK-886 (1, 2, or 4 μg/kg of body weight) dissolved in 5% DMSO, or an injection of this vehicle. One hour later, an osmotic stimulus consisting of an intraperitoneal (i.p.) injection of hypertonic saline (2 M NaCl) in a volume corresponding to10% body weight was given. An i.p. injection of isotonic saline (0.01 M NaCl) served as control for the osmotic stimulus. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for hematocrit, plasma osmolality and plasma AVP measurement. 2.5. Determination of hematocrit and plasma osmolality Hematocrit was measured by centrifugation and plasma osmolality by freezing-point depression (Precision System, INC., USA). 2.6. Radioimmunoassay for vasopressin A radioimmunoassay (RIA) for AVP was performed as previously described [19]. Briefly, plasma samples (1.0 ml) were extracted using the acetone/petroleum ether method, lyophilized and stored at − 70 °C until analysis. Standard reagents and incubation protocols
7
were used for the peptide assays. AVP measurements were done using a commercial antiserum (Peninsula Laboratories) at a final dilution of 1:40,000 in a phosphate buffer (0.062 M Na2HPO4, pH 7.5, containing 0.013 M Na2EDTA and 0.5% BSA). The antiserum is specific for AVP and essentially shows no cross-reactivity with other known peptides. For peptide labeling, 125I was purchased from a commercial supplier (Amersham). A non-equilibrium assay was used with an incubation volume of 500 μl and an incubation time of 4 days at 4 °C. Bound hormone was separated from unbound by a secondary antibody produced in the laboratory where the RIA was performed. The minimum detection limit was 0.9 pg/ml and the coefficients of inter- and intra-assay variation were 11% and 7%, respectively. 2.7. Data analysis The data are reported as mean ± S.E.M. Analysis of variance (ANOVA) and a post hoc Student–Newman–Keuls (SNK) test were used to reveal statistical differences among treatment and stimulus. For further analyses, Student's t-tests were used to directly compare groups. Values of P b 0.05 were considered statistically significant. Raw RIA data were logit transformed before testing. 3. Results Following the intraperitoneal injection of hypertonic saline, an increase was seen, as expected, in both osmolality (321 ± 4 vs. 287 ± 2 control, P b 0.001) and AVP plasma levels (12.7 ± 1.9 vs 1.3 ± 0.1 control, P b 0.001), already 30 min after the injection. An increase in the hematocrit (40 ± 0.7 vs 36 ± 1.5 control, P = 0.030) was also noted, but this being at a lower level than the one necessary to trigger an AVP release. The i.c.v. injection of 5% DMSO, serving as vehicle to dissolve the FLAP and LTC4 synthase inhibitor MK-886, did not affect the levels of plasma AVP, osmolality, or hematocrit seen in animals injected with hypertonic or isotonic saline. An i.c.v. injection of 2 μl of MK-886 given 1 h prior to the injection of isotonic saline also did not cause any alteration in the hematocrit, osmolality, or AVP plasma levels. But when this drug was given at doses of 2.0 or 4.0 μg/kg prior to the osmotic stimulus we noted a further significant increase in AVP plasma levels (F(3,66) = 31,62, P b 0.001) while hematocrit and osmolality were not affected (Fig. 1). 4. Discussion The present study was designed to examine whether LTs may modulate the osmotic stimulus-induced AVP release. We noted that the central injection of the LT synthesis blocker MK-886 further augmented the plasma AVP levels beyond those normally seen after intraperitoneal injection of hypertonic saline in rats. This leads us to infer that central leukotriene production may contribute to attenuating the AVP release that normally occurs in response to an osmotic stimulus, in the sense of preventing an overshooting of this humoral response. To our knowledge this is the first time that central LTs could be connected to AVP secretion during such a stimulus. A neuroendocrine role for LTs was first suggested by the colocalization of gonadotropin-releasing hormone (GnRH) and LTC4 in neurons of the median eminence in rats [12]. Subsequent in vitro studies showed that the release of luteinizing hormone (LH) from pituitary cells in response to GnRH was partly mediated by LTs, and this response appeared to be LTC4-specific, since LTB4 did not alter the release of luteinizing hormone [12]. Further studies revealed that LTC4 synthase is selectively localized in the hypothalamic and extrahypothalamic vasopressin systems, suggesting the involvement of cys-LTs in neuroendocrine system and, especially vasopressinergic neural functions [18]. An increase in plasma osmolality is a strong stimuli for AVP release [4], and the increase in osmolality seen in our study (more than the 4%) was considered adequate for triggering AVP secretion.
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J.F. Santos et al. / Regulatory Peptides 179 (2012) 6–9
Even though we could see an increase in the hematocrit accompanying the increase in plasma AVP levels, the magnitude of hypovolemia observed was judged as being too low to explain the increase in AVP plasma levels seen in our experimental design. Furthermore, MK-886 pretreatment did not affect hematocrit levels, thus indicating that the enhanced increase in AVP levels in response to hypertonic saline injection was not a consequence of a decrease in volemia. Although we did not analyze LTC4 synthase content in this study, we had obtained evidence in a prior study that an i.c.v. injection of MK-886 reduces the content of this enzyme in the respective brain regions during a sepsis condition [10]. But interestingly, in this pathophysiological condition, the MK-886 injection caused a reduction in AVP secretion in the early phase of sepsis, whereas in the physiological condition of an osmotic stimulus AVP secretion was further enhanced. This makes sense, as in a physiological condition, endocrine responses involved in homeostasis always require a careful balance, and in this situation leukotrienes would actually prevent an overshoot in the AVP release by hypothalamic neurons. In contrast, in the pathological condition of sepsis, hypotension occurs in combination with a strong inflammatory response, so the organism may use inflammatory mediators, including leukotrienes, in an attempt to regain control of blood pressure. Further investigating the dual role of centrally produced leukotrienes in blood pressure control should, thus, certainly be a goal worthwhile to pursue. 5. Conclusion The results of this study lead us to conclude that leukotrienes play a role in AVP secretion induced by an osmotic stimulus in rats. In this situation they actually seem to attenuate the humoral response, in contrast to their role seen in sepsis, where they further activate AVP release. So far we have no definitive explanation for this differential AVP response to the i.c.v. MK-886 injection, but we hypothesize that during sepsis vasopressin secretion is caused by both inflammatory mediators and hypotension, which together activate one pathway, whereas in the hyperosmotic condition, the activation of vasopressinergic neurons would involve a different pathway. An osmotic stimulus is typically not associated with a high production of nitric oxide, cytokines, or leukotrienes, as seen during sepsis conditions. So, while leukotrienes seem to activate vasopressin secretion during sepsis, these mediators seem to be inhibitory during an osmotic stimulus. Acknowledgments The authors thank Nadir M. Fernandes, Marina Holanda and Mariana Rossin Martinez for the technical assistance. We also thank Drs. José Antunes-Rodrigues and Lucila L. K. Elias from the Department of Physiology, Faculty of Medicine of Ribeirão Preto, USP, for providing the infrastructure for the RIA analyses. Financial support from FAPESP and CNP is gratefully acknowledged. Fig. 1. Effect of a central administration of the 5-lipoxygenase-activating protein (FLAP) and LTC4 synthase inhibitor MK-886, or its vehicle (5%DMSO) on hematocrit (upper panel), plasma osmolality (middle panel) and plasma vasopressin levels (lower) panel in response to an intraperitoneal injection of hypertonic (2 M NaCl) or isotonic saline ( 0.01 M NaCl). The number of animals analyzed in each group was 5 to 7. The results are expressed as means ± SEM. * P b 0.05 compared to the isotonic saline group. + P b 0.05 compared to the vehicle (5% DMSO) group. § P b 0.05 compared within the group.
As osmolality did not increase when MK-886 pretreated rats were given an osmotic stimulus, it is further inferred that the additional increase in AVP levels seen in this situation cannot be attributed to a change in osmolality. When looking for alternatives we first considered hypovolemia, which is another stimulus for AVP secretion [20].
References [1] Luckman SM, Dyball RE, Leng G. Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity. J Neurosci 1994;14:4825-30. [2] Holmes CL, Patel BM, Russell JA, Walley KR. Physiology of vasopressin relevant to management of septic shock. Chest 2001;120:989–1002. [3] Dunn FL, Brennan TJ, Nelson AE, Robertson GL. The role of blood osmolality and volume in regulating vasopressin secretion in the rat. J Clin Invest 1973;52: 3212-9. [4] Cunningham Jr ET, Sawchenko PE. Reflex control of magnocellular vasopressin and oxytocin secretion. Trends Neurosci 1991;14:406-11. [5] Pittman QJ, Chen X, Mouihate A, Hirasawa M, Martin S. Arginine vasopressin, fever and temperature regulation. Prog Brain Res 1998;119:383-92. [6] Callahan MF, Thore CR, Sundberg DK, Gruber KA, O'Steen K, Morris M. Excitotoxin paraventricular nucleus lesions: stress and endocrine reactivity and oxytocin mRNA levels. Brain Res 1992;597:8–15.
J.F. Santos et al. / Regulatory Peptides 179 (2012) 6–9 [7] Kasting NW, Mazurek MF, Martin JB. Endotoxin increases vasopressin release independently of known physiological stimuli. Am J Physiol 1985;248:E420-4. [8] Naito Y, Fukata J, Shindo K, Ebisui O, Murakami N, Tominaga T, Nakai Y, Mori K, Kasting NW, Imura H. Effects of interleukins on plasma arginine vasopressin and oxytocin levels in conscious, freely moving rats. Biochem Biophys Res Commun 1991;174:1189-95. [9] Giusti-Paiva A, Ruginsk SG, de Castro M, Elias LL, Carnio EC, Antunes-Rodrigues J. Role of nitric oxide in lipopolysaccharide-induced release of vasopressin in rats. Neurosci Lett 2003;346:21-4. [10] Athayde LA, Oliveira-Pelegrin GR, Nomizo A, Faccioli LH, Rocha MJ. Blocking central leukotrienes synthesis affects vasopressin release during sepsis. Neuroscience 2009;160:829-36. [11] Wolfe LS. Eicosanoids: prostaglandins, thromboxanes, leukotrienes, and other derivatives of carbon-20 unsaturated fatty acids. J Neurochem 1982;38:1–14. [12] Hulting AL, Lindgren JA, Hokfelt T, Eneroth P, Werner S, Patrono C, Samuelsson B. Leukotriene C4 as a mediator of luteinizing hormone release from rat anterior pituitary cells. Proc Natl Acad Sci U S A 1985;82:3834-8. [13] Peters-Golden M, Brock TG. 5-lipoxygenase and FLAP. Prostaglandins Leukot Essent Fatty Acids 2003;69:99–109. [14] Peters-Golden M, Henderson Jr WR. Leukotrienes. N Engl J Med 2007;357: 1841-54.
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[15] Lammers CH, Schweitzer P, Facchinetti P, Arrang JM, Madamba SG, Siggins GR, Piomelli D. Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling. J Neurochem 1996;66:147-52. [16] Lindgren JA, Hokfelt T, Dahlen SE, Patrono C, Samuelsson B. Leukotrienes in the rat central nervous system. Proc Natl Acad Sci U S A 1984;81:6212-6. [17] Miyamoto T, Lindgren JA, Hokfelt T, Samuelsson B. Formation of lipoxygenase products in the rat brain. Adv Prostaglandin Thromboxane Leukot Res 1987;17B:929-33. [18] Shimada A, Satoh M, Chiba Y, Saitoh Y, Kawamura N, Keino H, Hosokawa M, Shimizu T. Highly selective localization of leukotriene C4 synthase in hypothalamic and extrahypothalamic vasopressin systems of mouse brain. Neuroscience 2005;131:683-9. [19] Correa PB, Pancoto JA, de Oliveira-Pelegrin GR, Carnio EC, Rocha MJ. Participation of iNOS-derived NO in hypothalamic activation and vasopressin release during polymicrobial sepsis. J Neuroimmunol 2007;183:17-25. [20] Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 1993;328: 1471-7.
Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats☆ Josilene Fioravanti dos Santos a, Gabriela Ravanelli de Oliveira-Pelegrin a, Letícia Antunes Athayde a, b, Maria José Alves da Rocha a,⁎ a b
Departamento de Morfologia, Estomatologia e Fisiologia da Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Faculdade de Saúde Ibituruna, Montes Claros, MG, Brazil
a r t i c l e
i n f o
Article history: Received 18 April 2012 Received in revised form 1 July 2012 Accepted 27 August 2012 Available online 4 September 2012 Keywords: Antidiuretic hormone Neuroendocrine system Hyperosmolality FLAP inhibitor
a b s t r a c t Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2 μl of the LT synthesis inhibitor MK-886 (1, 2, or 4 μg/kg), or vehicle (DMSO 5%), 1 h before an intraperitoneal injection of hypertonic saline (NaCl 2 M) or isotonic saline (NaCl 0.01 M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P b 0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P b 0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Vasopressin (AVP), also known as antidiuretic hormone, is a nonapeptide synthetized by magnocellular neurons, primarily located in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, but also found in the circularis nucleus situated between the SON and PVN. The axons of these neurons project to the neurohyophysis, where the hormone is stored and, following adequate stimuli, secreted into the circulation [1,2]. The most important stimulus for AVP secretion is hyperosmolality [3], but other non-osmotic stimuli, viz. hypovolemia, hypotension, hypoxia, hypercapnia, pain, nausea, and fever can also lead to an increase in plasma hormone levels [2,4–6]. AVP release is also augmented by endotoxins, prostaglandins and interleukin-1 [7–9]. Recently, we reported an increase in AVP secretion occurring during the early phase of sepsis that correlated with the production of leukotrienes in the central nervous system [10].
☆ Conflict of Interest: All authors declare that there are no conflicts of interest. ⁎ Corresponding author at: Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n, 14049‐900 Ribeirão Preto, SP, Brazil. Tel.: +55 16 3602 3974; fax: +55 16 3633 0999. E-mail address: [email protected] (M.J.A. Rocha). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.08.012
Leukotrienes (LTs) are produced in several organs, including the central nervous system [11,12], being derived from arachidonic acid via the 5-lipoxygenase (5-LO) pathway. The enzyme 5-LO, in conjunction with its helper protein 5-LO-activating protein (FLAP), catalyzes the reaction that transforms arachidonic acid into the unstable intermediate 5-hydroxyperoxy-6,8,11,14-eicosatetranoic acid (5-HPETE), which is then rapidly converted to LTA4. This is also an unstable intermediate that can be either hydrolyzed to form LTB4, or conjugated with glutathione by LTC4 synthase to produce the cysteinylleukotrienes (cys-LTs) LTC4, LTD4 and LTE4 [13,14]. 5-LO and FLAP are found in neurons of several brain regions, including the hippocampus, cerebellum, thalamus, hypothalamus, and brain stem [15]. LTB4 is produced rather uniformly throughout the entire rat brain, with the highest concentrations of LTC4 being detected in the hypothalamus and the median eminence [16,17]. LTC4 synthase was shown to be selectively localized in neurons of the PVN and SON of the hypothalamus, as well as in the suprachiasmatic and retrochiasmatic nuclei in mice. Such LTC4 synthase localization seems to be specific for vasopressinergic neurons, since oxytocinergic neurons did not show LTC4 synthase immunoreactivity, suggesting that the enzyme is particularly involved in AVP functions within the neuroendocrine system [18]. While an osmotic stimulus is a typical physiological condition for AVP secretion, sepsis conditions also lead to a major increase in AVP secretion [7–9], but in a pathological background, not driven by an osmotic stimulus. Hence, we asked whether the relationship between LTs and
J.F. Santos et al. / Regulatory Peptides 179 (2012) 6–9
AVP, observed under this pathophysiological condition, would also hold during hyperosmolality, and hitherto we investigated the effects of a centrally administered LT synthesis blocker on AVP release during hyperosmolality in rats. 2. Material and methods 2.1. Animals Wistar male rats (200–300 g) obtained from the Animal Care Facility of Universidade de São Paulo (USP), Campus Ribeirão Preto were used in the present experiments. The rats were housed in a controlled temperature (25 ± 1°C) and photoperiod (12:12 h night:day cycle) conditions, with food (Nuvilab CR-1, NUVITAL) and water available ad libitum. All experimental protocols were performed according to the guidelines of the Ethics Committee of USP (CEUA)Campus Ribeirão Preto. 2.2. Drug The 5-lipoxygenase-activating protein (FLAP) and LTC4 synthase inhibitor MK-886, chemically known as 3-[1-(p-chlorobenzyl)-5(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (Merck), was dissolved in 5% dimethylsulfoxide (DMSO). 2.3. Cannulation procedure The animals were anesthetized with 2, 2, 2-tribromoethanol (Across Organics, 250 mg/kg i.p.) and fixed in a stereotaxic frame. A stainless guide cannula (0.7 mm outer diameter) was introduced into the right lateral ventricle (coordinates: A: − 0.8 mm, L: 1.4 mm, D: 3.2–3.7 mm). The cannula was attached to the bone via stainless steel screws and acrylic cement. A tight-fitting style was kept inside the guide cannula to prevent occlusion. After surgery, the animals were treated with 100,000 units of benzyl–penicillin and allowed to recover for 5–7 days. 2.4. Experimental protocol Intracerebroventricular (i.c.v.) injections were performed using a 10 μl Hamilton syringe and a dental injection needle (200 μm outer diameter; Mizzy, Brazil). The volume of each injection was 2 μl for all protocols. Injections were given over a period of 1 min, and an additional minute was allowed to elapse before the injection needle was withdrawn from the guide cannula, so as to avoid reflux. The animals received an i.c.v. injection of MK-886 (1, 2, or 4 μg/kg of body weight) dissolved in 5% DMSO, or an injection of this vehicle. One hour later, an osmotic stimulus consisting of an intraperitoneal (i.p.) injection of hypertonic saline (2 M NaCl) in a volume corresponding to10% body weight was given. An i.p. injection of isotonic saline (0.01 M NaCl) served as control for the osmotic stimulus. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for hematocrit, plasma osmolality and plasma AVP measurement. 2.5. Determination of hematocrit and plasma osmolality Hematocrit was measured by centrifugation and plasma osmolality by freezing-point depression (Precision System, INC., USA). 2.6. Radioimmunoassay for vasopressin A radioimmunoassay (RIA) for AVP was performed as previously described [19]. Briefly, plasma samples (1.0 ml) were extracted using the acetone/petroleum ether method, lyophilized and stored at − 70 °C until analysis. Standard reagents and incubation protocols
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were used for the peptide assays. AVP measurements were done using a commercial antiserum (Peninsula Laboratories) at a final dilution of 1:40,000 in a phosphate buffer (0.062 M Na2HPO4, pH 7.5, containing 0.013 M Na2EDTA and 0.5% BSA). The antiserum is specific for AVP and essentially shows no cross-reactivity with other known peptides. For peptide labeling, 125I was purchased from a commercial supplier (Amersham). A non-equilibrium assay was used with an incubation volume of 500 μl and an incubation time of 4 days at 4 °C. Bound hormone was separated from unbound by a secondary antibody produced in the laboratory where the RIA was performed. The minimum detection limit was 0.9 pg/ml and the coefficients of inter- and intra-assay variation were 11% and 7%, respectively. 2.7. Data analysis The data are reported as mean ± S.E.M. Analysis of variance (ANOVA) and a post hoc Student–Newman–Keuls (SNK) test were used to reveal statistical differences among treatment and stimulus. For further analyses, Student's t-tests were used to directly compare groups. Values of P b 0.05 were considered statistically significant. Raw RIA data were logit transformed before testing. 3. Results Following the intraperitoneal injection of hypertonic saline, an increase was seen, as expected, in both osmolality (321 ± 4 vs. 287 ± 2 control, P b 0.001) and AVP plasma levels (12.7 ± 1.9 vs 1.3 ± 0.1 control, P b 0.001), already 30 min after the injection. An increase in the hematocrit (40 ± 0.7 vs 36 ± 1.5 control, P = 0.030) was also noted, but this being at a lower level than the one necessary to trigger an AVP release. The i.c.v. injection of 5% DMSO, serving as vehicle to dissolve the FLAP and LTC4 synthase inhibitor MK-886, did not affect the levels of plasma AVP, osmolality, or hematocrit seen in animals injected with hypertonic or isotonic saline. An i.c.v. injection of 2 μl of MK-886 given 1 h prior to the injection of isotonic saline also did not cause any alteration in the hematocrit, osmolality, or AVP plasma levels. But when this drug was given at doses of 2.0 or 4.0 μg/kg prior to the osmotic stimulus we noted a further significant increase in AVP plasma levels (F(3,66) = 31,62, P b 0.001) while hematocrit and osmolality were not affected (Fig. 1). 4. Discussion The present study was designed to examine whether LTs may modulate the osmotic stimulus-induced AVP release. We noted that the central injection of the LT synthesis blocker MK-886 further augmented the plasma AVP levels beyond those normally seen after intraperitoneal injection of hypertonic saline in rats. This leads us to infer that central leukotriene production may contribute to attenuating the AVP release that normally occurs in response to an osmotic stimulus, in the sense of preventing an overshooting of this humoral response. To our knowledge this is the first time that central LTs could be connected to AVP secretion during such a stimulus. A neuroendocrine role for LTs was first suggested by the colocalization of gonadotropin-releasing hormone (GnRH) and LTC4 in neurons of the median eminence in rats [12]. Subsequent in vitro studies showed that the release of luteinizing hormone (LH) from pituitary cells in response to GnRH was partly mediated by LTs, and this response appeared to be LTC4-specific, since LTB4 did not alter the release of luteinizing hormone [12]. Further studies revealed that LTC4 synthase is selectively localized in the hypothalamic and extrahypothalamic vasopressin systems, suggesting the involvement of cys-LTs in neuroendocrine system and, especially vasopressinergic neural functions [18]. An increase in plasma osmolality is a strong stimuli for AVP release [4], and the increase in osmolality seen in our study (more than the 4%) was considered adequate for triggering AVP secretion.
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Even though we could see an increase in the hematocrit accompanying the increase in plasma AVP levels, the magnitude of hypovolemia observed was judged as being too low to explain the increase in AVP plasma levels seen in our experimental design. Furthermore, MK-886 pretreatment did not affect hematocrit levels, thus indicating that the enhanced increase in AVP levels in response to hypertonic saline injection was not a consequence of a decrease in volemia. Although we did not analyze LTC4 synthase content in this study, we had obtained evidence in a prior study that an i.c.v. injection of MK-886 reduces the content of this enzyme in the respective brain regions during a sepsis condition [10]. But interestingly, in this pathophysiological condition, the MK-886 injection caused a reduction in AVP secretion in the early phase of sepsis, whereas in the physiological condition of an osmotic stimulus AVP secretion was further enhanced. This makes sense, as in a physiological condition, endocrine responses involved in homeostasis always require a careful balance, and in this situation leukotrienes would actually prevent an overshoot in the AVP release by hypothalamic neurons. In contrast, in the pathological condition of sepsis, hypotension occurs in combination with a strong inflammatory response, so the organism may use inflammatory mediators, including leukotrienes, in an attempt to regain control of blood pressure. Further investigating the dual role of centrally produced leukotrienes in blood pressure control should, thus, certainly be a goal worthwhile to pursue. 5. Conclusion The results of this study lead us to conclude that leukotrienes play a role in AVP secretion induced by an osmotic stimulus in rats. In this situation they actually seem to attenuate the humoral response, in contrast to their role seen in sepsis, where they further activate AVP release. So far we have no definitive explanation for this differential AVP response to the i.c.v. MK-886 injection, but we hypothesize that during sepsis vasopressin secretion is caused by both inflammatory mediators and hypotension, which together activate one pathway, whereas in the hyperosmotic condition, the activation of vasopressinergic neurons would involve a different pathway. An osmotic stimulus is typically not associated with a high production of nitric oxide, cytokines, or leukotrienes, as seen during sepsis conditions. So, while leukotrienes seem to activate vasopressin secretion during sepsis, these mediators seem to be inhibitory during an osmotic stimulus. Acknowledgments The authors thank Nadir M. Fernandes, Marina Holanda and Mariana Rossin Martinez for the technical assistance. We also thank Drs. José Antunes-Rodrigues and Lucila L. K. Elias from the Department of Physiology, Faculty of Medicine of Ribeirão Preto, USP, for providing the infrastructure for the RIA analyses. Financial support from FAPESP and CNP is gratefully acknowledged. Fig. 1. Effect of a central administration of the 5-lipoxygenase-activating protein (FLAP) and LTC4 synthase inhibitor MK-886, or its vehicle (5%DMSO) on hematocrit (upper panel), plasma osmolality (middle panel) and plasma vasopressin levels (lower) panel in response to an intraperitoneal injection of hypertonic (2 M NaCl) or isotonic saline ( 0.01 M NaCl). The number of animals analyzed in each group was 5 to 7. The results are expressed as means ± SEM. * P b 0.05 compared to the isotonic saline group. + P b 0.05 compared to the vehicle (5% DMSO) group. § P b 0.05 compared within the group.
As osmolality did not increase when MK-886 pretreated rats were given an osmotic stimulus, it is further inferred that the additional increase in AVP levels seen in this situation cannot be attributed to a change in osmolality. When looking for alternatives we first considered hypovolemia, which is another stimulus for AVP secretion [20].
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