Vol. 281, Issue 4, R1224-R1231, October 2001
Cardiovascular changes induced by central hypertonic saline
are accompanied by glutamate release in awake rats
Qing-Hua
Jin1,
Yuto
Ueda2,
Yuta
Ishizuka2,
Takato
Kunitake1, and
Hiroshi
Kannan1
1 Department of Physiology and 2 Department of
Psychiatry, Miyazaki Medical College, Miyazaki 889 - 1692, Japan
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ABSTRACT |
To elucidate
neurochemical mechanisms responsible for cardiovascular responses
induced by central salt loading, we directly perfused the
paraventricular nucleus (PVN) of the hypothalamus region with
hypertonic saline (0.3 or 0.45 M) by using an in vivo brain
microdialysis technique. We then measured the extracellular concentrations of glutamate in the PVN region in conscious rats along
with the blood pressure and heart rate. Blood pressure, heart rate, and
glutamate levels were increased by perfusion of 0.45 M saline; however,
they did not change by perfusion of 0.3 M saline. Next, we examined the
possible involvement of glutamate in the cardiovascular responses
induced by hypertonic saline. Dizocilpine, a noncompetitive antagonist
of the N-methyl-D-aspartate (NMDA) receptor,
attenuated the increases of blood pressure and heart rate, although
6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA
receptor, did not affect the blood pressure and heart rate. Our results
show that local perfusion of the hypothalamic PVN region with
hypertonic saline elicits a local release of glutamate, which may act
via NMDA-type glutamate receptors to produce cardiovascular responses.
paraventricular nucleus; blood pressure; heart rate; microdialysis
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INTRODUCTION |
THE OSMOTIC
HOMEOSTASIS and ionic balance of the body are mainly regulated by
neural and humoral factors through the central nervous system (CNS).
Despite its clinical and physiological significance, however, little is
known about the underlying cellular and molecular mechanisms by which
the osmotic and ionic balances of the CNS are maintained in conscious
and freely moving animals. Salt loading is a potent stimulus for
drinking, vasopressin (AVP) secretion, and increasing arterial blood
pressure. Acute saline injection seems to activate osmoreceptive
structures, including those in the subfornical organ and the
anteroventral third ventricle (AV3V region) (17, 39),
subsequently stimulating AVP secretion and neurons related to
cardiovascular and behavioral responses in the hypothalamus and brain
stem (50). In addition, a number of studies have pointed
to the importance of the hypothalamic paraventricular nucleus (PVN) in
the regulation of body fluid homeostasis and in the central control of
cardiovascular function (36, 46). The PVN is involved in
the control of not only the neurohypophysial system but also the
adenohypophysial system and autonomic nervous outflow
(21). Several authors, employing electrophysiological methods, have shown that, in addition to the supraoptic nucleus (SON),
the PVN is a region where neurons sensitive to changes in osmolality
and/or NaCl concentration are located (9, 13, 26, 31, 40).
Recent research has also shown that systemic administration of
hypertonic saline activates cells in the PVN and SON (16, 28,
41). Moreover, an increased c-Fos expression in the PVN after
dehydration and salt loading further confirmed the role of this area in
the regulation of body fluid balance (12, 39). Thus the
PVN appears to be an important regulatory region of the hypothalamus
that plays a critical role in the modulation of cardiovascular,
neuroendocrine, and behavioral responses during osmotic challenges.
It has been reported that direct stimulation of the PVN and SON with a
high-NaCl perfusion medium via microdialysis probes resulted in
increased AVP and oxytocin release both locally into extracellular
fluid and into systemic circulation (14, 15, 25, 29, 38).
Similar releases of angiotensin (43), amino acids, and
dopamine (18) after local perfusion of hypertonic saline
have also been reported, and the findings have been interpreted as
evidence for the roles of these substances in the central osmotic and
ionic balance. However, the roles of endogenous neurochemicals in the
PVN responsible for cardiovascular responses induced by salt loading
remain to be elucidated. Glutamate (Glu) has been recognized as an
important neurotransmitter/neuromodulator in the CNS, mediating most of
the excitatory synaptic transmission. An injection of Glu into the PVN
raised blood pressure and heart rate (HR) accompanied with increases in
plasma catecholamines (30). These findings raise the
possibility that Glu may be involved in the responses induced by
central hypertonic saline stimulation.
In some cases, anesthesia, well known for its profound effect on
cardiovascular and autonomic function, reverses the responses (21, 32). Microdialysis is a useful method for
pharmacological assessment and manipulation of the microenvironment in
the brain of conscious and freely moving animals, in which changes in
osmolality or NaCl concentration, extracellular amino acids, and
related mediator levels can be manipulated and measured
(2). Therefore, in the present study, we have used a
microdialysis technique to investigate the release of Glu in response
to local stimulation with hypertonic saline and to monitor
cardiovascular responses in conscious and freely moving rats. In
addition, to test whether the observed Glu release is involved in the
cardiovascular responses, N-methyl-D-aspartate
(NMDA) and non-NMDA receptor antagonists were coadministered with
hypertonic saline stimulation, and the cardiovascular responses that
were induced by hypertonic saline were examined.
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METHODS AND MATERIALS |
Animals and surgical procedures.
Male Wistar rats, weighing 350-450 g at the start of the
experiments, were used in our study. The experiments were performed in
accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Bethesda, MD] and were approved by the
Committee on Animal Care of Miyazaki Medical College. The rats were
anesthetized with pentobarbital sodium (50 mg/kg ip) 5 days before the
experiments. An arterial catheter (SP-31 polyethylene tubing
heat-coupled to SP-50) was inserted into the surgically exposed
abdominal aorta for measurement of arterial blood pressure (BP) and HR
and tunneled under the skin to exit at the nape of the neck. A guide
cannula (0.50-mm ID, 0.70-mm OD) for the placement of a microdialysis probe was stereotaxically implanted 2.0 mm above the PVN according to
the atlas of Paxinos and Watson (42) and sealed with a
dummy cannula after implantation. The stereotaxic coordinates were 1.7 mm posterior to the bregma, 0.3 mm lateral to the midline, and 6.7 mm
ventral to the dural surface. To determine whether the direct effects
of hypertonic saline (HTS) were specific to the PVN, the guide cannula
was implanted into the outside of the PVN region (1 mm rostral to the
PVN, n = 2; 1 mm lateral to the PVN, n = 3) as an anatomic control group. A head plate for the fixation of the
arterial catheter and a guide cannula were fitted to the skull by
dental cement.
Experimental procedure.
On the day before the experiment, the animals were anesthetized with
isoflurane (2.5-3.0% in 100% oxygen), and the dummy cannulas were replaced with microdialysis probes. To reach the PVN region, the
tip of the probe covered with a 2.0-mm length of hollow fibers (200-µm OD, cellulose acetate membrane, cut-off 4.8 × 104 mol wt; Terumo, Japan) was set to extend 2 mm beyond
the guide shaft, and a similar operation was performed in the anatomic
control group. So that they would get used to the environment in which the experiment would later be performed, the animals were placed in
cylindrical metabolic cages (28-cm diameter × 28-cm height) that
were controlled by computers. This system can protect the arterial
catheter and tubes connected to the probe from the twist induced by the
movement of rats, allowing for long-term recording without twisting. On
the day of the experiments, the collection of dialysates for the Glu
and cardiovascular measurements were carried out while the animals were
permitted to move about freely in their cages. The microdialysis probe
was perfused with modified Ringer solution (147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2; pH 6.5) at a constant rate of 1 µl/min using a
microinfusion pump. The perfusate from the PVN region was collected
manually in an Eppendorf tube every 20 min. After a 2-h stabilization
period, two consecutive dialysate samples were collected to measure the
baseline Glu levels. All samples of collected dialysates were kept at
80°C for later analysis. The arterial catheter was connected to a
pressure transducer (P23 ID, Gould, Singapore) to monitor BP, while the
HR was counted by BP wave. The BP and HR were monitored simultaneously
and recorded on a thermal pen-writing recorder (RJG-4122, Nihon Kohden,
Japan) and on an FM magnetic tape recorder (RM-7000, Sony, Tokyo,
Japan). The mean arterial pressure (MAP) was obtained by electronic
averaging of the pulsatile arterial pressure signal.
The Glu levels were measured by using high-performance liquid
chromatography with electrochemical detection (HPLC-ECD). Before applying the HPLC-ECD method, an o-phthalaldehyde (OPA)
solution (40 mM) was made by adding 13.5 mg of OPA and 10 µl of
2-mercaptoethanol to 2.5 ml of 0.1 M K2CO3
buffer (pH 9.5) with 10% ethanol. The solution was then stored at
4°C and diluted in 0.1 M K2CO3 to yield a 4 mM OPA solution just before detection. The dialysate (18 µl) was
mixed with 5 µl of the 4 mM OPA solution and allowed to react for 3 min at 25°C incubation. After completing the reaction, 20 µl of the
reaction mixture was applied to HPLC with an Eicompak MA-5 ODS column
(4.6-mm ID × 150 mm; Eicom, Tokyo, Japan). Detection was
accomplished with an ECD (Eicom) with +700 mV Ag/AgCl
electrodes. The elution buffer for the Glu was 0.1 M phosphate
buffer, 30% methanol, and 0.5 mM EDTA (pH 6.5).
At the end of each experiment, the rats were killed with an overdose
injection of pentobarbital sodium, and their brains were fixed in 10%
neutral-buffered formalin. The implantation site of the dialysis probe
was verified histologically in 40-µm coronal sections after cresyl
violet staining.
Reagents.
HTS with two different tonicities, 0.3 and 0.45 M (the osmolalities
were 591 and 900 mosmol/kgH2O, respectively), was
used in this experiment to observe the responses. The PVN region was perfused with the HTS through the microdialysis probe at a constant rate of 1 µl/min for 20 min. Physiological saline (0.15 M NaCl) was
used in this experiment as a control. In addition, 0.6 M mannitol (Nacalai Tesque, Kyoto, Japan) dissolved in the physiological saline
(the osmolality is 904 mosmol/kgH2O) was perfused in the PVN via the microdialysis probe for 20 min, and 1 mM NMDA (Nacalai Tesque) dissolved in the modified Ringer solution was perfused for 10 min.
Dizocilpine (MK-801; Sigma), an NMDA-receptor antagonist, was dissolved
in the modified Ringer solution (1 mM), and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Sigma), a non-NMDA-receptor
antagonist, was dissolved in a 0.1 N NaOH solution (1 mM). The drugs
were then stored at 4°C. To measure the effect of these drugs on the
responses induced by HTS stimulation, both drugs were diluted with 0.45 M NaCl to 100 µM, and the mixtures were administered via the
microdialysis probe. The same vehicles used to dilute the drugs were
given at the same concentration in the 0.45 M saline, and this solution served in this experiment as the vehicle control.
Statistical analysis.
All data are expressed as means ± SE, and statistical analysis
was performed using ANOVA followed by Fisher's protected least significant difference test. To provide a temporal description of the
cardiovascular responses, the area under the curve (AUC) was also
calculated (1). A comparison of the maximal changes in BP
and HR and in AUC between the PVN group and the anatomic control group was carried out by a paired Student's t-test.
A probability level of P < 0.05 was considered
statistically significant.
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RESULTS |
In the present study, the baseline levels are the average of
baseline values before stimulation in each parameter. The baseline levels for MAP, HR, and Glu are shown in Table
1, with no significant differences
between the groups. (See Fig. 2, top, for the exact positioning of the probe.)
Figure 1 shows the effect on MAP and HR
of direct infusion of HTS into the PVN region for 20 min through a
dialysis probe. The local perfusion of 0.45 M saline elicited
significant increases in MAP and HR from the baseline levels of
96.7 ± 3.6 mmHg and 285.3 ± 3.5 beats/min to the maximums
of 114.4 ± 2.4 mmHg and 394.0 ± 10.7 beats/min,
respectively. The MAP and HR began to increase 12 min after starting
the perfusion and returned to the basal levels ~15 min after the
perfusion was stopped. However, the 0.3 M saline did not cause
significant increases in MAP and HR, although the maximums reached
99.8 ± 3.7 mmHg and 336.5 ± 18.1 beats/min, respectively
(baseline levels in this group were 93.6 ± 1.9 mmHg and
297.6 ± 9.7 beats/min, respectively).
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Fig. 1.
Effects of direct infusion of hypertonic saline (HTS)
into the paraventricular nucleus (PVN) through microdialysis probes on
mean arterial pressure (MAP; A) and heart rate (HR;
B). In Figs. 1, 3, and 5-7, MAP and HR are shown by
changed amounts from baseline levels, and horizontal bars show period
of perfusion. Data are means ± SE. * P < 0.05 compared with 0.15 M NaCl group; + P < 0.05 compared with 0.3 M NaCl group.
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To investigate whether the increases in BP and HR in response to local
administration of HTS were specific to the PVN, perfusion of 0.45 M
saline into a region outside the PVN [~1 mm rostral (n = 2) or lateral (n = 3)] was
performed (Fig. 2, middle and bottom). In this anatomic control group, BP and HR did not
show significant increases after 0.45 M saline perfusion, although the
maximal changes reached 7.2 ± 2.7 mmHg and 39.2 ± 22.2 beats/min, respectively. The maximal changes in the HTS perfusion of
the PVN region were 20.9 ± 2.6 mmHg and 115.9 ± 8.7 beats/min, respectively, which were strikingly larger than the anatomic
control group.
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Fig. 2.
Photomicrographs of a typical placement of the unilateral
microdialysis probe in the PVN (top) and rostral
(middle) and lateral to the PVN (bottom). Arrows
indicate tip of the microdialysis probe. SCh, suprachiasmatic
nucleus.
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In addition, a 0.6 M mannitol solution dissolved in the 0.15 M saline
was used to determine whether the cardiovascular responses to direct
HTS perfusion into the PVN region were caused by a change in ions,
sodium chloride, or osmolality. The mannitol solution, its osmolality
approximately equivalent to 0.45 M saline, caused little increase in BP
and HR after the perfusion was stopped (Fig. 3). The maximal changes were only
4.7 ± 2.5 mmHg and 42.1 ± 14.9 beats/min, respectively. As
shown in Fig. 3, the responses of BP and HR to the mannitol were
markedly less than they were to NaCl, and their durations were also
shorter than they were in the NaCl group.
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Fig. 3.
Comparison between 0.45 M NaCl and 0.6 M mannitol groups
in responses of MAP (A) and HR (B) induced by
direct perfusion of the PVN. Data are means ± SE.* P < 0.05 compared with baseline levels; + P < 0.05 compared with 0.6 M mannitol group.
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The effect of local stimulation of PVN with HTS on Glu levels in the
PVN region is summarized in Fig. 4. The
Glu levels in the PVN region showed an immediate increase by the local
administration of 0.45 M saline, which reached to 249.8 ± 54.7%
of the basal level. The elevated Glu levels remained after the
perfusion and almost returned to the basal levels at 40 min after the
perfusion was stopped. The direct perfusion of 0.3 M saline increased
the Glu levels to 118.4 ± 7.4% of the basal level, but there was
no significant difference compared with the control group with 0.15 M
NaCl.
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Fig. 4.
Effects on glutamate (Glu) level in the PVN by direct
infusion of HTS into the PVN through microdialysis probes. Glu levels
are expressed as percentages of baseline levels. Horizontal bar shows
period of perfusion. Data are means ± SE. * P < 0.05 compared with 0.15 M NaCl group; + P < 0.05 compared with 0.3 M NaCl group.
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Figure 5 shows the effects of MK-801 on
the responses of MAP and HR that were induced by 0.45 M saline.
Coadministration of MK-801 (100 µM) with HTS significantly attenuated
the increased responses of MAP and HR. The AUC in the MAP and HR was
changed from 314.0 ± 70.7 mmHg · m and 1,791.6 ± 465.4 beats/min · m of the vehicle control group to 85.8 ± 21.1 mmHg · m and 533.3 ± 206.6 beats/min · m
(both P < 0.05), respectively. On the other hand, the
responses of MAP and HR, induced by direct perfusion of 0.45 M saline
into the PVN, were not influenced by CNQX (100 µM; Fig.
6). The AUC in the MAP and HR in the CNQX
group was 209.9 ± 23.7 mmHg · m and 1,119.8 ± 149.9 beats/min · m, and in the vehicle group it was 295.0 ± 73.7 mmHg · m and 1,320.3 ± 396.2 beats/min · m,
respectively (both P > 0.05).
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Fig. 5.
Effects of MK-801 (100 µM) on HTS-induced increases in
MAP (A) and HR (B). Data are means ± SE.
* P < 0.05 compared with baseline levels; + P < 0.05 compared with vehicle group.
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Fig. 6.
Effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX;
100 µM) on HTS-induced increases in MAP (A) and HR
(B). Data are means ± SE. * P < 0.05 compared with baseline levels.
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Finally, effects of direct perfusion of NMDA into the PVN region on BP
and HR were examined. BP and HR increased strikingly by perfusion of 1 mM NMDA. The maximal changes reached ~25.0 ± 3.6 mmHg and
132.4 ± 11.8 beats/min, respectively, and the increases remained
at high levels for 15 min after the NMDA perfusion was stopped (Fig.
7).
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Fig. 7.
Effect on MAP (A) and HR (B) of
direct infusion of N-methyl-D-aspartate (NMDA)
into the PVN through a dialysis probe. Data are means ± SE.
* P < 0.05 compared with vehicle group.
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DISCUSSION |
This study demonstrates that dialysis with hypertonic saline in
the PVN region results in increased BP and HR accompanied with an
increase in the Glu level in the dialysates obtained from the PVN.
These changes are related to the tonicity of the hypertonic saline. Any
consideration of the effects of hypertonic saline solution requires
recognition of whether either osmotic or ion-specific factors are
involved. In the PVN, some neurons show excitation and others show
inhibition in response to hypertonic saline and other osmotic stimuli
in vitro and in vivo (11, 13, 36, 40, 52). On the other
hand, in vivo local microdialysis of 1 M hypertonic saline elicited
oxytocin release in the PVN, whereas dialysis with equimolar mannitol
did not (14). Present work showed that equiosmotic
mannitol perfusion in the PVN showed a much lesser cardiovascular
effect than the hypertonic saline solution in conscious rats (Fig. 3).
Therefore, the results observed in the present studies with hypertonic
saline solution might be much more dependent on an ion effect, i.e.,
Na+ or Cl
, than on an osmotic effect. In this
regard, Eriksson and colleagues (10) found in the goat
that antidiuretic and thirst responses could be induced by
intracerebroventricular (icv) infusion of hypertonic NaCl but not by
solutions of equivalent osmolarity made from sucrose in water. They
proposed that a periventricular sodium-sensitive receptor system,
located in the vicinity of the third ventricle, was responsible for the
regulation of antidiuresis, thirst, and renal Na excretion. However,
McKinley and colleagues (33) revealed that icv
infusion of hypertonic sucrose in sheep can elicit thirst and
antidiuresis if the solution contains sufficient amounts of NaCl to
prevent the osmotic dilution of cerebrospinal fluid (CSF) Na. These
results provided support for the involvement of osmoreceptors. Whether
sodium and/or an osmoreceptor is involved in the cardiovascular
responses that are induced by salt loading remains to be elucidated.
Because cardiovascular responses induced by local perfusion of
hypertonic saline in the PVN region were more prominent than those
induced by mannitol in the present study, activation of NaCl receptors
rather than osmoreceptors might be involved in the cardiovascular
responses induced by hypertonic saline.
Considerable evidence exists that extracellular concentrations of
putative amino acid neurotransmitters are also affected by osmotic
and/or NaCl changes in the microenvironment (4, 47, 49).
It is known from immunocytochemical and physiological studies that
glutamatergic synaptic terminals innervate neurons in the PVN and
control their activity (7, 8, 34, 48). In addition, the
injection of Glu into the PVN evoked the increases of MAP, HR, and
plasma catecholamines (30), and the unilateral infusion of
the PVN region with Glu produced a great increase of plasma oxytocin
level (15).
Therefore, we asked whether the release of Glu on local stimulation
with hypertonic saline solutions is involved in the cardiovascular responses. If so, blockade of glutamatergic synaptic transmission with
antagonists could modify the cardiovascular responses. We found an
increase in extracellular Glu concentration after local perfusion of
hypertonic saline. Furthermore, the NMDA-receptor blocker MK-801, but
not the non-NMDA-receptor antagonist CNQX, attenuated cardiovascular
responses after local perfusion of the PVN with hypertonic saline, and
local injection of NMDA into the PVN region evoked increases in BP and
HR. Glu excites parvocellular neurons in the PVN (8), some
of which project directly to the preganglionic sympathetic neurons in
the intermediolateral cell column of the spinal cord (44,
46). Electrical and chemical stimulation of the PVN increases BP
and renal sympathetic nerve activity in conscious rats
(21). Therefore, released Glu in the PVN might be involved
in the cardiovascular responses induced by hypertonic saline.
Hypertonic saline stimulation of the PVN and SON was reported to
increase AVP and oxytocin in the nuclei, with a maximum during poststimulation (14, 25, 28, 37, 38). In contrast to these
studies on hypothalamic oxytocin and AVP release, we observed that the
stimulated release of Glu in the PVN was quantitatively related to the
increase in tonicity, which increased immediately during the hypertonic
saline stimulation and returned gradually to baseline when the stimulus
was withdrawn. These findings suggest that a mechanism different from
the one operating for AVP and oxytocin release is responsible for Glu
release in the PVN region under hypertonic saline loading.
The mechanism that triggers the hypertonic saline-induced release of
amino acids is not known. One plausible explanation is that the amino
acids are released as neurotransmitters in response to direct NaCl
and/or osmotic stimulation of the neurons of their origin
(18). In this regard, Glu, 
-aminobutyric acid, and
glycine can be released from nerve terminals through their carrier by exchanging with extracellular amino acids sharing the same transport system (22, 27). Therefore, it is unclear which of the
released substances may trigger a possible secondary amino acid
substance. Another possible explanation could involve pH changes due to
activation of the plasmalemmal sodium/hydrogen exchanger
(20) or sodium/calcium exchange mechanisms, causing the
release of intracellular calcium from internal stores such as
mitochondria (6), resulting in facilitation of
exocytosis. It has been known for many years that increases in
the frequency of spontaneous miniature end-plate potentials at a
neuromuscular junction occur after exposure to high osmotic pressure
(35). Despite continued investigation, an explanation for
this mechanism remains to be revealed (19, 23). However,
the question arises whether the measured extracellular concentrations
of Glu within the PVN originated from neuronal or glial
release. Both glial cells and neurons contain Glu, and the
release could have come from either cellular compartment. The possible
release of Glu from glia rather than, or in addition to, that from
neuronal axon terminals raises the possibility of the reversal of
normal glial Glu uptake by transporters due to disturbance of the ionic
concentration of the extracellular milieu. A recent study by Landgraf
and colleagues (18) demonstrated that the changes in
extracellular amino acid concentration within the PVN are calcium
dependent for some amino acids but not for others. Thus it can be
presumed that local perfusion with hypertonic saline solution evokes an
increase in Glu release in the PVN through multiple pathways.
Excitatory amino acid (EAA) receptors have been implicated in
cardiovascular and body fluid regulation at the brain stem and hypothalamus levels (24, 45, 50). These ionotropic EAA
receptors are classified as NMDA and non-NMDA
(
-amino-3-hydroxy-5-methyl4-isoxazole propionic acid
and kainate) (5). The noncompetitive
NMDA-receptor antagonist MK-801 could significantly suppress
the increases in BP and HR induced by local application of hypertonic
saline. This suggests that Glu transmission through an NMDA receptor in
the PVN is involved in the neural mechanism whereby hypertonic saline stimuli provoke cardiovascular activation. The non-NMDA-receptor antagonist CNQX had no effect on cardiovascular responses, suggesting that the effects observed were specific for an NMDA subtype of the Glu
receptor. Other findings also point to NMDA being involved in the
central regulation of body fluid balance (50, 51), such
as, for example, icv administration of NMDA-stimulated dipsogenic responses in pigeons (3).
Perspectives
The phenomenon of hypertonic saline-induced release of amino acids
such as Glu appears to be constant throughout many areas of the CNS
(18), although the sensitivity to hypertonic saline solution might be different on the basis of areas examined. However, cardiovascular responses induced by injection of hypertonic saline through microdialysis probes were presumed to be site specific. This
assumption can be supported by the following. Cardiovascular responses
were not induced or greatly attenuated after hypertonic NaCl perfusion
into areas 1.0 mm rostral to or 1.0 mm lateral to the PVN, suggesting a
localized site of action. However, using microdialysis probes to
administer or collect substances, we cannot be certain 1)
from what volume of tissue the substances released are collected,
2) into what volume of tissue or extracellular space the
substances administered can diffuse, and 3) what the effective concentrations of drugs administered will be at any point in
this sphere of influence. Therefore, we have to be guarded about
claiming very localized effects or claiming that concentrations are physiological.
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ACKNOWLEDGEMENTS |
The hollow fibers were a gift from Terumo (Tokyo, Japan).
This research was supported in part by Grants-in-Aid for Scientific Research 10557009 and 11470019 (to H. Kannan) from the Japan Ministry of Education, Science, Sports, and Culture.
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FOOTNOTES |
Address for reprint requests and other correspondence: H. Kannan, Dept. of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan (E-mail:
kannan{at}post.miyazaki-med.ac.jp).
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.
Received 28 December 2000; accepted in final form 20 June 2001.
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