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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS
1Department of Physiology and Functional Genomics and 2McKnight Brain Institute, University of Florida, Gainesville, Florida 32610
Submitted 25 March 2003 ; accepted in final form 6 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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and CaMKII in rat septum and hypothalamus. These data suggest that
signaling molecules involved in ANG II-induced responses in vitro are also
relevant in physiological responses elicited by ANG II in the whole animal
model. calcium/calmodulin-dependent protein kinase II; intracellular signaling; intracerebroventricular; third cerebroventricle; carbachol
The above physiological and behavioral effects of ANG II are mediated by
rapid receptor-mediated alterations in neuronal activity, resulting in release
of neuropeptides and neurotransmitters, which then modulate other neurons. In
vitro studies from our laboratory have used neurons cultured from the
hypothalamus and brain stem of newborn rats to elucidate the cellular and
intracellular actions that underlie the ANG II-mediated changes in neuronal
activity. These studies demonstrated that ANG II elicits a positive
chronotropic effect via AT1 receptors
(35,
37). Specifically, these
experiments have shown that ANG II increases total calcium current
(ICa), decreases the voltage-dependent delayed rectifier
(IKv)- and A-type (IA) K+
currents, and consequently increases neuronal firing rate
(34,
37,
38). The specific
intracellular signaling pathways that are involved in these AT1
receptor-mediated effects have also been investigated, and the results
indicate that ANG II modulation of neuronal K+ and Ca2+
currents involves stimulation of phospholipase C and activation of subsequent
Ca2+-dependent signaling molecules
(7,
34,
40). Specifically, these data
suggest that the ANG II-stimulated increases in neuronal firing rate and
decreases in IKv involve activation of both protein kinase
C (PKC) and Ca2+/calmodulin dependent protein kinase II (CaMKII)
(34,
35,
40), while the ANG
II-stimulated increase in ICa appears to only require
activation of PKC (34,
38). Moreover, recent
experiments indicate the involvement of PKC- in the ANG II-stimulated
decrease in IKv
(33)
While the neuronal culture model has been useful in identifying the intracellular signaling components involved in the rapid cellular actions of ANG II, it is important to establish that these mechanisms operate in vivo. In situ electrophysiological studies have demonstrated that ANG II via the AT1 receptor alters neuronal K+ current and firing rate in hypothalamic nuclei, similar to the effects observed in cell culture (9). However, these in vivo studies have not investigated the intracellular signaling molecules involved in the centrally mediated effects of ANG II. The goal of the present study was to ascertain whether the intracellular signaling molecules identified as important in the ANG II-mediated changes in neuronal activity in vitro are also important in the brain.
One of the most striking actions of ANG II in the brain is the
AT1 receptor-mediated stimulation of water intake, an effect that
is one component of this peptide's regulatory actions on fluid balance
(2,
5,
10,
21,
24,
28). Intracerebroventricular
injection of ANG II elicits a rapid and acute drinking response that occurs
within 
1 min, similar to the time course necessary for ANG II-mediated
changes in membrane ionic currents and neuronal activity (1-3 min).
Furthermore, this effect involves activation of neuronal AT1
receptors in the subfornical organ (SFO), paraventricular nucleus (PVN), and
median preoptic nucleus (MnPO)
(10,
12,
13,
19,
21,
22,
27,
30). In the experiments
described here, we assess the roles of PKC and CaMKII in the drinking response
produced by injection of ANG II into the lateral cerebroventricle (icv) and
third ventricle (i3v) of Sprague-Dawley (SD) rats. The results indicate that
1) central injection of ANG II activates PKC- and CaMKII in
the septal and hypothalamic regions of the SD rat brain; and 2) the
dipsogenic response produced by icv or i3v injection of ANG II involves
activation of both PKC- and CaMKII.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ANG II, KN-93, carbamylcholine chloride (carbachol), and anti-rabbit
peroxidase-conjugated secondary antibody were all purchased from Sigma (St.
Louis, MO). Chelerythrine chloride and Go-6976 were obtained from BioMol
(Plymouth Meeting, PA). Rabbit anti-PKC-, rabbit anti-PKC-
I, and
rabbit anti-phosphoCaMKII- were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Mouse anti-paxillin was purchased from BD Transduction
Laboratories (San Jose, CA), and anti-mouse peroxidase-conjugated secondary
antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove,
PA). Stainless steel guide cannulas and microinjectors were special ordered
from Plastics One (Roanoke, VA). Protein assay dye, 10% Tris·HCl
Criterion gels, and nitrocellulose were purchased from BioRad Laboratories
(Hercules, CA). Western Lightning Chemiluminescence Reagent Plus was purchased
from Perkin Elmer Life Sciences (Boston, MA).
Animals
Adult male rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed individually in light-controlled facilities. Animals were fed ad libitum and had free access to water. All procedures were approved by the University of Florida Institutional Animal Use and Care Committee.
Surgical Procedure
Adult male rats (200-250 g) were anesthetized with ketamine (30 mg/kg body wt im) and xylazine (6 mg/kg body wt im). Once fully anesthetized, rats were placed in a Kopf stereotaxic frame, a midline incision was made on the top of the head, and the skull was cleaned. For icv injections, a stainless steel guide cannula (22 gauge) was placed into the right lateral cerebroventricle using the following stereotaxic coordinates: bregma -1.30 mm, lateral 1.50 mm, from skull -4.50 mm (26). Since many of the ANG II-responsive nuclei surround the third ventricle, i3v injections were made using a stainless steel guide cannula (26 gauge) placed into the third cerebroventricle according to the following stereotaxic coordinates: bregma -2.56 mm, lateral 0.00 mm, from skull -10.00 mm (26). Postsurgery, rats were injected with banamine (1.1 mg/kg body wt im) and allowed to recover for 1 wk.
Injection Protocols
To confirm correct placement of the guide cannula, rats were injected icv or i3v with ANG II (10 ng/2 µl) and monitored for a drinking response for 30 min postinjection. Additionally, if the protocol allowed, rat brains were dissected to further verify that the cannula was correctly placed. The dose of ANG II employed here is at the lower end of the range used in similar studies and consistently yielded a rapid and vigorous drinking response. These routes of administration allow ANG II and all pharmacological inhibitors to reach AT1 receptors within the SFO and PVN. Rats with a positive drinking response (>6 ml of water in 30-min time period) were entered into the study. For the remainder of the study, rats with icv or i3v cannulas randomly received either injections of 0.9% saline (2 µl), ANG II (10 ng/2 µl), inhibitor (2 µl), or an inhibitor (2 µl) pretreatment (15 min) followed by ANG II (10 ng/2 µl) until each rat had received all four treatments. A similar protocol was used for another group of icv-cannulated rats, except carbachol (200 ng/2 µl) was substituted for ANG II as the dipsogen. At least 2 days separated each injection. All injections were given between 7:30 AM and 12:00 PM to control for differences in water intake throughout the day. After completion of one inhibitor sequence, all rats were injected with ANG II (10 ng/2 µl) to verify that a positive drinking response still existed and to ensure the inhibitors had no long-term effects on the ANG II-induced drinking response. Rats were either killed or used in further protocols. A total of 109 male SD rats were used to complete all these studies.
Analysis of PKC and CaMKII Activities
Changes in the activity of PKC- and PKC-I were assessed within
the septum-hypothalamus and brain stem (medulla) regions after icv injection
of ANG II. Activation of PKC was determined by measuring the translocation of
PKC protein from the cytosolic to membrane fractions within cell extracts.
This was achieved by Western blot analysis of PKC- and PKC-I
proteins in both the cytosolic and membrane fractions of the cellular
extracts, an approach widely used by others
(16,
17). Simultaneous changes in
the activity of CaMKII after icv injection of ANG II were assessed by Western
blot analysis of phosphorylated CaMKII-, the active form of this enzyme
within the cytosolic fraction. Detailed methodologies for these procedures
were as follows.
Extraction of cytosolic and membrane proteins. Rats demonstrating a positive drinking response were injected with either ANG II (10 ng/2 µl) or 0.9% saline (2 µl) and monitored for water intake. Five minutes postinjection, rats were killed, and their brains were removed. Brains were placed in ice-cold PBS, and the septum and hypothalamus regions (according to bregma: rostral to caudal 0.0 to -5.5 mm; dorsal to ventral 2.5 to 10.2 mm) (26) and brain stem (according to bregma: rostral to caudal -8.0 to -15.0 mm; dorsal to ventral 7.0 to 11.0 mm) (26) were dissected and frozen on dry ice. Frozen brains were stored at -80°C until they could be processed.
The frozen septal-hypothalamic regions and brain stems were weighed and homogenized on ice in 5 vol (5 x weight in g) of extraction buffer (pH 7.5) containing (in mM) 20 Tris·HCl, 150 NaCl, 2 EDTA, 1 EGTA, 1 PMSF, and 1 NaVO4. To remove cellular debris, samples were centrifuged at 1,000 g for 10 min at 4°C followed by centrifugation of the supernatant at 45,000 g for 20 min at 4°C. The supernatant was saved as the cytosolic fraction and stored at -80°C. The pellet was resuspended in extraction buffer supplemented with 0.5% Triton X-100 and were shaken for 60 min at 4°C. The resuspended pellet was centrifuged at 45,000 g for 20 min at 4°C, and the supernatant was saved as the membrane fraction and stored at -80°C. Protein concentrations for both cytosolic and membrane fractions were determined using a Bradford-based protein assay.
Western Blot analysis of PKC, phosphoCaMKII, and paxillin
proteins. Laemmli sample buffer (1.5x) was added to the membrane
and cytosolic fractions (10 µg of protein for septalhypothalamic region and
5 µg of protein for the brain stem) and boiled for 5 min at 100°C.
SDS-PAGE and Western blot analyses were performed as described previously
(3,
15). Briefly, proteins were
separated using 10% Tris·HCl gels and then transferred to
nitrocellulose. Western blot analysis was performed according to protocol from
Santa Cruz Biotechnology. Anti-PKC- (1:10,000), anti-PKC-I
(1:10,000), and anti-phosphoCaMKII- (1:500) primary antibodies and
anti-rabbit secondary antibody (1:16,000) were used to detect PKC- (80
kDa), PKC-I (80 kDa), and phosphorylated CaMKII- (50 kDa). To
ensure equal loading of samples, anti-paxillin primary antibody (1:10,000) and
anti-mouse secondary antibody (1:10,000) were used to detect paxillin (68
kDa), a protein not affected by ANG II treatment. Bands were visualized using
Western Lightning Chemiluminescence Reagent Plus and were quantified using a
GS-710 Densitometer and the Quantity One protein analysis package (BioRad
Laboratories). Data are presented as arbitrary units.
Data Analysis
All results are expressed as means ± SE. Statistical significance was determined using either a repeated-measures one-way ANOVA, one-way ANOVA, or the Mann-Whitney rank sum test. If the ANOVA yielded statistical significance (P < 0.05), pairwise comparisons were performed using the Bonferroni t-test. Differences were significant at P < 0.05; n refers to the number of animals.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Intracerebroventricular injection of ANG II (10 ng/2 µl) via the icv route stimulated a significant increase in water intake within 5 min (Fig. 1). Furthermore, this dipsogenic response was sustained over the next 10 min and then tapered off (Fig. 1). The total volume of water consumed 30 min after ANG II (10 ng/2 µl) injection was significantly greater than the total volume ingested 30 min after 0.9% saline (2 µl) injection (2,600% above control; Fig. 1). Likewise, i3v injection of ANG II (10 ng/2 µl) produced a significant increase in water intake (1,800%) compared with saline-treated (0.9%, 2 µl) control rats.
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Having established the drinking responses to icvand i3v-injected ANG II, we
determined whether inhibition of PKC modified these responses. The data shown
in Fig. 2A clearly
show that injection of the general PKC inhibitor chelerythrine chloride
(0.5-50 µM, 2 µl) into the lateral cerebroventricle (icv) reduced the
ANG II (10 ng/2 µl icv)-stimulated drinking response in a dose-dependent
manner (21, 42, and 61% respectively). Similarly, i3v injection with
chelerythrine chloride (50 µM, 2 µl) significantly attenuated the
drinking response (33%) produced by ANG II (10 ng/2 µl) injected via
the same route (Fig.
2B). Compared with saline-treated (0.9%, 2 µl)
controls, central injection of chelerythrine chloride (0.5-50 µM, 2 µl)
alone had no effect on water intake over the 30-min time period
(Fig. 2, A and
B). Furthermore, after icv or i3v injection of
chelerythrine chloride (0.5-50 µM, 2 µl), it was observed that rats were
still active and alert (i.e., rats roamed the cage, groomed themselves, and
would approach the water bottle randomly).
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Because in vitro studies demonstrated that PKC- is important in ANG
II-mediated effects on neuronal IKv
(20), we determined if the ANG
II-mediated drinking response also involved activation of this PKC isozyme.
Figure 2C shows that
Go-6976 (2.3 nM, 2 µl), a PKC inhibitor specific for PKC- at this
dose (20), attenuated the ANG
II-induced drinking response (32%). Go-6976 (2.3 nM, 2 µl) alone did not
alter water intake compared with rats injected with 0.9% saline (2 µl;
Fig. 2C) nor were
there any gross behavior effects (i.e., rats were active and alert). These
data suggest that the PKC- isozyme is involved in the ANG II-mediated
drinking response.
Previous studies have demonstrated that central injection of the muscarinic
acetylcholine receptor agonist carbachol elicits a drinking response, and this
effect involves activation of an inhibitory G protein
(31). To verify that results
of inhibition of PKC were specific to the ANG II-induced drinking response,
the effects of chelerythrine chloride on the dipsogenic response of carbachol
were also investigated. Compared with sa- line-treated (0.9%; 2 µl) control
rats, icv injection of carbachol (200 ng/2 µl) increased water intake by
400% over a 60-min time period (Fig.
2D). However, this dipsogenic effect of carbachol was not
affected by prior icv injection of the rats with the PKC inhibitor
chelerythrine chloride (50 µM, 2 µl;
Fig. 2D).
ANG II Increases PKC- Activity in the Rat Septum and
Hypothalamus
To further substantiate a role for PKC- in the ANG II-induced
drinking response, we determined whether the PKC- isozyme was activated
in the septum and hypothalamus and brain stem after icv injection of ANG II.
In addition, we studied the effects of ANG II on PKC-I activity for the
sake of comparison. Injection of ANG II (10 ng/2 µl, 5 min) icv stimulated
a translocation of PKC- from the cytosol to the membrane in a
hypothalamic block containing the SFO, PVN, and MnPO
(Fig. 3A). This
translocation was indicated by 1) an 77% increase in the levels
of PKC- protein in the membrane fraction of ANG II-treated rats
compared with saline (0.9%; 2 µl) controls, and 2) a 65% decrease
in the ratio of cytosolic vs. membrane PKC- protein levels in the ANG
II-treated rats (Fig.
3A). Conversely, injection of ANG II (10 ng/2 µl, 5
min) icv had no significant effect on the activity of PKC- in the brain
stem (Fig. 3B), as
indicated by a lack of significant translocation from the cytosol to membrane
in ANG II-treated rats. Furthermore, icv injection of ANG II had no
significant effect on the activation of PKC-I in the septum-hypothalamus
(mean ± SE ratio of cytosolic vs. membrane PKC-I protein levels:
saline 72.1 ± 42.4; ANG II 130.4 ± 97.8) and brain stem (mean
± SE ratio of cytosolic vs. membrane PKC-I protein levels: saline
12.4 ± 1.91; ANG II 21.7 ± 7.92). This indicates that the action
of ANG II is specific for PKC-. As a gel loading control, levels of
paxillin were assessed in both the cytosol and membrane fractions of the
septumhypothalamus and brain stem. In all conditions, paxillin protein levels
were similar (Fig. 3).
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Role of CaMKII in the ANG II-Induced Drinking Response
Studies in neuronal cultures have also indicated that along with PKC, CaMKII is involved in the ANG II modulation of neuronal IKv and firing (33-35, 37, 40). In the next set of experiments, we determined whether CaMKII was important for the ANG II-induced drinking response. Injection of the CaMKII inhibitor KN-93 (10 µM, 2 µl) either icv or i3v significantly attenuated the drinking response produced by ANG II (10 ng/2 µl) administered via the same route (38 and 33%, respectively; Fig. 4, A and B). Importantly, central injection of KN-93 (10 µM, 2 µl) alone had no effect on water intake or behavior compared with central injection of 0.9% saline (2 µl; Fig. 4, A and B). In addition, this effect of KN-93 (10 µM, 2 µl) was specific to the ANG II-induced drinking response because it did not alter the increase in water intake elicited by icv injection of carbachol (200 ng/2 µl; Fig. 4C).
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Because our studies demonstrated that both PKC- and CaMKII play a
role in the ANG II-stimulated drinking response, the next set of experiments
was performed to determine if inhibition of both PKC and CaMKII would abolish
the ANG II drinking response completely. Coinjection (2 µl) of both
chelerythrine chloride (50 µM) and KN-93 (10 µM) significantly reduced
the ANG II (10 ng/2 µl)-stimulated drinking response (23%), but the effect
of each inhibitor was not additive (Fig.
5).
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ANG II Increases CaMKII Activity in the Rat Septum and Hypothalamus
To substantiate the above findings, the next set of experiments
investigated the activation of CAMKII in the hypothalamus and brain stem after
central injection of saline and ANG II. ANG II (10 ng/2 µl, 5 min) injected
icv increased the activity of CaMKII-, as determined by the levels of
phosphorylated CaMKII-, in the septum-hypothalamus
(Fig. 6) by 92%.
Phosphorylated CaMKII- protein was not detectable in the brain stem of
0.9% saline- or ANG II-treated rats (data not shown). The levels of paxillin
protein were similar under each treatment condition, indicating that there was
equal loading of each sample onto the gel
(Fig. 6).
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Role of Phosphatidylinositol 3-Kinase and c-Jun NH2-Terminal Kinase in the ANG II-Induced Drinking Response
In vitro studies from our laboratory have also demonstrated that ANG II elicits cellular and physiological responses that are delayed (>30 min) when compared with the ANG II-stimulated changes in neuronal activity (<5 min) (33). Additionally, studies have demonstrated that these ANG II-mediated longer-term effects involve a variety of signaling molecules, which include phosphatidylinositol 3-kinase (PI3-K) and c-Jun NH2-terminal kinase (JNK) (14, 33, 39). Temporally, because activation of PI3-K and JNK occurs after the changes in neuronal activity and the induction of the drinking response (14, 33, 39), it is not expected that inhibition of these molecules would alter the ANG II-stimulated dipsogenic response. Indeed, as Fig. 7 demonstrates, icv injection of either the selective PI3-K inhibitor LY-294002 (10 µM, 2 µl; Fig. 7A) or the pharmacological JNK inhibitor II (100 nM, 2 µl; Fig. 7B) did not significantly reduce the increase in water intake elicited by ANG II (10 ng/2 µl) injected via the same route.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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and CaMKII. As indicated in
Fig. 1, icv injection of ANG II
stimulates a rapid and vigorous drinking response (5 min) that is
sustained for 10-15 min and then begins to taper off with random bursts
occurring for the remainder of the 30 min. A similar response profile is
observed with i3v-injected ANG II. The beginning of the ANG II-induced
drinking time course is similar to the time course for ANG II modulation of
K+ and Ca2+ currents (1-3 min). Because many
physiological responses elicited by the brain are due to changes in neuronal
activity, it is logical that the same mechanisms involved in ANG II modulation
of neuronal activity in vitro are involved in ANG II-mediated responses in
vivo. Indeed, central injection of the general PKC antagonist chelerythrine
chloride significantly reduces the ANG II-induced drinking response,
indicating a role for PKC. Further investigation into the PKC isozyme involved
indicated that PKC- is involved in the ANG II-mediated drinking
response, similar to the ANG II modulation of IKv and
neuronal activity (34,
35,
40). Furthermore, these data
suggest that the ANG II drinking response involves activation of PKC-
in the septum and hypothalamus but not the brain stem. However, these data
indicate that other PKC isozymes may be involved in the ANG II-stimulated
dipsogenic effect because Go-6976 does not completely inhibit the drinking
response elicited by ANG II. Additionally, similar to its role in the ANG II
modulation of IKv and neuronal activity
(34,
35,
40), CaMKII plays a role in
the ANG II-mediated drinking response because central injection of the CaMKII
antagonist KN-93 reduces the ANG II-induced dipsogenic effect. These results
also suggest that activation of CaMKII- in the septum and hypothalamus
is partially responsible for the ANG II-induced drinking response. Observations made during these studies also indicate that antagonism of PKC and CaMKII inhibit the drinking response due to icv injection of ANG II in the first 10 min, but not the random bursts. Thus it may be hypothesized that one of these kinases (i.e., PKC) is important for the initiation of the drinking response elicited by ANG II, while another kinase (i.e., CaMKII) may be involved in maintaining the drinking response for the first 10 min. Therefore, it is plausible to conclude that inhibition of both signaling molecules would inhibit the dipsogenic response completely. However, coadministration of PKC and CaMKII antagonists only partially inhibits the ANG II-induced drinking response. These results were intriguing because they differ from those found in vitro, which demonstrated that inhibition of both PKC and CaMKII completely abolishes the ANG II-stimulated decrease in IKv and stimulation of neuronal activity (40). There are several conceivable explanations that may explain the differences between the in vivo and in vitro systems. One possibility may be that PKC and CaMKII are both equally involved in the same aspects of the ANG II-mediated drinking response; thus inhibition of both enzymes will not completely inhibit the ANG II-mediated effect. A second thought may be that these two enzymes are sequentially activated (i.e., PKC is upstream of CaMKII or vice versa), and thus coinhibition is not additive because they are both part of the same signaling network. The data also could suggest that while both enzymes are involved in the ANG II-induced drinking response, PKC and CaMKII are not the only two kinases important in this ANG II-mediated effect. For this reason, experiments were performed to investigate other signaling molecules that may be involved in the ANG II-induced drinking response. These data show that inhibition of PI3-K and JNK reduces the drinking response, but this effect is not significant. This could suggest that PI3-K and JNK play a very minor role in the ANG II-stimulated dipsogenic response. However, this also suggests that there are other signaling molecules in addition to PKC and CaMKII that are more significantly involved in this ANG II-mediated response, and these still are yet to be determined. Another explanation for this difference between the cell culture system and the in vivo model may be the administration of the drug. In the cell culture system, the individual drugs are diluted separately and then individually added to the dish. On the other hand, the drugs for the in vivo experiments were made as an inhibitor cocktail for icv injection. The two chemical compounds may interact with each other, thus reducing their relative potencies. Finally, this difference between results in the in vitro and in vivo system may be due to the fact that the whole animal brain is much more complicated than the simplified cell culture system. Inhibition of one signaling molecule in the brain may elicit one effect, while inhibition of two signaling molecules at the same time may elicit a totally different response. This difference supports the necessity to verify that ANG II-mediated actions discovered through the use of in vitro systems also occur in the whole animal model.
The results presented here also demonstrate that activation of PKC and CaMKII is specific to the dipsogenic response elicited by ANG II because inhibition of PKC and CaMKII has no effect on the carbachol-induced drinking response. Moreover, these results also verify that the reduction in the ANG II-mediated dipsogenic response is due to the inhibition of PKC and CaMKII by the chelerythrine chloride and KN-93, respectively, and not by secondary effects of the inhibitor on the alertness and activity of the rat. Additionally, these results suggest that different intracellular signaling molecules are involved in the ANG II-mediated and carbachol-induced drinking responses. This is consistent with the previously published study that demonstrated that the dipsogenic response elicited by carbachol involves an inhibitory G protein (31). Furthermore, these results support studies presented in the literature that demonstrated that the ANG II-mediated dipsogenic response does not require activation of cholinergic pathways because the acetylcholine antagonist atropine has no effect on the ANG II-induced drinking response (10).
Another important observation made during these studies was the activation of PKC and CaMKII in the septum-hypothalamus regions but not in the brain stem. This is of interest because nuclei of the brain stem such as the nucleus tractus solitarius are known to be important in ANG II-mediated physiological responses. There are several different interpretations that could be made. First, it could suggest that the septum and hypothalamus are more important in the ANG II-induced dipsogenic response than the brain stem. However, a more plausible interpretation is that different intracellular signaling molecules are activated by ANG II in cells in the septum and hypothalamus than in the brain stem. This means that PKC and CaMKII are the signaling molecules predominantly activated in the septal-hypothalamic regions while different signaling molecules are activated by this peptide in the brain stem.
In summary, these initial experiments are important because they
demonstrate for the first time that intracellular signaling molecules involved
in ANG II-mediated cellular actions are also involved in physiological
responses due to this peptide. While the in vitro cell culture system is
beneficial because it allows investigators to study individual neurons in a
controlled environment, it has its limitations. Due to these limitations, it
is important to verify that the knowledge regarding AT1
receptor-mediated cellular signaling discovered in vitro is relevant in ANG
II-mediated physiological responses in the whole animal model. These data
demonstrate that the findings from in vitro cell culture systems on
AT1 receptor-mediated activation of intracellular signaling
pathways can be extrapolated to the physiological responses regulated by ANG
II. Specifically, these results provide the first evidence that PKC-
and CaMKII in the brain are involved in the ANG II-mediated drinking
response.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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) or
saline (0.9%, 2 µl; 
), and water intake was measured at 5, 10, 15,
and 30 min. Graph presents mean ± SE water intake at each time point
for 9 rats. Data are cumulative. *P < 0.05 from saline
at all time points; 
P < 0.05 from ANG II (5 min).
