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Oxytocin antisense reduces salt intake in the baroreceptor-denervated rat
ELSEVIER
Regulatory Peptides 59 (1995) 261-266
Oxytocin antisense reduces salt intake in the baroreceptor-denervated rat Mariana Morris *, Ping Li, Cindy Barrett, Michael F. Callahan Department of Physiology and Pharmacology and the Hypertension Center, Bowman Gray School of Medicine of WakeForest University, Winston-Salem, NC 27157, USA Received 26 May 1995; accepted 27 June 1995
Abstract Experiments were performed to evaluate the role of central oxytocin (OT) in the inhibition of salt intake produced by sinoaortic denervation (SAD). The effect of OT antisense treatment on 24 h intake of 2% NaCI in SAD and sham-operated (SO) rats was determined. PVN injection of unmodified antisense oligodeoxynucleotides (ODNs) to OT mRNA decreased intake of 2% NaCI in SAD, but not SO rats. Salt consumption was 22 + 4 ml after the injection of control ODN as compared to 8 ___4 ml after the OT antisense injection (P < 0.05). SAD animals also demonstrated an increased plasma OT response to salt loading, an elevation from 3.2 + 0.7 to 6.9 + 0.8 pg/ml. In contrast, salt ingestion produced no significant change in plasma OT in the SO group. The increased endocrine response in the SADs occurred even though salt intake was lower in this group. There were no group differences in plasma electrolytes or posterior pituitary OT content. Results show that OT antisense specifically inhibits salt intake in the denervated rat, suggesting that the central oxytocinergic axis stimulates sodium drive in this experimental model. Keywords: Hypothalamus; Paraventricular nucleus; Posterior pituitary; Dipsogenesis, cardiovascular
1. Introduction Salt intake and excretion are regulated by interactions between neural and endocrine factors. Oxytocin (OT), a peptide produced by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, is thought to be one of ~Ihe key mediators. Low doses of OT produce natriuresis while OT antagonists decrease urinary sodium excretion [1-3]. With regard to ingestive behavior, OT attenuates salt intake pro-
* Corresponding author. Fax: + 1 (910) 7168501.
duced by volume depletion while OT antagonists increase the salt intake induced by naloxone and angiotensin [4,5]. The baroreceptor-denervated animal provides an experimental model o f altered neurohypophyseal function. Denervation produces rapid changes in nuclear peptide and amine concentrations as well as increases in plasma vasopressin (VP) secretion [6,7]. There is also a heightened sensitivity to sodium stimulation as demonstrated by increased responses to acute and chronic salt loading [8,9]. Ingestion of a hypertonic NaC1 solution resulted in greater increases in both peptide secretion and peptide m R N A expression even though the SAD animals consumed
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 9 4 - 1
262
M. Morris et al. /Regulatory Peptides 59 (1995) 261-266
less salt than the controls [9]. Likewise, salt intake induced by volume depletion was greatly attenuated in denervated animals [10]. The data points to the idea that interruption of baroreceptor input to the brain produces a state of salt sensitivity. Recent studies have employed central antisense treatment as a means of investigating the involvement of peptide systems in physiological responses [11]. The advantage of antisense ODNs is that they can be targeted to nonhomologous regions of the mRNA, offering specificity which is not available with peptide receptor antagonists. Antisense ODNs to OT and VP were shown to have acute and specific effects on stimulus induced changes in heart rate, hormone secretion, neuronal firing and milk ejection [12-141. This new approach was applied to the study of baroreceptor modulation of salt consumption. In view of the hypothesized role of OT in the regulation of fluid/electrolyte balance, experiments were performed to evaluate the effect of PVN administration of OT antisense ODNs on sodium chloride intake in sham-operated and baroreceptor-denervated rats.
2. Materials and methods Male Sprague-Dawley rats (300-400 g) were housed individually on a 12/12 light/dark cycle with free access to water and laboratory chow. Under ketamine/xylazine anesthesia (10:50 mg/kg, i.m.) the baroreceptor nerves were surgically interrupted by stripping the carotid sinus and resecting the superior laryngeal nerve, a 1.0 mm section of the sympathetic chain posterior to the superior cervical ganglion and the aortic depressor nerves. The sham ol~eration (SO) included dissection of the neck muscles, but no manipulation of the nerves. The studies were conducted 3 - 4 weeks after surgical denervation. At this time post-surgery, the animals have regained weight and are in normal fluid balance [7]. The animals were anesthetized with ketamine/xylazine (10:50 mg/kg, i.m.) and bilateral guide cannulas were implanted dorsal to the PVN. The cannulas were formed from cutoff 25 ga stainless steel needles. The cannulas were secured to the skull with stainless steel screws and dental cement and closed with polyethylene (PE) closures.
The stereotaxic coordinates with a level skull were 1.8 mm posterior to bregma, 0.6 mm lateral from the sagittal sinus and 7.1 mm ventral to the skull. The intake of 2% NaC1 was measured following the PVN injection of OT antisense or mixed-base ODNs. ODNs were dissolved in sterile saline (0.9% NaC1) and slowly injected into the PVN at a dose of 2 ~ g / 0 . 3 /xl per side. The ODNs were injected using a 30 ga needle which extended 1 mm beyond the end of the cannula. The animals were given 2% NaC1 as the sole drinking fluid for a 24 h period after the injection. Unmodified ODNs were synthesized by the DNA Synthesis Core Laboratory (Comprehensive Cancer Center of Bowman Gray School of Medicine). An ODN (18-mer) complimentary to the region around the putative translation start codon of rat OT mRNA was used. The sequence of the ODN is 5'-CAG CAA GCG AGA CTG GGG-3' for OT and 5'-ATG-GACTGT-CGA-AGG-TI'C-3' for mixed base. This OT antisense has been previously used and found effective in modifying stress responses in the rat [12]. A second experiment was conducted to evaluate the effect of NaC1 intake on plasma and posterior pituitary OT content and plasma sodium and osmolality in SAD and SO rats. The rats were given 2% NaC1 or water to drink for an 18 h period. The animals were decapitated with collection of blood samples in heparinized tubes on ice. The posterior pituitaries were removed and frozen on dry ice and stored at -80°C. The tissue was sonicated in methanol/acetic acid (98%/0.02 N) and peptide content measured in the supernatant. Plasma samples were extracted using acetone precipitation and petroleum ether extraction. Plasma and tissue extracts were measured for OT by a sensitive and specific radioimmunoassay [9]. Data was analyzed using two-way ANOVA with the Newmann-Keuls post hoc test. Significance was taken as P < 0.05.
3. Results PVN injection of OT antisense ODN in the baroreceptor-denervated group produced a decrease in salt intake as compared to a mixed-base control (Fig. 1; P < 0.05). Mean intake of 2% NaC1 was 22
M. Morris et al. / Regulatory Peptides 59 (1995) 261-266 60 [ 50 I
263 Fluid Intake
Plasma Oxytocin
lmtisense Treatment EIMixed BaseBBOxytocin
40 ~.~ 40 -
- - * - -
E
30 ,T 10 10 0 0
Control
Control
SAD
SAD
Control
Fig. 2. Plasma oxytocin and fluid intake in SAD and SO male rats. Rats were given 2% NaC1 or water to drink for 18 h. n = 7 and 6 for the water and salt SAD groups, respectively, and n = 4 and 5 for the water and salt SO groups, respectively. * P < 0.05.
Fig. 1. Effect o f PVN injection of oxytocin antisense or control mixed-base ODNs on 2% NaCI intake (24 h) in sinoaortic denerrated (SAD) or sham-operated male rats. Unmodified ODNs (2.0 /zg/0.3 /~1) were injected bilaterally into the PVN o f conscious animals, n = 5 and 6 for the SAD rats given OT and mixed base, respectively, and n = 7 and 4 for the SO rats given OT and mixed base, respectively. * = P < 0.05.
osmolality, or posterior pituitary OT content between the groups (Table 1). There was a significant effect of treatment on plasma sodium, but no differences between the groups, indicating that the animals received a similar sodium challenge (Table 1).
vs. 8 m l / 2 4 h (mixed base vs. OT antisense). This was in contrast to the results in the SO group in which there was no difference in salt intake in response to the ODN treatment (29 vs. 38 m l / 2 4 h, mixed base vs. OT antisense). A second experiment was performed to evaluate the endocrine, plasma electrolyte and intake responses to salt loading in the SAD and SO groups. SAD rats consumed less NaC1 yet they demonstrated a greater increase in plasma OT than the SO animals (Fig. 2). The mean intake of 2% NaCI in the SAD animals was 18 ml a,; compared to 35 ml in the SO group. Plasma OT was significantly higher in the SAD animals which received salt as compared to water, 3.2 vs. 6.9 pg/ml. Salt intake produced no change in plasma OT in the SO group. There were no significant differences in body weight, plasma
4. Discussion Experiments were performed to evaluate the role of central OT in the repressed sodium chloride intake observed after baroreceptor denervation [9,10]. The results demonstrate that PVN injection of OT antisense ODN produced a decrease in salt intake in the SAD. Since denervated animals show increased OT responses to sodium stimulation, the results suggest that central OT systems are inhibitory to salt intake in this model. While the aortic baroreceptor system is best known for its role in the regulation of blood pressure/heart
Table 1 Effect of 24 h salt loading in SAD and sham-operated rats Group
Treatment
Body weight
(g) Sham Sham SAD SAD
Water Salt Water Salt
354.8 362.5 365.8 341.0
+ + + +
15.5 13.4 6.6 10.7
Data presented as mean + S.E.M.; * P < 0.05, effect of treatment.
SAD
Pituitary
Plasma sodium * (mEq/l)
osmolality (mosm/kg)
oxytocin (ng/PP)
145.9 151.7 147.8 150.6
302.6 312.8 307.0 311.8
45.9 28.4 49.7 30.9
-I- 3.7 -1- 1.2 + 1.3 + 0.8
+ 6.8 + 3.2 + 2.8 -I- 2.7
+ 6.0 -I- 6.3 + 14.7 + 2.7
264
M. Morris et al. /Regulatory Peptides 59 (1995) 261-266
rate interactions, it is also critical in the control of fluid and endocrine balance. The hypothalamic PVN and SON receive input from baroreceptor nerves, serving to relay information on volume and blood pressure status. Interruption of afferent input via surgical interruption of the baroreceptor nerves produces prominent effects on water/electrolyte and endocrine balance. SAD causes acute adipsia, aphagia, hypertension, increased pressor responses to central stimulation and increases in plasma vasopressin [7,15]. Denervation is also associated with salt sensitivity as demonstrated by the increased VP and OT responses to acute and chronic salt loading [8,9]. The chronic stimulus increased both peptide secretion and peptide mRNA expression in the face of reduced saline intake [9]. There is evidence that central OT systems are inhibitory to salt intake [16]. Thus, the driving hypothesis for the current study was that increased OT responses in the SAD were responsible for the reduction in salt intake in this model. However, the present results are at variance with this idea since PVN injection of OT antisense significantly reduced, rather than increased, salt intake in the SAD. There was no significant difference between the effects of the mixed base and OT ODNs in the control group, further demonstrating the specificity of the antisense effect. Additionally, the denervated animals showed evidence of increased endocrine responses with salt consumption producing an increase in plasma OT even though intake was lower in SADs than in the controls. Studies which have investigated the role of OT in the control of salt appetite have relied on the use of OT peptides or peptide antagonists. Using various experimental models, results show that OT inhibits salt consumption while the OT antagonist increases intake. For example, volume depletion induced by polyethylene glycol treatment produced an increase in salt appetite which was attenuated by OT [4]. OT antagonists produced an increase in sodium consumption in animals treated with naloxone, an opiate antagonist which increases OT secretion [4]. A point which should be considered in the interpretation of these studies is that they employ large doses of peptide or antagonist (/xg amounts). The specificity of the antagonists, particularly at high doses, has been questioned [17]. The OT antagonist has effects
on VP and vice versa for the VP antagonist. Furthermore, centrally injected peptides have effects which are dependent on the dosage range [18]. Indeed, OT was found to stimulate rather than inhibit sodium intake when it was administered chronically at a low dose [19]. Another explanation for the difference in the findings may be related to the mode of stimulation. A recent study by Blackburn and colleagues demonstrated that OT antagonists had no effect on salt intake induced by hypertonic saline [20]. The antagonist was only effective in modulating the response to hypertonic mannitol. The investigators hypothesized that there are basic differences between osmotic and sodium sensors in terms of responses and CNS interactions. It is possible that OT has both stimulatory and inhibitory effects, dependent on where and how it is released or the types of receptors which are activated. The changes which are seen in the baroreceptor-denervated animal may be related to selective effects on sodium-responsive systems. Since baroreceptor denervation produces a generalized reduction in salt intake, the excitatory OT tone must be balanced by inhibitory input which predominates in this situation. It has been suggested that brain centers regulate salt intake and that these are influenced by barosensitive input. Thornton and colleagues reported that a mild hypotension (without hypovolemia) produced salt appetite which was not mediated by central angiotensin [21]. Likewise, a reduction in blood pressure coupled with volume depletion produced an increase in salt intake [22]. The neural pathways mediating these effects may be via baroreceptor afferent input to the brainstem and higher centers. This would be supported by our findings of changes in salt intake and responsiveness after baroreceptor denervation. These results further illustrate the usefulness of the antisense ODN approach in the study of physiological systems. They demonstrate that the acute application of an antisense to a neuropeptide has prominent effects on gustatory behavior which is dependent on neural organization. There is accumulating evidence that antisense deoxynucleotides have effects in vivo which are consistent with their functional roles [11]. Antisense to VP and OT administered centrally affect water balance, peptide secretion, peptide content, milk ejection, heart rate and
M. Morris et al. / Regulatory Peptides 59 (1995) 261-266
behavior [12,14,23]. The physiological actions are observed most often with stimulus-induced behaviors. For example, angiotensin receptor antisense had no effects on basal water intake while it blocked the dipsogenic and VP responses to angiotensin II [24,25]. Direct injection of OT antisense into the neurosecretory nuclei abolished stress-induced tachycardia [12] and suckling-induced milk ejection [14]. The VP response to osmotic stimulation was inhibited by the application of VP antisense to the perfused hypothalamic/posterior pituitary explant [26]. Likewise, in the present study, antisense effects were seen only in the surgically altered animals, a situation in which the hypothalamic/neurohypophyseal axis is activated [9]. The antisense ODN actions often occur rapidly (within hours) and are observed with a single injection [12,14,23,25]. While the mechanisms of antise,nse action are not completely understood, for neuroendocrine systems they may be related to changes in a rapidly replaced pool of receptors or peptides. There is evidence that CNS adminstration of VP or OT antisense produced rapid changes in peptide content and immunochemical staining in localized brain regions [12,23]. Although there is no information on the effect of antisense treatment on peptide biosynthesis, results show that newly synthesized peptides are rapidly transported and secreted [27]. In addition there could be interactions between the genetic machinery and cellular responsiveness as suggested by in vitro studies of antisense effects on osmotic-induced VP secretion [26]. In conclusion, antisense techniques were applied to the study of baroreceptor control of sodium chloride consumption. The results revealed that OT antisense directly injected into the PVN region had specific effects on intake in the baroreceptor-denervated rat, a model of salt sensitivity. The data provide further evidence that baroreceptor nerves are important in the regulation of sodium appetite perhaps via interactions with the hypothalamic OT axis.
References [1] Cort, J.H., Rudinger, J., Lichardus, B. and Hagemann, I. Effects of oxytocin antagonists on the saluresis accompanying carotid occlusion, Am. J. Physiol., 210 (1966) 162-168.
265
[2] Huang, W., Lee, S.-L. and Sjoquist, M., Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaC1, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 268 (1995) R634-R640. [3] Verbalis, J.G., Mangione, M.P. and Stricker, E.M., Oxytocin produces natriuresis in rats at physiological plasma concentrations, Endocrinology, 128 (1992) 1317-1322. [4] Blackburn, R.E., Stricker, E.M. and Verbalis, J.G., Central oxytocin meciates inhibition of sodium appetite by naloxone in hypovolemic rats, Neuroendocrinology, 56 (1992) 255263. [5] Blackburn, R.E., Demko, A.D., Hoffman, G.E., Stficker, E.M. and Verbalis, J.G., Central oxytocin inhibition of angiotensin-induced salt appetite in rats, Am. J. Physiol. (Regulatory Integrative Comp. Physiol), 263 (1992) R1347-R13531 [6] Alexander, N. and Morris, M., Effects of chronic sinoaortic denervation on central vasopressin and catecholamine systems, Am. J. Physiol., 255 (1988) R768-R773. [7] Alexander, N. and Morris, M., Increased plasma vasopressin in sinoaortic denervated rats, Neuroendocrinology, 42 (1986) 361-367. [8] Morris, M. and Alexander, N., Baroreceptor influences on oxytocin and vasopressin secretion, Hypertension, 13 (1989) 110-114. [9] Rocha, M.J.A., Callahan, M.F., Sundberg, D.K. and Morris, M., Sinoaortic denervation alters the molecular and endocrine responses to salt loading, Neuroendocrinology, 57 (1993) 729-737. [10] Thunhorst, R.L., Lewis, S.J. and Johnson, A.K., Effects of sinoaortic baroreceptor denervation on depletion-induced salt appetite, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 267 (1994) R1043-R1049. [11] Morris, M. and Lucion, A.B., Antisense oligonucleotides in the study of neuroendocrine systems, J. Neuroendocrinol., (1995) in press. [12] Morris, M., Li, P., Callahan, M.F and Lucion, A.B., Central oxytocin mediates stress-induced tachycardia, J. Neuroendocrinol., (1995) in press [13] Neumann, I. and Pittman, Q.J., An oxytocin (OXT) antisense oligonucleotide (AS) infused into the supraoptic nucleus (SON) rapidly alters the excitability of OXT neurons in lactating rats, Soc. Neurosci. Abstr., 40 (1994) 1538. [14] Neumann, I., Porter, D.W.F., Landgraf, R. and Pittman, Q.J., Rapid effect on suckling of an oxytocin antisense oligonucleotide administered into rat supraoptic nucleus, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 267 (1994) R852-R858. [15] Barton, K.W., Trapani, A.J., Gordon, F.J. and Brody, M.J., Baroreceptor denervation profoundly enhances cardiovascular responses to central angiotensin II, Am. J. Physiol., 257 (1989) H314-H323. [16] Stricker, E.M. and Verbalis, J.G., Hormones and behavior: the biology of thirst and sodium appetite, Am. Sci., 76 (1988) 261-267. [17] Manning, M. and Sawyer, W.H., Discovery, development, and some uses of vasopressin and oxytocin antagonists, J. Lab. Clin. Med., 114 (1989) 617-632.
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M. Morris et al. / Regulatory Peptides 59 (1995) 261-266
[18] Sonntag, M., Schalike, W. and Brattstrom, A., Cardiovascular effects of vasopressin micro-injections into the nucleus tractus solitarii in normotensive and hypertensive rats, J. Hypertens., 8 (1990) 417-421. [19] Gruber, K.A., Eskridge, S.L. and Callahan, M,F., The central vasopressin and/or oxytocin systems may be neural substrates for the regulation of sodium appetite, Soc. Neurosci. Abstr., 13 (1987) 1169. [20] Blackburn, R.E., Samson, W.K., Fulton, R.J., Stricker, E.M. and Verbalis, J.G., Central oxytocin inhibition of salt appetite in rats: evidence for differential sensing of plasma sodium and osmolality, Proc. Natl. Acad. Sci. USA, 90 (1993) 10380-10384. [21] Thornton, S.N., Sanchez, A. and Nicolaidis, S., An angiotensin-independent, hypotension-induced, sodium appetite in the rat, Physiol. Behav., 57 (.1995) 555-561. [22] Thunhorst, R.L. and Johnson, A.K., Regin-angiotensin, arterial blood pressure, and salt appetite in rats, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 366 (1994) R458R465.
[23] Skutella, T., Probst, J.C., Engelmann, M., Wotjak, C.T., Landgraf, R. and Jirikowski, G.F.. Vasopressin antisense oligonucleotide induces temporary diabetes insipidus in rats, J. Neuroendocdnol., 6 (1994) 121-125. [24] Sakai, R.R., He, P.F., Yang, X.D., Ma, L.Y., Guo, Y.F., Reilly, J.J., Moga, C.N. and Fluharty, S.F., Intracerebroventricular administration of AT1 receptor antisense oligonucleotides inhibits the behavioral actions of angiotensin II, J. Neurochem., 62 (1994) 2053-2056. [25] Meng, H., Wielbo, D., Gyurko, R. and Phillips, M.I., Antisense oligonucleotide to AT 1 receptor mRNA inhibits central angiotensin induced thirst and vasopressin, Regul. Pept., 54 (1994) 543-551. [26] Ludwig, M., Morris, M. and Sladek, C.D., Vasopressin antisense specifically inhibits osmotic stimulation of vasopressin, but not oxytocin release, Soc. Neurosci. Abstr., (1995) in press. [27] Gainer, H., Same, Y., Brownstein, M.J., Biosynthesis and axonal transport of neurohypophyseal proteins and peptides, J. Cell Biol., 73 (1977) 366-381.
Regulatory Peptides 59 (1995) 261-266
Oxytocin antisense reduces salt intake in the baroreceptor-denervated rat Mariana Morris *, Ping Li, Cindy Barrett, Michael F. Callahan Department of Physiology and Pharmacology and the Hypertension Center, Bowman Gray School of Medicine of WakeForest University, Winston-Salem, NC 27157, USA Received 26 May 1995; accepted 27 June 1995
Abstract Experiments were performed to evaluate the role of central oxytocin (OT) in the inhibition of salt intake produced by sinoaortic denervation (SAD). The effect of OT antisense treatment on 24 h intake of 2% NaCI in SAD and sham-operated (SO) rats was determined. PVN injection of unmodified antisense oligodeoxynucleotides (ODNs) to OT mRNA decreased intake of 2% NaCI in SAD, but not SO rats. Salt consumption was 22 + 4 ml after the injection of control ODN as compared to 8 ___4 ml after the OT antisense injection (P < 0.05). SAD animals also demonstrated an increased plasma OT response to salt loading, an elevation from 3.2 + 0.7 to 6.9 + 0.8 pg/ml. In contrast, salt ingestion produced no significant change in plasma OT in the SO group. The increased endocrine response in the SADs occurred even though salt intake was lower in this group. There were no group differences in plasma electrolytes or posterior pituitary OT content. Results show that OT antisense specifically inhibits salt intake in the denervated rat, suggesting that the central oxytocinergic axis stimulates sodium drive in this experimental model. Keywords: Hypothalamus; Paraventricular nucleus; Posterior pituitary; Dipsogenesis, cardiovascular
1. Introduction Salt intake and excretion are regulated by interactions between neural and endocrine factors. Oxytocin (OT), a peptide produced by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, is thought to be one of ~Ihe key mediators. Low doses of OT produce natriuresis while OT antagonists decrease urinary sodium excretion [1-3]. With regard to ingestive behavior, OT attenuates salt intake pro-
* Corresponding author. Fax: + 1 (910) 7168501.
duced by volume depletion while OT antagonists increase the salt intake induced by naloxone and angiotensin [4,5]. The baroreceptor-denervated animal provides an experimental model o f altered neurohypophyseal function. Denervation produces rapid changes in nuclear peptide and amine concentrations as well as increases in plasma vasopressin (VP) secretion [6,7]. There is also a heightened sensitivity to sodium stimulation as demonstrated by increased responses to acute and chronic salt loading [8,9]. Ingestion of a hypertonic NaC1 solution resulted in greater increases in both peptide secretion and peptide m R N A expression even though the SAD animals consumed
0167-0115/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 9 4 - 1
262
M. Morris et al. /Regulatory Peptides 59 (1995) 261-266
less salt than the controls [9]. Likewise, salt intake induced by volume depletion was greatly attenuated in denervated animals [10]. The data points to the idea that interruption of baroreceptor input to the brain produces a state of salt sensitivity. Recent studies have employed central antisense treatment as a means of investigating the involvement of peptide systems in physiological responses [11]. The advantage of antisense ODNs is that they can be targeted to nonhomologous regions of the mRNA, offering specificity which is not available with peptide receptor antagonists. Antisense ODNs to OT and VP were shown to have acute and specific effects on stimulus induced changes in heart rate, hormone secretion, neuronal firing and milk ejection [12-141. This new approach was applied to the study of baroreceptor modulation of salt consumption. In view of the hypothesized role of OT in the regulation of fluid/electrolyte balance, experiments were performed to evaluate the effect of PVN administration of OT antisense ODNs on sodium chloride intake in sham-operated and baroreceptor-denervated rats.
2. Materials and methods Male Sprague-Dawley rats (300-400 g) were housed individually on a 12/12 light/dark cycle with free access to water and laboratory chow. Under ketamine/xylazine anesthesia (10:50 mg/kg, i.m.) the baroreceptor nerves were surgically interrupted by stripping the carotid sinus and resecting the superior laryngeal nerve, a 1.0 mm section of the sympathetic chain posterior to the superior cervical ganglion and the aortic depressor nerves. The sham ol~eration (SO) included dissection of the neck muscles, but no manipulation of the nerves. The studies were conducted 3 - 4 weeks after surgical denervation. At this time post-surgery, the animals have regained weight and are in normal fluid balance [7]. The animals were anesthetized with ketamine/xylazine (10:50 mg/kg, i.m.) and bilateral guide cannulas were implanted dorsal to the PVN. The cannulas were formed from cutoff 25 ga stainless steel needles. The cannulas were secured to the skull with stainless steel screws and dental cement and closed with polyethylene (PE) closures.
The stereotaxic coordinates with a level skull were 1.8 mm posterior to bregma, 0.6 mm lateral from the sagittal sinus and 7.1 mm ventral to the skull. The intake of 2% NaC1 was measured following the PVN injection of OT antisense or mixed-base ODNs. ODNs were dissolved in sterile saline (0.9% NaC1) and slowly injected into the PVN at a dose of 2 ~ g / 0 . 3 /xl per side. The ODNs were injected using a 30 ga needle which extended 1 mm beyond the end of the cannula. The animals were given 2% NaC1 as the sole drinking fluid for a 24 h period after the injection. Unmodified ODNs were synthesized by the DNA Synthesis Core Laboratory (Comprehensive Cancer Center of Bowman Gray School of Medicine). An ODN (18-mer) complimentary to the region around the putative translation start codon of rat OT mRNA was used. The sequence of the ODN is 5'-CAG CAA GCG AGA CTG GGG-3' for OT and 5'-ATG-GACTGT-CGA-AGG-TI'C-3' for mixed base. This OT antisense has been previously used and found effective in modifying stress responses in the rat [12]. A second experiment was conducted to evaluate the effect of NaC1 intake on plasma and posterior pituitary OT content and plasma sodium and osmolality in SAD and SO rats. The rats were given 2% NaC1 or water to drink for an 18 h period. The animals were decapitated with collection of blood samples in heparinized tubes on ice. The posterior pituitaries were removed and frozen on dry ice and stored at -80°C. The tissue was sonicated in methanol/acetic acid (98%/0.02 N) and peptide content measured in the supernatant. Plasma samples were extracted using acetone precipitation and petroleum ether extraction. Plasma and tissue extracts were measured for OT by a sensitive and specific radioimmunoassay [9]. Data was analyzed using two-way ANOVA with the Newmann-Keuls post hoc test. Significance was taken as P < 0.05.
3. Results PVN injection of OT antisense ODN in the baroreceptor-denervated group produced a decrease in salt intake as compared to a mixed-base control (Fig. 1; P < 0.05). Mean intake of 2% NaC1 was 22
M. Morris et al. / Regulatory Peptides 59 (1995) 261-266 60 [ 50 I
263 Fluid Intake
Plasma Oxytocin
lmtisense Treatment EIMixed BaseBBOxytocin
40 ~.~ 40 -
- - * - -
E
30 ,T 10 10 0 0
Control
Control
SAD
SAD
Control
Fig. 2. Plasma oxytocin and fluid intake in SAD and SO male rats. Rats were given 2% NaC1 or water to drink for 18 h. n = 7 and 6 for the water and salt SAD groups, respectively, and n = 4 and 5 for the water and salt SO groups, respectively. * P < 0.05.
Fig. 1. Effect o f PVN injection of oxytocin antisense or control mixed-base ODNs on 2% NaCI intake (24 h) in sinoaortic denerrated (SAD) or sham-operated male rats. Unmodified ODNs (2.0 /zg/0.3 /~1) were injected bilaterally into the PVN o f conscious animals, n = 5 and 6 for the SAD rats given OT and mixed base, respectively, and n = 7 and 4 for the SO rats given OT and mixed base, respectively. * = P < 0.05.
osmolality, or posterior pituitary OT content between the groups (Table 1). There was a significant effect of treatment on plasma sodium, but no differences between the groups, indicating that the animals received a similar sodium challenge (Table 1).
vs. 8 m l / 2 4 h (mixed base vs. OT antisense). This was in contrast to the results in the SO group in which there was no difference in salt intake in response to the ODN treatment (29 vs. 38 m l / 2 4 h, mixed base vs. OT antisense). A second experiment was performed to evaluate the endocrine, plasma electrolyte and intake responses to salt loading in the SAD and SO groups. SAD rats consumed less NaC1 yet they demonstrated a greater increase in plasma OT than the SO animals (Fig. 2). The mean intake of 2% NaCI in the SAD animals was 18 ml a,; compared to 35 ml in the SO group. Plasma OT was significantly higher in the SAD animals which received salt as compared to water, 3.2 vs. 6.9 pg/ml. Salt intake produced no change in plasma OT in the SO group. There were no significant differences in body weight, plasma
4. Discussion Experiments were performed to evaluate the role of central OT in the repressed sodium chloride intake observed after baroreceptor denervation [9,10]. The results demonstrate that PVN injection of OT antisense ODN produced a decrease in salt intake in the SAD. Since denervated animals show increased OT responses to sodium stimulation, the results suggest that central OT systems are inhibitory to salt intake in this model. While the aortic baroreceptor system is best known for its role in the regulation of blood pressure/heart
Table 1 Effect of 24 h salt loading in SAD and sham-operated rats Group
Treatment
Body weight
(g) Sham Sham SAD SAD
Water Salt Water Salt
354.8 362.5 365.8 341.0
+ + + +
15.5 13.4 6.6 10.7
Data presented as mean + S.E.M.; * P < 0.05, effect of treatment.
SAD
Pituitary
Plasma sodium * (mEq/l)
osmolality (mosm/kg)
oxytocin (ng/PP)
145.9 151.7 147.8 150.6
302.6 312.8 307.0 311.8
45.9 28.4 49.7 30.9
-I- 3.7 -1- 1.2 + 1.3 + 0.8
+ 6.8 + 3.2 + 2.8 -I- 2.7
+ 6.0 -I- 6.3 + 14.7 + 2.7
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M. Morris et al. /Regulatory Peptides 59 (1995) 261-266
rate interactions, it is also critical in the control of fluid and endocrine balance. The hypothalamic PVN and SON receive input from baroreceptor nerves, serving to relay information on volume and blood pressure status. Interruption of afferent input via surgical interruption of the baroreceptor nerves produces prominent effects on water/electrolyte and endocrine balance. SAD causes acute adipsia, aphagia, hypertension, increased pressor responses to central stimulation and increases in plasma vasopressin [7,15]. Denervation is also associated with salt sensitivity as demonstrated by the increased VP and OT responses to acute and chronic salt loading [8,9]. The chronic stimulus increased both peptide secretion and peptide mRNA expression in the face of reduced saline intake [9]. There is evidence that central OT systems are inhibitory to salt intake [16]. Thus, the driving hypothesis for the current study was that increased OT responses in the SAD were responsible for the reduction in salt intake in this model. However, the present results are at variance with this idea since PVN injection of OT antisense significantly reduced, rather than increased, salt intake in the SAD. There was no significant difference between the effects of the mixed base and OT ODNs in the control group, further demonstrating the specificity of the antisense effect. Additionally, the denervated animals showed evidence of increased endocrine responses with salt consumption producing an increase in plasma OT even though intake was lower in SADs than in the controls. Studies which have investigated the role of OT in the control of salt appetite have relied on the use of OT peptides or peptide antagonists. Using various experimental models, results show that OT inhibits salt consumption while the OT antagonist increases intake. For example, volume depletion induced by polyethylene glycol treatment produced an increase in salt appetite which was attenuated by OT [4]. OT antagonists produced an increase in sodium consumption in animals treated with naloxone, an opiate antagonist which increases OT secretion [4]. A point which should be considered in the interpretation of these studies is that they employ large doses of peptide or antagonist (/xg amounts). The specificity of the antagonists, particularly at high doses, has been questioned [17]. The OT antagonist has effects
on VP and vice versa for the VP antagonist. Furthermore, centrally injected peptides have effects which are dependent on the dosage range [18]. Indeed, OT was found to stimulate rather than inhibit sodium intake when it was administered chronically at a low dose [19]. Another explanation for the difference in the findings may be related to the mode of stimulation. A recent study by Blackburn and colleagues demonstrated that OT antagonists had no effect on salt intake induced by hypertonic saline [20]. The antagonist was only effective in modulating the response to hypertonic mannitol. The investigators hypothesized that there are basic differences between osmotic and sodium sensors in terms of responses and CNS interactions. It is possible that OT has both stimulatory and inhibitory effects, dependent on where and how it is released or the types of receptors which are activated. The changes which are seen in the baroreceptor-denervated animal may be related to selective effects on sodium-responsive systems. Since baroreceptor denervation produces a generalized reduction in salt intake, the excitatory OT tone must be balanced by inhibitory input which predominates in this situation. It has been suggested that brain centers regulate salt intake and that these are influenced by barosensitive input. Thornton and colleagues reported that a mild hypotension (without hypovolemia) produced salt appetite which was not mediated by central angiotensin [21]. Likewise, a reduction in blood pressure coupled with volume depletion produced an increase in salt intake [22]. The neural pathways mediating these effects may be via baroreceptor afferent input to the brainstem and higher centers. This would be supported by our findings of changes in salt intake and responsiveness after baroreceptor denervation. These results further illustrate the usefulness of the antisense ODN approach in the study of physiological systems. They demonstrate that the acute application of an antisense to a neuropeptide has prominent effects on gustatory behavior which is dependent on neural organization. There is accumulating evidence that antisense deoxynucleotides have effects in vivo which are consistent with their functional roles [11]. Antisense to VP and OT administered centrally affect water balance, peptide secretion, peptide content, milk ejection, heart rate and
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behavior [12,14,23]. The physiological actions are observed most often with stimulus-induced behaviors. For example, angiotensin receptor antisense had no effects on basal water intake while it blocked the dipsogenic and VP responses to angiotensin II [24,25]. Direct injection of OT antisense into the neurosecretory nuclei abolished stress-induced tachycardia [12] and suckling-induced milk ejection [14]. The VP response to osmotic stimulation was inhibited by the application of VP antisense to the perfused hypothalamic/posterior pituitary explant [26]. Likewise, in the present study, antisense effects were seen only in the surgically altered animals, a situation in which the hypothalamic/neurohypophyseal axis is activated [9]. The antisense ODN actions often occur rapidly (within hours) and are observed with a single injection [12,14,23,25]. While the mechanisms of antise,nse action are not completely understood, for neuroendocrine systems they may be related to changes in a rapidly replaced pool of receptors or peptides. There is evidence that CNS adminstration of VP or OT antisense produced rapid changes in peptide content and immunochemical staining in localized brain regions [12,23]. Although there is no information on the effect of antisense treatment on peptide biosynthesis, results show that newly synthesized peptides are rapidly transported and secreted [27]. In addition there could be interactions between the genetic machinery and cellular responsiveness as suggested by in vitro studies of antisense effects on osmotic-induced VP secretion [26]. In conclusion, antisense techniques were applied to the study of baroreceptor control of sodium chloride consumption. The results revealed that OT antisense directly injected into the PVN region had specific effects on intake in the baroreceptor-denervated rat, a model of salt sensitivity. The data provide further evidence that baroreceptor nerves are important in the regulation of sodium appetite perhaps via interactions with the hypothalamic OT axis.
References [1] Cort, J.H., Rudinger, J., Lichardus, B. and Hagemann, I. Effects of oxytocin antagonists on the saluresis accompanying carotid occlusion, Am. J. Physiol., 210 (1966) 162-168.
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[2] Huang, W., Lee, S.-L. and Sjoquist, M., Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaC1, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 268 (1995) R634-R640. [3] Verbalis, J.G., Mangione, M.P. and Stricker, E.M., Oxytocin produces natriuresis in rats at physiological plasma concentrations, Endocrinology, 128 (1992) 1317-1322. [4] Blackburn, R.E., Stricker, E.M. and Verbalis, J.G., Central oxytocin meciates inhibition of sodium appetite by naloxone in hypovolemic rats, Neuroendocrinology, 56 (1992) 255263. [5] Blackburn, R.E., Demko, A.D., Hoffman, G.E., Stficker, E.M. and Verbalis, J.G., Central oxytocin inhibition of angiotensin-induced salt appetite in rats, Am. J. Physiol. (Regulatory Integrative Comp. Physiol), 263 (1992) R1347-R13531 [6] Alexander, N. and Morris, M., Effects of chronic sinoaortic denervation on central vasopressin and catecholamine systems, Am. J. Physiol., 255 (1988) R768-R773. [7] Alexander, N. and Morris, M., Increased plasma vasopressin in sinoaortic denervated rats, Neuroendocrinology, 42 (1986) 361-367. [8] Morris, M. and Alexander, N., Baroreceptor influences on oxytocin and vasopressin secretion, Hypertension, 13 (1989) 110-114. [9] Rocha, M.J.A., Callahan, M.F., Sundberg, D.K. and Morris, M., Sinoaortic denervation alters the molecular and endocrine responses to salt loading, Neuroendocrinology, 57 (1993) 729-737. [10] Thunhorst, R.L., Lewis, S.J. and Johnson, A.K., Effects of sinoaortic baroreceptor denervation on depletion-induced salt appetite, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 267 (1994) R1043-R1049. [11] Morris, M. and Lucion, A.B., Antisense oligonucleotides in the study of neuroendocrine systems, J. Neuroendocrinol., (1995) in press. [12] Morris, M., Li, P., Callahan, M.F and Lucion, A.B., Central oxytocin mediates stress-induced tachycardia, J. Neuroendocrinol., (1995) in press [13] Neumann, I. and Pittman, Q.J., An oxytocin (OXT) antisense oligonucleotide (AS) infused into the supraoptic nucleus (SON) rapidly alters the excitability of OXT neurons in lactating rats, Soc. Neurosci. Abstr., 40 (1994) 1538. [14] Neumann, I., Porter, D.W.F., Landgraf, R. and Pittman, Q.J., Rapid effect on suckling of an oxytocin antisense oligonucleotide administered into rat supraoptic nucleus, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 267 (1994) R852-R858. [15] Barton, K.W., Trapani, A.J., Gordon, F.J. and Brody, M.J., Baroreceptor denervation profoundly enhances cardiovascular responses to central angiotensin II, Am. J. Physiol., 257 (1989) H314-H323. [16] Stricker, E.M. and Verbalis, J.G., Hormones and behavior: the biology of thirst and sodium appetite, Am. Sci., 76 (1988) 261-267. [17] Manning, M. and Sawyer, W.H., Discovery, development, and some uses of vasopressin and oxytocin antagonists, J. Lab. Clin. Med., 114 (1989) 617-632.
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[18] Sonntag, M., Schalike, W. and Brattstrom, A., Cardiovascular effects of vasopressin micro-injections into the nucleus tractus solitarii in normotensive and hypertensive rats, J. Hypertens., 8 (1990) 417-421. [19] Gruber, K.A., Eskridge, S.L. and Callahan, M,F., The central vasopressin and/or oxytocin systems may be neural substrates for the regulation of sodium appetite, Soc. Neurosci. Abstr., 13 (1987) 1169. [20] Blackburn, R.E., Samson, W.K., Fulton, R.J., Stricker, E.M. and Verbalis, J.G., Central oxytocin inhibition of salt appetite in rats: evidence for differential sensing of plasma sodium and osmolality, Proc. Natl. Acad. Sci. USA, 90 (1993) 10380-10384. [21] Thornton, S.N., Sanchez, A. and Nicolaidis, S., An angiotensin-independent, hypotension-induced, sodium appetite in the rat, Physiol. Behav., 57 (.1995) 555-561. [22] Thunhorst, R.L. and Johnson, A.K., Regin-angiotensin, arterial blood pressure, and salt appetite in rats, Am. J. Physiol. (Regulatory Integrative Comp. Physiol.), 366 (1994) R458R465.
[23] Skutella, T., Probst, J.C., Engelmann, M., Wotjak, C.T., Landgraf, R. and Jirikowski, G.F.. Vasopressin antisense oligonucleotide induces temporary diabetes insipidus in rats, J. Neuroendocdnol., 6 (1994) 121-125. [24] Sakai, R.R., He, P.F., Yang, X.D., Ma, L.Y., Guo, Y.F., Reilly, J.J., Moga, C.N. and Fluharty, S.F., Intracerebroventricular administration of AT1 receptor antisense oligonucleotides inhibits the behavioral actions of angiotensin II, J. Neurochem., 62 (1994) 2053-2056. [25] Meng, H., Wielbo, D., Gyurko, R. and Phillips, M.I., Antisense oligonucleotide to AT 1 receptor mRNA inhibits central angiotensin induced thirst and vasopressin, Regul. Pept., 54 (1994) 543-551. [26] Ludwig, M., Morris, M. and Sladek, C.D., Vasopressin antisense specifically inhibits osmotic stimulation of vasopressin, but not oxytocin release, Soc. Neurosci. Abstr., (1995) in press. [27] Gainer, H., Same, Y., Brownstein, M.J., Biosynthesis and axonal transport of neurohypophyseal proteins and peptides, J. Cell Biol., 73 (1977) 366-381.