INTRODUCTION

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CENTRAL OSMOREGULATORY processes comprise a complex pattern of behavioral and other physiological effects such as water intake, changes in mean arterial blood pressure, renal sodium and water excretion or retention, and hormonal changes (3). Vasopressin and oxytocin, major effector peptides in the control of fluid and salt homeostasis, are synthesized in neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). The hypothalamic structures are interconnected via neuronal pathways with forebrain areas, such as organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), and median preoptic area (MnPO), regions that are known to contain osmosensitive sites (3, 24). Stimulation of these osmoreceptors results in the neural activation of PVN and SON, leading to the release of vasopressin and oxytocin from the pituitary gland into the circulation.

A connection between central osmoreceptor-mediated and angiotensinergic mechanisms has been postulated, since angiotensin immunoreactivity and angiotensin receptors have been found in the above-mentioned brain areas (13, 21). The osmoregulatory responses following centrally applied hyperosmolar saline are very similar to those following intracerebroventricularly injected angiotensin II. Furthermore, increase in mean arterial blood pressure, water intake, natriuresis, and vasopressin release have been shown to be partially mediated via the angiotensin AT₁ receptor (13, 33, 34, 39). The angiotensinergic system is believed to interact with other neurotransmitter systems in the maintenance of body fluid homeostasis, blood pressure control, and pituitary hormone secretion. In the regulation of these processes, central acetylcholine seems to play an important role. An intensive cholinergic innervation and choline binding sites have been demonstrated in forebrain structures (2, 35). Stimulation of periventricular cholinoceptors affects body fluid and cardiovascular homeostasis similarly to hyperosmolar saline. Blockade of muscarinic receptors with atropine has been demonstrated to antagonize osmotically induced vasopressin secretion, natriuresis, and drinking (14, 19, 27). Together, these data indicate the involvement of angiotensinergic as well as cholinergic pathways in central osmoregulation.

Immunohistochemical mapping of the inducible transcription factors (ITF), proteins encoded by specific immediate-early genes, has often been used to determine the spatial and temporal distribution of neuronal activation. After peripherally applied hyperosmolar saline, an expression of c-Fos has been observed in OVLT, MnPO, SFO, and the hypothalamic PVN and SON as well as in the brain stem areas, nucleus of the solitary tract (NTS) and area postrema (AP) (6, 12, 17, 29), which are also involved in cardiovascular and osmoregulatory processes. In addition, the expression of c-Jun, another leucine zipper protein, has been reported in the MnPO, PVN, and SON following intraperitoneal injections of hyperosmolar saline (7). In contrast to the leucine zipper proteins Fos and Jun, Krox-24 (also known as NGFI-A, Egr-1, Tis 8, Zif 268, or Zenk) belongs to another group of ITF, the zinc finger proteins, and its expression has not been determined so far. The ITF have been postulated to act as third messengers in the signal transduction cascade from receptor via second messengers to the nucleus and, thereby, to be involved in the transcriptional regulation of genes. Individual classes of ITF might be induced by various membrane receptor systems, and a distinct expression pattern of ITF has been implicated in the differential regulation of gene transcription. Therefore, the expression and distribution pattern of ITF such as c-Fos, c-Jun, JunB, JunD, and Krox-24 in the brain following intracerebroventricular injections of hyperosmolar saline in conscious rats has been determined, and the coexpression with vasopressin and oxytocin, which would imply the corresponding genes as putative target genes of the ITF, has been investigated. To test the hypothesis that ITF expression is mediated via different membrane receptor systems according to osmotically induced vasopressin release, water intake, and natriuresis, we investigated the involvement of angiotensinergic and/or muscarinergic pathways by examining the effects of central pretreatment with the angiotensin AT₁ receptor antagonist losartan and the muscarinic receptor antagonist atropine in the expression of ITF following intracerebroventricular injection of hyperosmolar saline.

	MATERIALS AND METHODS

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Animals. Male Wistar rats weighing 250-300 g (Dr. Karl Thomae, Biberach, Germany) were kept under controlled conditions with respect to temperature, humidity, and light periodicity (12:12-h light-dark cycle). The animals were fed a standard diet (Altromin maintenance diet 1320 containing 0.2% sodium and 0.3% chloride; Altromin, Lage/Lippe, Germany) and were allowed free access to water.

Surgical methods. For intracerebroventricular injections, chronic cannulas (PP20, Portex) were implanted into the right lateral ventricle of the brain under chloral hydrate anesthesia (400 mg/kg body weight ip) using the coordinates 0.6 mm caudal and 1.3 mm lateral to bregma and 5.0 mm below the skull surface. The cannulas were fixed in position by dental cement and anchored to the skull by three stainless steel screws. After surgery, rats were housed individually for a 1-wk postoperative period and handled daily to minimize unspecific stress-dependent expression of ITF until the day of experiment.

Experimental protocol. The experiments were performed in conscious animals between 9:00 AM and 1:00 PM. All injections of hyperosmolar saline were given intracerebroventricularly in a volume of 1 µl flushed with 4 µl physiological saline (0.15 M NaCl). Rats were randomly divided into five groups. In the first set of experiments, rats of group 1 were injected once with either 0.15 (n = 3), 0.20 (n = 5), 0.30 (n = 4), or 0.60 M NaCl (n = 5). Animals of all other groups received two intracerebroventricular injections with an interval of 5 min between treatments. Group 2 was pretreated with 0.15 M NaCl followed by a second injection of either 0.15 (n = 3), 0.20 (n = 4), 0.30 (n = 4), or 0.60 M saline (n = 4). Group 3 received an injection of the AT₁ receptor antagonist losartan (10 nmol) followed by an injection of either 0.15 (n = 3), 0.20 (n = 4), 0.30 (n = 5), or 0.60 M NaCl (n = 5). Group 4 was pretreated with 20 nmol losartan and subsequently injected with either 0.15 (n = 2) or 0.60 M NaCl (n = 4). Group 5 was pretreated with the muscarinic receptor antagonist atropine (15 nmol) followed by an injection of either 0.15 (n = 3), 0.20 (n = 4), 0.30 (n = 4), or 0.60 M NaCl (n = 4). Doses of hypertonic saline (0.20, 0.30, and 0.60 M NaCl), losartan (10 nmol), and atropine (15 nmol) were chosen in accordance with previous experiments (13, 14, 33, 34).

Ninety minutes after the last injection, rats were deeply anesthetized with chloral hydrate (400 mg/kg body weight) and transcardially perfused with 200 ml phosphate-buffered saline followed by 200 ml of 4% paraformaldehyde solution in phosphate buffer. The brains were removed, postfixed in the same fixative overnight at 4°C, and, subsequently, cryoprotected with 30% sucrose solution at 4°C.

Immunocytochemistry. Coronal cryostat sections (40 µm) were processed free floating for immunocytochemistry. The sections were incubated with polyclonal antibodies against c-Fos (1:8,000), c-Jun (1:30,000), JunB (1:4,000), JunD (1:8,000), and Krox-24 (1:6,000) for 72 h at 4°C. Immunoreactivity was visualized by the biotin-avidin complex peroxidase reaction (Vectastain, Vector Laboratories) as described previously (11). Generation and specificity of the primary antibodies have been demonstrated by immunoprecipitation and preabsorption of the antisera with different antigens (11, 16).

For double labeling, c-Fos, c-Jun, and Krox-24 immunocytochemistry preceded the vasopressin (1:4,000) and oxytocin (1:2,000) immunoreactivity. Vasopressin and oxytocin immunoreactivity were visualized by the avidin-biotin complex peroxidase reaction using Vector SG or VIP Vector (Vector Laboratories) as chromogen.

Quantification and statistical analysis. To quantify immunoreactivity in AP, NTS, OVLT, MnPO, SFO, PVN (magno- and parvocellular part), and SON, photographs of sections at the same level of each brain region were taken. The level of each region was identified according to the atlas of rat brain of Paxinos and Watson (30). For quantification, the number of stained neurons of each brain structure was counted by two observers in a blinded manner. The AP, NTS, MnPO, PVN, and SON was counted on photographs of two sections from each rat brain, the OVLT on photographs of one section. No attempt was made to quantify the intensity of the staining. The mean number of labeled neurons ± SD was calculated for each region and group. Statistical analysis was performed using ANOVA followed by Student-Newman-Keuls test (SPSS) comparing changes in ITF expression in each brain region subsequent to different concentrations of hyperosmolar saline (group 1: comparison across treatment conditions) or comparing the effects of pretreatment with angiotensin and muscarinic receptor antagonists on hyperosmolar saline-induced ITF expression (group 2 vs. groups 3, 4, and 5). Results were considered to be significantly different when P < 0.05.

Drugs. Losartan was a generous gift from R. Smith, DuPont-Merck Pharmaceutical, Wilmington, DE. Atropine and sodium chloride were purchased from Sigma-Aldrich Chemie, Deisenhofen, Germany. Sodium chloride and losartan were dissolved in 0.9% saline and stored at 20°C until use. Atropine was dissolved in 0.9% NaCl on the day of the experiment. The antiserum against vasopressin was obtained from Chemicon International, Temecula, CA, and the antiserum against oxytocin from Peninsula Laboratories, Belmont, CA.

	RESULTS

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Basal staining. All antisera produced basal stainings in brain areas such as cortex and striatum; there was a substantial basal staining of Krox-24 in cortex and caudate putamen. Scattered immunoreactivity of c-Fos, c-Jun, and Krox-24 was also observed in the hippocampus, the nucleus of the amygdala, and the suprachiasmatic nucleus. In AP, OVLT, MnPO, SFO, PVN, and SON, no basal expression of the ITF, c-Fos, JunB, and Krox-24 was observed, whereas in the NTS there was basal expression of c-Jun. JunD showed a substantial basal staining in the investigated areas that was not altered on treatment with hyperosmolar saline.

Effect of intracerebroventricular injections of hyperosmolar (0.20, 0.30, and 0.60 M NaCl) saline on ITF expression in the brain. Whereas the ITF JunB and JunD were not induced following periventricular stimulation with hyperosmolar saline, the expression of the ITF c-Fos, c-Jun, and Krox-24 was induced in five distinct forebrain areas evidenced by staining of the proteins in neuronal nuclei. In the OVLT, c-Fos and c-Jun were expressed in a few neurons after intracerebroventricular injections of hyperosmolar saline. This expression was not dose dependent. After 0.60 M NaCl, Krox-24 expression was observed in the OVLT (Table 1). In the MnPO, c-Fos and c-Jun were dose-dependently expressed after intracerebroventricular injections of 0.20, 0.30, and 0.60 M NaCl. Expression of Krox-24 was dose-dependently induced after 0.30 and 0.60 M NaCl icv (Table 1). In the SFO, hyperosmolar saline (intracerebroventricularly) induced a dose-dependent expression of c-Fos and c-Jun (Fig. 1, Table 1). Krox-24 was not expressed in the SFO (Table 1). In the PVN, the expression of c-Fos and c-Jun was dose-dependently induced after intracerebroventricular saline injections. Krox-24 was dose-dependently induced following 0.30 and 0.60 M saline (Table 1). In the SON, treatment with intracerebroventricular hyperosmolar saline induced a dose-dependent expression of c-Fos and c-Jun. Krox-24 was dose-dependently induced after 0.30 and 0.60 M NaCl (Table 1). In the brain stem areas, AP and NTS, there was no expression of c-Fos, c-Jun, or Krox-24 after intracerebroventricular injections of hyperosmolar saline.

View larger version (114K): [in this window] [in a new window]   Fig. 1.   Representative photomicrographs showing dose-dependent expression of c-Jun after intracerebroventricular injections of NaCl in coronal sections of subfornical organ (SFO): 0.15 M NaCl as control (A), 0.20 M NaCl (B), 0.30 M NaCl (C), or 0.60 M NaCl (D).

Effect of pretreatment with the AT₁ receptor antagonist losartan on hyperosmolar saline-induced ITF expression in the brain. In all investigated areas, the AT₁ receptor antagonist losartan (10 nmol icv) reduced the expression of c-Fos and c-Jun induced by 0.20 and 0.30 M NaCl icv. After 0.60 M NaCl, the expression of c-Fos and c-Jun was not significantly inhibited by either 10 or 20 nmol losartan icv. Krox-24 expression after 0.30 and 0.60 M NaCl was not attenuated by losartan (10 and 20 nmol icv) in all brain regions investigated. In control experiments, losartan applied intracerebroventricularly at the above doses before 0.15 M NaCl did not induce any ITF expression (Figs. 2 and 3).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Diagram of effect of pretreatment with AT1 receptor antagonist losartan (10 and 20 nmol icv) on the hyperosmolar saline (0.20, 0.30, and 0.60 M NaCl icv)-induced inducible transcription factor (ITF) expression in median preoptic area (MnPO; A), SFO (B), paraventricular nucleus of the hypothalamus (PVN; C), and supraoptic nucleus (SON; D). Data were given as average number of labeled neurons (mean ± SD) * P < 0.05 vs. controls (0.15 M NaCl + 0.20 M NaCl or 0.15 M NaCl + 0.30 M NaCl).

View larger version (73K): [in this window] [in a new window]   Fig. 3.   Representative photomicrographs of c-Fos immunostaining in coronal sections of SON in rats showing effect of pretreatment with AT1 receptor antagonist losartan (intracerebroventricular) on hyperosmolar saline (intracerebroventricular)-induced c-Fos expression. Rats were pretreated with 0.15 M NaCl + 0.15 M NaCl (A), losartan (10 nmol) + 0.15 M NaCl (B), 0.15 M NaCl + 0.20 M NaCl (C), losartan (10 nmol) + 0.20 M NaCl (D), 0.15 M NaCl + 0.30 M NaCl (E), losartan (10 nmol) + 0.30 M NaCl (F), 0.15 M NaCl + 0.60 M NaCl (G), losartan (10 nmol) + 0.60 M NaCl (H), losartan (20 nmol) + 0.60 M NaCl (I), or losartan (20 nmol) + 0.15 M NaCl (J).

Effect of the muscarinic receptor antagonist atropine on hyperosmolar saline-induced ITF expression in the brain. Pretreatment with atropine (15 nmol icv) markedly reduced the c-Fos, c-Jun, and Krox-24 expression in response to intracerebroventricular 0.30 and 0.60 M saline in the MnPO, PVN, and SON but did not significantly affect the 0.20 M NaCl-induced ITF expression. In the SFO, atropine did not alter the expression of c-Fos and c-Jun in response to 0.30 and 0.60 M NaCl. In control experiments, atropine injected intracerebroventricularly before 0.15 M NaCl had no effect on ITF expression (Figs. 4 and 5).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Diagram of effect of pretreatment with muscarinic receptor antagonist atropine (15 nmol icv) on the hyperosmolar saline (0.20, 0.30, and 0.60 M NaCl icv)-induced expression of c-Fos, c-Jun, and Krox-24 in MnPO (A), SFO (B), PVN (C), and SON (D). Data were given as average number of labeled neurons (mean ± SD). * P < 0.05 vs. controls (0.15 M NaCl + 0.30 M NaCl or 0.15 M NaCl + 0.60 M NaCl).

View larger version (98K): [in this window] [in a new window]   Fig. 5.   Representative photomicrographs of Krox-24 immunostaining in coronal sections of PVN in rats showing effect of pretreatment with muscarinic receptor antagonist atropine (15 nmol icv) on hyperosmolar saline (intracerebroventricular)-induced Krox-24 expression. Rats were pretreated with 0.15 M NaCl + 0.15 M NaCl (A), atropine + 0.15 M NaCl (B), 0.15 M NaCl + 0.20 M NaCl (C), atropine + 0.20 M NaCl (D), 0.15 M NaCl + 0.30 M NaCl (E), atropine + 0.30 M NaCl (F), 0.15 M NaCl + 0.60 M NaCl (G), or atropine + 0.60 M NaCl (H).

Colocalization of c-Fos and c-Jun with vasopressin and oxytocin in magnocellular neurons of the PVN and SON. Double labeling was designed to show brown nuclear staining for ITF immunoreactivity and blue or red cytoplasmatic staining for the immunoreactivity of vasopressin and oxytocin, respectively. After intracerebroventricular injections of 0.60 M NaCl, immunohistochemical double staining revealed coexpression of many magnocellular c-Fos- and c-Jun-labeled neurons of the PVN and SON with vasopressin and oxytocin, respectively. Krox-24 was colocalized with oxytocin in magnocellular neurons of the PVN and SON, but not vasopressin, after 0.60 M NaCl (Fig. 6, A-C).

View larger version (61K): [in this window] [in a new window]   Fig. 6.   Photomicrographs showing colocalization (arrows) of nuclear c-Fos, c-Jun, and Krox-24 with cytoplasmatic vasopressin or oxytocin in magnocellular neurons of the PVN and SON following intracerebroventricular injection of 0.6 M NaCl. A: coexpression of c-Fos with vasopressin in magnocellular neurons of PVN. B: coexpression of c-Jun with vasopressin in magnocellular neurons of PVN. C: coexpression of Krox-24 with oxytocin in magnocellular neurons of SON.

	DISCUSSION

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This is the first study describing the expression and distribution pattern of five different ITF, c-Fos, c-Jun, JunB, JunD, and Krox-24, in the brain following central osmotic stimulation with hyperosmolar saline in conscious rats. Furthermore, this study demonstrates the involvement of central angiotensinergic and muscarinergic pathways in the osmotically induced expression of these ITF.

For osmotic stimulation, saline injections were given intracerebroventricularly to bypass peripherally induced osmoregulatory responses. In addition, stress, pain, and changes in blood pressure, reactions related to the intraperitoneal injection procedure of hyperosmolar saline (17), could thus be avoided. Osmotic challenge increases glucose utilization, an index of functional activity, in the same brain regions in which ITF expression was observed: OVLT, MnPO, SFO, PVN, and SON (15). Thus expression of ITF can be regarded as a marker of neuronal activation and allows us to gain further information about the pathways that participate in central osmoregulation. Counting the number of immunopositive cells does not present an absolute quantitative measurement of the protein levels: the amount of protein within a neuron does not necessarily correspond to the intensity of the staining, and, second, the absence of immunoreactivity is not tantamount to a lack of ITF expression, since the amount of the ITF may be below the detection limit. However, changes in ITF expression levels can be judged as relative alterations compared with controls. Despite its shortcomings, the counting method has become a well-documented tool to (semi-) quantify ITF expression (12, 18, 36).

Expression pattern of ITF under nonstimulated and stimulated conditions. Whereas all investigated ITF showed basal expression, only the expression of c-Fos, c-Jun, and Krox-24, but not JunB and JunD, was induced following periventricular stimulation with hyperosmolar saline, indicating the ability of neurons to react to external stimuli with alteration of the gene expression leading to a distinct expression pattern. Consistent with the data of this study, the analysis of the activator protein-1 (AP-1) complex, a dimer of Fos and Jun proteins, following peripherally applied hyperosmolar saline revealed an absence of JunB as well as a lack of JunD expression (41). The absence of JunD induction in the present study is probably due to a permanent activation of the JunD promoter, resulting in a high constitutive expression throughout the brain. JunD has been postulated as a housekeeping protein controlling the expression of genes involved in the maintenance of basal physiological processes (4). Ongoing physiological processes are probably related to the expression of all investigated ITF under nonstimulated conditions. Basal expression of Krox-24 exceeded that of c-Fos, suggesting a differential transcriptional regulation of these genes under nonstimulated conditions (10). We also propose a differential regulation of the leucine zipper and zinc finger proteins following stimulation, since Krox-24 was only expressed after increasing concentrations of saline (0.30 and 0.60 M NaCl). Moreover, the Krox-24 expression was only sensitive to muscarinic and not angiotensin receptor blockade in the present study. Our observations concerning the distinct expression pattern of the ITF belonging to the leucine zipper and zinc finger protein families as well as their individual recruitment by angiotensin and muscarinic receptors suggest a highly differentiated regulation of target genes following central osmotic stimulation.

Distribution pattern of ITF under nonstimulated and stimulated conditions. Under nonstimulated conditions, immunoreactivity of the investigated ITF was observed in brain areas such as cortex, striatum, caudate putamen, hippocampus, amygdala, and nucleus suprachiasmaticus. Consistent with the assumption that basal expression contributes to inputs from, e.g., autonomic nervous system or intracerebral networks, the basal expression of the ITF occurred in brain regions involved in the processing of somato- and viscerosensory or collateral and integrative information (10).

Periventricular osmotic stimulation evoked a discrete pattern of ITF expression in distinct brain structures, the OVLT, the MnPO, the SFO, the PVN, and the SON, regions known to be involved in osmoregulation. ITF expression might be induced directly in these brain regions. Electrophysiological experiments indicate that these brain areas contain neurons that are intrinsically sensitive to hyperosmolar solutions (3, 12). On the other hand, since the location of osmo- and/or sodium-sensitive sites has been proposed in brain areas lacking a blood-brain barrier (3, 24), a hierarchy of ITF activation through neuronal pathways originating in the lamina terminalis and terminating in the PVN and SON seems also possible. This assumption is supported by the fact that an extensive neural network interconnects the OVLT and SFO with the PVN and SON directly or indirectly via the MnPO (3, 24). Furthermore, lesions of OVLT, MnPO, and SFO have been shown to interfere with osmoregulatory responses (3, 12, 40). Local osmotic stimulation of the OVLT or MnPO induces an excitation of magnocellular neurosecretory cells within the PVN and SON, resulting in the release of vasopressin and oxytocin (3).

The absence of c-Fos expression in the NTS and AP in the present study does not contradict the presence of c-Fos expression in response to peripherally administered hyperosmolar saline. Hemodynamic and osmotic information is transduced from peripheral volume-, baro-, and osmoreceptors and/or sodium sensors via the NTS to the MnPO. Even if signaling from these receptors could be excluded, peripherally applied hyperosmolar saline induces c-Fos expression in brain stem neurons (12). In electrophysiological studies, an increase in electrical activity in NTS neurons following peripherally applied hyperosmolar saline has been demonstrated, even in the absence of inputs from circumventricular osmoreceptors and/or sodium sensors of the forebrain (12). Thus autonomous osmo- and/or sodium-sensitive sites in the medulla independent of osmo- and/or sodium-sensitive sites in the forebrain have been suggested (12). Therefore, the ITF expression in the NTS and the adjacent AP is probably dependent on the peripheral application of hyperosmolar saline and is not induced by intracerebroventricular injection of osmotic stimuli.

Despite differences in brain stem areas, the ITF showed strikingly congruent distribution patterns following central and peripheral application of hyperosmolar saline, suggesting central mechanisms in osmoregulation. Since brain areas activated by hyperosmolar saline correspond to those regions activated by angiotensin II and acetylcholine, we investigated in the second part of this study the involvement of angiotensinergic as well as cholinergic pathways.

Effect of pretreatment with the AT₁ receptor antagonist losartan on hyperosmolar saline-induced ITF expression in the brain. The presented data demonstrate that central AT₁ receptors participate in mild hyperosmolar saline (0.20 and 0.30 M)-induced ITF expression. Excitatory angiotensin II-containing pathways between the SFO, PVN, and SON and angiotensinergic pathways between SFO and MnPO have been described (20, 21). Furthermore, immunohistochemical and binding studies have revealed the presence of angiotensin II-immunoreactive cells and nerve terminals with angiotensin II receptor sites in these regions (13, 21), which implies that angiotensin II itself may act as a neurotransmitter or neuromodulator in central osmocontrol. In line with this assumption, water deprivation or local osmotic stimulation of the PVN has been shown to evoke the release of angiotensin II (18) and angiotensin III (28) into the PVN (9, 31). The expression pattern of central hyperosmolar saline- and angiotensin II-induced c-Fos and c-Jun in the brain reveals a striking similarity (29). The angiotensin II-induced ITF expression was also blocked by pretreatment with the AT₁ receptor antagonist losartan (18, 36). Furthermore, physiological responses following central hyperosmolar stimulation, such as release of vasopressin, drinking, natriuresis, and increase in mean arterial blood pressure, were shown to be partially mediated via the AT₁ receptor (13, 33, 34, 39). Consistent with our data, losartan inhibited 0.20 and 0.30 but not 0.60 M NaCl-induced vasopressin release and natriuresis, suggesting the engagement of a second nonangiotensinergic mechanism following NaCl solutions of higher osmolarity (13, 32, 34).

Effect of the muscarinic receptor antagonist atropine on hyperosmolar saline-induced ITF expression in the brain. In the present study, the ITF expression in response to 0.30 and 0.60 M NaCl but not 0.20 M NaCl was inhibited by pretreatment with atropine in all investigated brain regions except the SFO, suggesting the involvement of cholinoceptors in central osmoregulation.

Neuroanatomical and autoradiographic studies demonstrated the intense cholinergic innervation as well as choline binding sites in forebrain structures (2, 35). Stimulation of cholinoceptors has been shown to induce c-Fos expression in the same brain regions as angiotensin II (23). The involvement of cholinergic mechanisms in antidiuresis subsequent to osmo- and/or sodium receptor stimulation has also been demonstrated (5). In agreement with the results of this study, vasopressin secretion in response to intracerebroventricular 0.30 and 0.60 M NaCl stimulation was antagonized by atropine intracerebroventricularly, whereas 0.20 M NaCl-induced vasopressin release was not (14, 32). In addition, the increase of the firing rate of neurosecretory cells in the PVN by intracarotid injections of 0.60 M NaCl was inhibited by atropine (1). Finally, natriuresis and thirst following osmotic stimulation were attenuated by pretreatment with atropine (19, 27).

Despite pharmacological differences, there is evidence of overlapping of the angiotensinergic and the cholinergic system in osmocontrol. Interactions of both systems in cardiovascular regulation have also been reported (28). This interplay might be a result of an interference of both signaling pathways resulting in a differential regulation of target genes' transcription.

Colocalization of c-Fos and c-Jun with vasopressin and oxytocin in magnocellular neurons of the PVN and SON. Following central hyperosmolar saline stimulation, numerous magnocellular neurons of the PVN and SON showed colocalization of c-Fos and c-Jun with vasopressin and oxytocin, respectively. The gene for vasopressin has been regarded as a putative target gene of the ITF c-Fos and c-Jun following peripherally applied hyperosmolar saline (6, 41). The promoter region of the vasopressin gene harbors a calcium/cAMP response element (CRE) (26). Fos and Jun proteins can bind to CRE consensus sequences as AP-1 homo- or heterodimers or as heterodimers with CRE binding protein and activating factor proteins (8, 38). These findings support the hypothesis that the vasopressin gene may constitute a target gene of c-Fos and c-Jun in response to centrally applied hyperosmolar saline. Dimerization of c-Fos and c-Jun to the AP-1 complex is very likely because the majority of neurons showed double labeling with Fos and Jun in the SON following peripherally administered hyperosmolar saline (7). However, because the Fos/Jun dimer can generally modulate transcriptional activity of genes with AP-1 binding sites in the promoter region, other AP-1-containing genes, e.g., nitric oxide synthase, cholecystokinin, tyrosine hydroxylase, and prodynorphin, are further candidates. Findings of Meister et al. (25) are in agreement with this assumption. These authors showed that salt loading induced an increase in vasopressin, oxytocin, thyrosine hydroxylase, dynorphin, and cholecystokinin mRNA levels in the PVN and SON and a decrease in the level of corresponding peptides in the posterior pituitary, suggesting both increased synthesis and release.

In magnocellular neurons of the PVN and SON, we also observed colabeling of Krox-24 with oxytocin following hyperosmolar saline. Consistent with our data, induction of Krox-24 mRNA in oxytocin neurons has been observed following peripheral osmotic stimulation (22). As a member of another class of transcription factors, Krox-24 probably recognizes discrete DNA binding sites different from the leucine zipper proteins, e.g., the EGR site. Binding of the different ITF with distinct recognition sites may influence the transcriptional regulation of genes and thereby enable a finely tuned expression of target genes.

Perspectives

Recruitment of individual receptor systems seems to result in a distinct ITF expression pattern. The differential expression of ITF is a prerequisite of a complex regulation of target genes. Although interactions of the leucine zipper proteins and the zinc finger protein in the control of target genes seem to be possible, Krox-24 may be rather involved in the regulation of its own set of late-response genes (Fig. 7). Further efforts have to be made to elucidate the complex mechanisms by which ITF control target gene activity.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Schematic illustration showing hypothetical involvement of angiotensin and muscarinic receptors in hyperosmolar saline-induced ITF expression in the central nervous system. AP, activator protein.