Vol. 273, Issue 4, R1492-R1500, October 1997
Lateral hypothalamic injection of
GABAA antagonist induces
gastric vagus-mediated hypocalcemia in the rat
Katsuhiko
Shiramine,
Shuji
Aou, and
Tetsuro
Hori
Department of Physiology, Faculty of Medicine, Kyushu University 60, Fukuoka 812-82, Japan
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ABSTRACT |
The
involvement of lateral hypothalamic area (LHA) neurons in the
regulation of blood calcium homeostasis was investigated in
unanesthetized rats. The microinjection of the 
-aminobutyric acid A
receptor antagonist bicuculline methiodide (BM, 4-40
ng · 0.5 µl
1 · 5 min1) into the LHA
decreased the blood concentration of ionized calcium. Total serum
calcium also decreased after the BM injection. This hypocalcemic effect
was eliminated by a bilateral vagotomy of the gastric branches. An
intravenous injection of atropine methyl bromide (a muscarinic
antagonist), nadolol (a 
-adrenergic blocker), or ranitidine (a
histamine H2 blocker) suppressed
the BM-induced hypocalcemia, whereas phenoxybenzamine (an
-adrenergic blocker) proved to be ineffective. Although the
intra-LHA injection of BM increased the serum gastrin, which is known
to have a hypocalcemic effect, neither secretin nor somatostatin
(gastrin-release inhibitors) blocked the hypocalcemic response. These
results suggest that the hypocalcemia observed after the excitation of
LHA neurons was mediated by muscarinic, -adrenergic, and histamine
H2 receptors through the gastric
vagus.
calcium homeostasis; stomach; histamine
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INTRODUCTION |
ALTHOUGH CALCIUM HOMEOSTASIS is well known to be
regulated by calciotropic hormones acting peripherally on bone, kidney,
and intestine, little is yet known about its central regulation
mechanisms. It has recently been shown that calcitonin and parathyroid
hormone (PTH) alter the activity of hypothalamic neurons through their actions on respective receptors (15, 26). Whereas an
intracerebroventricular injection of calcitonin reduces blood calcium
levels (6), an injection of PTH reverses the urethan-induced
hypocalcemia in a dose-dependent manner (15). Moreover, the electrical
stimulation of the lateral hypothalamic area (LHA), paraventricular
nucleus (PVN), or ventromedial nucleus of the hypothalamus (VMH)
decreases the concentration of blood calcium in anesthetized rats (1, 13). These findings, taken together, thus suggest that the
hypothalamus is involved in the regulation of calcium
homeostasis.
Furthermore, we found that the calcium-lowering effects of the
electrical stimulation of the LHA and VMH were specifically blocked by
a bilateral gastric vagotomy, whereas PVN stimulation-induced hypocalcemia was abolished by a vagotomy of the thyroid-parathyroid branches (13). The immobilization stress-induced hypocalcemia was thus
eliminated by VMH lesions (1, 2) as well as by a gastric vagotomy but
not by a vagotomy of the thyroid-parathyroid branches (14). In
addition, pretreatment with a muscarinic antagonist (atropine),
inhibitors of gastrin release (secretin and galanin), or antagonists of
histamine H2 receptor (cimetidine
and ranitidine) also blocked the stress-induced hypocalcemia (3, 14).
Moreover, not only a gastrectomy but also a fundectomy or antrectomy
abolished the hypocalcemic effect of immobilization (3). These findings suggest that the vagally mediated releases of gastrin and histamine in
the stomach may therefore participate in the calcium-lowering process
during immobilization stress.
Although hypocalcemia is observed after the electrical stimulation of
LHA in the anesthetized rat (13), it remains to be determined whether
hypocalcemia is induced in an unanesthetized condition either by the
activation of LHA neurons or by stimulation of nerve fibers passing
through the site. Using both pharmacological and surgical treatments,
we undertook the present study 1) to elucidate whether or not the excitation of LHA neurons decreases the
blood calcium concentration and 2)
to investigate its peripheral mechanisms in awake and freely behaving
rats.
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MATERIALS AND METHODS |
Animals.
Female Wistar rats weighing 210-270 g were used because female
rats have been shown to be more sensitive than male rats to attempts to
alter the blood calcium in experimental manipulations (16). The rats
were housed in groups of three or four in our closed colony and were
maintained on a 12:12-h light-dark cycle (light period, 0800-2000)
in a constant air-conditioned environment of 23 ± 1°C and 60 ± 10% relative humidity. They had free access to laboratory chow
and tap water.
Surgery.
The animals were anesthetized with pentobarbital sodium (30-50
mg/kg ip) and placed in a stereotaxic apparatus. A stainless steel
guide cannula (0.5 mm OD) was unilaterally (left side) implanted into a
position 1 mm above the LHA (anterior-posterior 6.20, lateral 1.8, depth 8.5-9.0) according to the atlas of Paxinos and
Watson (19). The stainless steel cannula was anchored firmly to screws in the skull by dental cement. Thereafter, some animals underwent a
selective vagotomy 7 days after implantation of the cannula. While rats
were under pentobarbital sodium anesthesia (30-50 mg/kg ip), the
vagal nerves were cut bilaterally at either the gastric, celiac, or
hepatic branches. In the sham-operated rats, the vagal nerves were
exposed at the respective sites but were not cut. At least 5 days were
allowed for postsurgical recovery. Subsequently, 2 days before the
experiment, while rats were under ketamine anesthesia (25 mg/kg), a
Silastic catheter (1.0 mm OD, 0.5 mm ID) filled with heparinized (20 IU/ml, Ciba Corning) physiological saline was inserted into either the
right jugular vein or the right femoral artery for sampling of blood.
Hypothalamic injection and measurements of blood ionized calcium
and pH and arterial blood pH,
PCO2, and
PO2.
All experiments were done with the rats in a freely moving condition.
The rats were fasted overnight (with only water available). A stainless
steel injection cannula filled with bicuculline methiodide (BM,
4-40 ng) or Ringer solution was inserted through the guide cannula
and placed in a position 1 mm beyond its tip. Because an injection of
glutamate (0.05-0.5 M in concentration, 0.5 µl in volume) did
not elicit hypocalcemia (our unpublished observation), we used BM to
activate LHA neurons with inhibition of -aminobutyric acidergic
inputs. After 120-150 min, the drug in a fluid volume of 0.5 µl
was administrated into the LHA slowly over a 5-min period. We intended
to use a relatively large volume (0.5 µl) to cover the entire LHA at
the level of VMH to avoid inconsistent responsiveness, as previously
reported (24).
Two baseline blood samples (0.15 ml each) were collected 30 min and
just before the LHA injection of drugs. If the difference between these
two baseline values of the blood concentration of ionized calcium was
>3.5%, an additional baseline blood sample was then collected after
another 30-min interval. If the baseline levels were still
unstable, no further blood sampling was performed. After we confirmed
stable basal calcium levels, the blood samples were collected 15, 30, 60, 90, and 120 min after the injection. The concentration of the
ionized calcium and pH in the whole blood was measured by ion-selective
electrodes (643 Ca2+/pH Analyzer,
Ciba Corning). PO2,
PCO2, and pH of arterial blood (0.25 ml each) were measured by a pH-blood gas analyzer (238, Ciba Corning).
Each measurement was duplicated, and the average value was used for the
data analysis.
Measurements of serum total calcium, blood sodium, potassium,
chloride, and serum gastrin.
In a separate series of experiments, a 5-ml blood sample was collected
30 min after the LHA administration of either BM or Ringer solution.
The concentration of sodium, potassium, and chloride in the whole blood
was measured by ion-selective electrodes (644 Na/K/Cl analyzer, Ciba
Corning). The concentration of total calcium and magnesium in the serum
was then measured by a calcium-magnesium meter (EDTA titration method,
Joko). The concentration of serum gastrin was then determined by a
radioimmunoassay (Gastrin RIA kit II, Dinabot) with use of blood
samples taken from the catheter 15 min after the LHA injection of
either BM or Ringer solution. Each measurement was done either done two
or three times, and the average value was then used for the data
analysis.
Pharmacological treatment.
Various pharmacological manipulations were made to identify the
peripheral mechanisms of the central BM-induced hypocalcemia. Atropine
methyl bromide (0.1 and 0.6 mg/kg, Sigma, St. Louis, MO) and secretin
(6 µg/kg, Peptide Institute) were injected intravenously 5 min before
BM injection. Phenoxybenzamine (PBZ, 3 mg/kg, Tokyo Chemical), nadolol
(2 mg/kg, Sigma), and ranitidine (5 mg/kg, Sigma) were administered
intravenously 20 min before the BM injection. These drugs were
dissolved in Ringer solution before use and were injected in a fluid
volume of 0.1 ml (atropine, ranitidine, and secretin) or 0.2 ml (PBZ
and nadolol). Somatostatin (1 µg · h1 · rat1,
Sigma) was dissolved in physiological saline and was then continuously administered for 25 min by intravenous drip infusion starting 5 min
before the BM injection.
Histology.
At the end of the experiments, 0.5 µl of Pontamine sky blue was
injected through the LHA cannula. The rats were deeply anesthetized with an overdose of pentobarbital sodium (60 mg/kg ip) and were transcardially perfused with 10% Formalin. The brain was cut into 120-µm serial frozen sections by a microtome and was stained with Neutral red to identify the site of injection histologically. Only the
data obtained from the animals that were found to have the tip of the
cannula positioned correctly in the LHA were used for the analyses.
Statistics.
Results are expressed as means ± SE. The changes in either the
blood calcium or pH levels were calculated by subtracting the blood
calcium or pH value just before the injection of BM or vehicle (time 0) from the values observed
after injection. Either the unpaired Student's
t-test or the one-way or two-way
analysis of variance (ANOVA) with post hoc Bonferroni test was used for
the statistical analyses. P values
<0.05 were considered statistically significant.
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RESULTS |
Changes in blood levels of ionized calcium and pH after BM injection
into LHA.
The basal blood concentration of ionized calcium of the awake rat 30 min before and just before the intra-LHA injection of BM or vehicle
ranged from 1.30 to 1.45 mM. There was no significant difference in
basal level (time 0) among the
vehicle-injected control group (1.39 ± 0.01 mM,
n = 5) and the BM-injected groups (4, 12, 30, and 40 ng; 1.37 ± 0.01, 1.40 ± 0.004, 1.40 ± 0.02, and 1.37 ± 0.02 mM, respectively;
n = 4 or 5). Although intra-LHA injection of 4 ng BM did not affect the concentration of blood ionized
calcium, the injection of 12, 30, and 40 ng of BM significantly decreased calcium concentration in a dose-dependent manner
[F(4,90) = 3.69, P < 0.001, 2-way ANOVA; Fig.
1A].
The blood calcium level significantly decreased 15 min after the start
of the BM injection (12-40 ng)
[F(4,18) = 5.05, P < 0.01, 1-way ANOVA, in comparison with that of the vehicle-injected control group], and then the decrease reached a peak 30 min after injection
[F(4,18) = 12.45, P < 0.001, 1-way ANOVA]. Blood
calcium returned to the basal level within 60 min.
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Fig. 1.
Effect of injection of bicuculline methiodide (BM) in lateral
hypothalamic area (LHA) on blood ionized calcium levels
( Ca2+,
A), ionized calcium levels adjusted
for pH 7.5 (Ca2+adj,
B), and blood pH (pH,
C). Each point represents mean ± SE. n, No. of rats/group.
* P < 0.05, ** P < 0.005 decrease below
control value in vehicle-injected rats for same time.
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In association with the hypocalcemia, the blood pH level increased dose
dependently after the BM injection
[F(4,90) = 5.51, P < 0.001; Fig.
1C]. The blood pH level reached
a peak 15 min after the start of the BM injection
[F(4,18) = 12.21, P < 0.001, 1-way ANOVA]. The
increase remained at statistically significant levels 30 min after the
injection [F(4,18) = 11.95, P < 0.001, 1-way ANOVA] and
then returned to the basal level 60 min after the injection.
Changes in arterial blood pH,
PCO2, and
PO2 after BM injection into
LHA.
Because the blood concentration of ionized calcium is known to be
affected by blood pH, we subsequently sought a theoretical basis for
the adjustment of the calcium data for pH 7.4. To determine the nature
of the BM injection-induced alkalosis, we measured pH,
PO2, and
PCO2 of the arterial blood of the BM
(30 ng)- and vehicle-injected groups. The arterial blood pH and
PCO2 of the BM-injected group were
significantly different from those of the vehicle-injected rats
[pH, F(1,50) = 11.06, P < 0.001;
PCO2,
F(1,50) = 5.22, P < 0.001; 2-way ANOVA;
Fig.
2A].
Fifteen minutes after the BM injection, the arterial blood pH increased
significantly to 7.54 ± 0.01 Torr (P < 0.05), and
PCO2 decreased significantly to 27.86 ± 0.91 Torr (P < 0.05) in
comparison with the values of the vehicle-injected group (pH, 7.48 ± 0.02; PCO2, 34.75 ± 1.89 Torr). Arterial blood pH and PCO2
returned to the basal levels 30 min after the injection. When
PCO2 and pH of the BM-injected group
at 0, 15, and 30 min after BM injection were plotted on the acid-base
chart (29), the values at 15 and 30 min were within the area of acute
hypocapnia, indicating respiratory alkalosis (Fig.
2B). There was no significant
difference in the arterial blood PO2
between the BM- and vehicle-injected groups (data not shown).
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Fig. 2.
Arterial blood PCO2 and pH
(A) and their plots on acid-base
chart (29) (B). Each point
represents mean ± SE. n, No. of
rats/group. * P < 0.05 difference from control value in vehicle-injected rats at same time.
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Changes in blood level of ionized calcium adjusted for pH 7.4, total
calcium level, and other blood electrolytes after BM injection into
LHA.
In the case of respiratory alkalosis, the blood
Ca2+ levels can be well adjusted
for pH to evaluate pH-independent changes in blood
Ca2+ with use of the following
equation (5)
The data thus adjusted for pH 7.4 revealed a dose-dependent
hypocalcemia after the BM injection
[F(4,90) = 2.05, P < 0.05, 2-way ANOVA]. The
pH-adjusted change in ionized calcium
(Ca2+adj) reached a minimum 30 min after
the injection. [F(4,18) = 4.74, P < 0.01, 1-way ANOVA;
Fig. 1B] and then recovered
gradually.
To further characterize the BM-induced hypocalcemia, we measured the
serum levels of total calcium and magnesium (Table
1). The total calcium level of the BM (30 ng)-injected group was significantly lower at 30 min after BM injection
than that of the vehicle-injected group
(P < 0.05), whereas the serum
magnesium levels did not differ between two groups (Table 1). Blood
concentrations of sodium and chloride of the BM-injected group did not
differ from those of the vehicle-injected group either. However, the
blood potassium level of the BM (30 ng)-injected group was
significantly lower than that of the vehicle-injected group
(P < 0.05).
Histological examination.
The results of the histological examination are summarized in Fig.
3. The hypocalcemic effect of the BM
injection was observed when the cannula tips were located within the
LHA. The injection of the vehicle into comparable sites in the LHA had
no hypocalcemic effect. The sites of BM injection in the animals used
to examine the effects of selective vagotomy or pharmacological
pretreatments were also located within this area (See Figs. 5, 7, and
9).
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Fig. 3.
Location of cannula tips in vehicle-injected rats
(left) and BM-injected rats
(right). Symbols represent different
levels of decreases in blood ionized calcium levels 30 min after LHA
stimulation in comparison with basal calcium levels measured before
stimulation. (×   0.03 mM, 0.04  0.07 mM,   0.08 mM). Drawings were made on the basis
of the atlas of Paxinos and Watson (19), with slight
modifications. Numbers at left bottom,
rostrocaudal distance (mm) from interaural line.
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Effects of selective subdiaphragmatic vagotomy on hypocalcemia
induced by BM injection into LHA.
To determine the involvement of the vagal nerves in BM
injection-induced hypocalcemia, we examined the effects of a selective vagotomy (Figs. 4 and
5). A gastric vagotomy almost completely abolished the hypocalcemia induced by the BM injection (Fig.
4A, left).
Ca2+ of the gastric-vagotomized
group 30 min after the injection (0.008 ± 0.010 mM,
n = 4) was significantly smaller than
that of the sham-operated group (0.072 ± 0.005 mM,
n = 4, P < 0.01). A gastric vagotomy also
attenuated the BM-induced alkalosis (Fig.
4C,
left). Although the pH did not
change significantly (pH, 0.003 ± 0.011) 30 min after the
BM injection in the gastric-vagotomized group, the pH in the
sham-operated group showed alkalosis (pH, 0.035 ± 0.010, P < 0.05). The pH-adjusted change in
ionized calcium (Ca2+adj) of the
gastric-vagotomized group (0.015 ± 0.010 mM) was also
significantly smaller than that of the sham-operated group
(0.052 ± 0.006 mM, P < 0.05; Fig. 4B,
left).
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Fig. 4.
Effects of selective vagotomy (Vagx) on LHA stimulation-induced
hypocalcemia and alkalosis. A:
Ca2+ from prestimulation basal
level 30 min after LHA stimulation. B:
changes in Ca2+adj 30 min after
stimulation. C: pH 30 min after
stimulation in comparison with prestimulation level. No. of rats in
each group indicated in bars.
* P < 0.05, ** P < 0.01.
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Fig. 5.
Location of cannula tips in sham-operated rats and Vagx rats. Symbols
represent different levels of decreases in blood ionized calcium levels
30 min after LHA stimulation in comparison with basal calcium levels
measured before stimulation. (× 0.03 mM, 0.04 0.07 mM, 0.08 mM).
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On the other hand, a hepatic vagotomy did not affect the BM
injection-induced hypocalcemia and alkalosis (Fig. 4). The
Ca2+ (0.050 ± 0.017 mM), Ca2+adj (0.038 ± 0.012 mM), and pH (0.024 ± 0.009) of the hepatic-vagotomized rats 30 min after the BM injection (n = 5) did
not significantly differ from those of sham-operated rats
(Ca2+, 0.070 ± 0.004 mM; Ca2+adj, 0.050 ± 0.006 mM; pH, 0.033 ± 0.008;
n = 6). A celiac vagotomy was also
ineffective in affecting the BM-induced hypocalcemia
(Ca2+, 0.047 ± 0.012 mM; Ca2+adj,
0.037 ± 0.015 mM; pH, 0.017 ± 0.007;
n = 6). There was no
significant difference in the changes in
Ca2+
[F(2, 14) = 1.23, P = 0.32, 1-way ANOVA], blood pH
[F(2,14) = 1.13, P = 0.35, 1-way ANOVA], and
Ca2+adj
[F(2,14) = 0.43, P = 0.66, 1-way ANOVA] among the
hepatic-, celiac-, and sham-operated groups (Fig. 4).
Effects of pharmacological treatments on BM injection-induced
hypocalcemia.
Because gastric vagal activation has been shown to promote the release
of acetylcholine, gastrin, and histamine, we investigated the effects
of various treatments with their antagonists and release inhibitors on
BM injection-induced hypocalcemia. There was no significant difference
in the basal level of blood Ca2+
between the drug-pretreated groups (ranging from 1.35 ± 0.01 to
1.41 ± 0.02 mM) and the vehicle-pretreated groups (ranging from
1.37 ± 0.01 to 1.41 ± 0.01 mM).
Intravenous injection of atropine methyl bromide (0.1 and 0.6 mg/kg),
which does not cross the blood-brain barrier, suppressed the induction
of the BM-induced hypocalcemia in a dose-dependent manner (Figs.
6 and 7). The
Ca2+ of rats pretreated with
atropine (0.1 mg/kg, 0.039 + 0.009 mM, n = 9, P < 0.05; 0.6 mg/kg, 0.018 ± 0.010 mM, n = 11, P < 0.005) showed significant
differences from that of the vehicle-pretreated control rats
(0.072 ± 0.009 mM, n = 5 and 0.070 ± 0.012 mM, n = 7, respectively). Atropinization also blocked changes in the blood pH
induced by a BM injection (0.1 mg/kg, 0.019 ± 0.006 vs. 0.038 ± 0.012, P < 0.01; 0.6 mg/kg,
0.003 ± 0.013 vs. 0.034 ± 0.007, P < 0.05). Although there was no
significant difference in the pH-adjusted
Ca2+ between the groups
pretreated with 0.1 mg/kg of atropine (0.042 ± 0.006 mM) and
vehicle (0.048 ± 0.004 mM), the 0.6 mg/kg treatment with
atropine suppressed the BM-induced decrease in pH-adjusted Ca2+ (0.6 mg/kg, 0.026 ± 0.006 mM vs. vehicle, 0.049 ± 0.007 mM; P < 0.05; Fig. 6).
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Fig. 6.
Effects of atropine methyl bromide (Atr, muscarinic receptor
antagonist), phenoxybenzamine (PBZ, -adrenergic blocker), and
nadolol (Nad, -adrenergic antagonist) on LHA stimulation-induced
hypocalcemia and alkalosis. A:
Ca2+ 30 min after LHA
stimulation from prestimulation basal level.
B:
Ca2+adj 30 min after stimulation.
C: pH 30 min after stimulation in
comparison with prestimulation level. Each bar represents mean ± SE. Open bars, vehicle-pretreated control. Hatched bars, drug
pretreated. No. of rats in each group indicated in each bar.
* P < 0.05, ** P < 0.005, *** P < 0.0001.
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Fig. 7.
Location of cannula tips in rats pretreated with vehicle or muscarinic
and/or adrenergic blockers. Symbols represent different levels
of decreases in blood ionized calcium levels 30 min after LHA
stimulation in comparison with basal calcium levels measured before
stimulation. (× 0.03 mM, 0.04 0.07 mM, 0.08 mM).
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It has been shown that the gastric vagus nerves contain adrenergic
fibers (7). We thus investigated the possible involvement of adrenergic
mechanisms. PBZ (3 mg/kg iv), an -adrenergic antagonist, failed to
affect the BM-induced hypocalcemia and alkalosis. When the
-adrenergic antagonist nadolol (2 mg/kg iv), which does not easily
cross the blood-brain barrier, was given, the degree of hypocalcemia
was significantly suppressed
(Ca2+, 0.013 ± 0.013 mM, P < 0.05;
Ca2+adj, 0.010 ± 0.011 mM,
P < 0.005;
n = 12) in comparison with the
vehicle-pretreated group (Ca2+,
0.059 ± 0.011 mM;
Ca2+adj, 0.041 ± 0.005 mM;
n = 7) without affecting alkalosis
(pH of nadolol-pretreated group, 0.033 ± 0.005; pH of
vehicle-pretreated group, 0.043 ± 0.012). When the rats
were pretreated with both nadolol (2 mg/kg iv) and atropine (0.6 mg/kg
iv), hypocalcemia was completely blocked (Ca2+ of rats pretreated with
nadolol and atropine, 0.002 ± 0.006 mM, P < 0.0001;
Ca2+adj, 0.006 ± 0.008 mM,
P < 0.05; n = 5 vs.
Ca2+ of rats pretreated with
vehicle, 0.083 ± 0.003 mM;
Ca2+adj, 0.053 ± 0.013 mM;
n = 3; Fig. 6).
Pretreatment with the histamine H2
receptor antagonist ranitidine (5 mg/kg iv) blocked the hypocalcemic
effect of BM injection (Ca2+,
0.031 ± 0.007 mM, n = 11, P < 0.05;
Ca2+adj, 0.006 ± 0.008 mM,
P < 0.05), but it did not affect the
alkalosis (pH, 0.036 ± 0.010; Figs.
8 and 9).
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Fig. 8.
Effects of histamine H2 blocker (ranitidine)
and gastrin-release inhibitors (secretin and somatostatin) on LHA
stimulation-induced hypocalcemia and alkalosis.
A:
Ca2+ 30 min after LHA
stimulation from prestimulation basal level.
B:
Ca2+adj 30 min after stimulation.
C: pH 30 min after stimulation in
comparison with prestimulation level. Each bar represents mean ± SE. Open bars, pretreated vehicle; hatched bars, drug pretreated. No.
of rats in each group indicated in each bar.
* P < 0.05.
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Fig. 9.
Location of cannula tips in rats pretreated with vehicle, histamine
H2 blocker, or gastrin-release inhibitors.
Symbols represent different levels of decreases in blood ionized
calcium levels 30 min after LHA stimulation in comparison with basal
calcium levels measured before stimulation. (× 0.03 mM,
0.04 0.07 mM, 0.08 mM).
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In contrast, secretin (6 µg/kg iv), an inhibitor of gastrin release,
did not affect Ca2+
(0.070 ± 0.006 mM, n = 5),
Ca2+adj (0.040 ± 0.011 mM,
n = 5), or pH (0.052 ± 0.013).
Somatostatin (1 µg/h), the most potent inhibitor of gastrin release,
which was infused intravenously for 25 min starting 5 min before BM
injection, also failed to eliminate hypocalcemia
(Ca2+, 0.047 ± 0.011 mM; Ca2+adj, 0.034 ± 0.013 mM) and alkalosis (pH, 0.023 ± 0.007, n = 7; Figs. 8 and 9).
Serum gastrin after BM injection into LHA.
The concentration of gastrin in the serum significantly increased after
the BM injection into the LHA (186.0 ± 25.4 pg/ml, n = 9) in comparison with that of the
vehicle-injected group (97.0 ± 20.1 pg/ml,
n = 7, P < 0.05; Fig.
10).
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Fig. 10.
Effect of BM injection in LHA on serum gastrin level. No. of rats in
each group indicated in each bar.
* P < 0.05.
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DISCUSSION |
The present study demonstrated that an intra-LHA injection of BM
induced hypocalcemia, and this effect was eliminated either by a
gastric vagotomy or pretreatment with either muscarinic, -adrenergic, or histaminergic H2 receptor
antagonist. The decrease in blood calcium levels (0.05-0.1 mM,
0.2-0.4 mg/dl) shown in the present study did not appear to be a
great change; similar hypocalcemic responses have been shown after
immobilization or an injection of hypocalcemic agents (1, 3, 13, 14,
20) that may be sufficient enough to trigger the calcium-regulating responses (~60-70% of the maximum response of PTH) as well as other physiological and psychological responses (2, 14).
The blood concentration of ionized calcium is known to depend on the
blood pH (21), which increases with the development of alkalosis. In
the present study, the BM injection into the LHA resulted in an
increase in blood pH that preceded a decrease in the blood calcium. In
the case of respiratory alkalosis, the blood ionized calcium levels can
be adjusted for blood pH (5). In the present study, arterial blood gas
analyses revealed that the changes in pH were caused by respiratory
alkalosis; we therefore calculated the blood level of
Ca2+ adjusted for pH 7.4. The
blood level of the Ca2+adj significantly
decreased 30 min after the BM injection in a dose-dependent manner. The
serum level of total calcium also significantly decreased. We thus
conclude that the hypocalcemic response is not a result of the
associated respiratory alkalosis.
Blood concentrations of sodium, chloride, and magnesium did not change
after the BM injection, although hypocalcemia and hypokalemia did
occur. This indicates that the effect of BM injection into the LHA on
the peripheral blood electrolytes is selective and not caused by any
nonspecific mechanisms such as dilution caused by fluid shift or water
retention. Although the mechanisms for the induction of hypokalemia
remain unknown, this is the first demonstration that the neuronal
activation of LHA elicits hypokalemia. It has been shown that the
hypothalamus may be involved in potassium metabolism through renal
electrolyte regulation and/or the production of endogenous
Na+-K+
adenosinetriphosphatase inhibitors in the hypothalamus (23). Several
clinical investigations have also suggested that brain injury induces
hypokalemia through the peripheral catecholaminergic systems (22).
Further study is necessary, however, to elucidate the precise
mechanisms and physiological significance of the central nervous
system, but these findings suggest that it is involved in the control
of not only the blood calcium level but also the blood potassium
concentration.
The hypocalcemic effect after the BM injection was observed when the
cannula tips were located within the area of the LHA, as shown in Fig.
3. In this study, the volume of the BM solution (0.5 µl) was used to
cover the entire LHA at the level of VMH. This volume might be
relatively large in comparison with some microinjection studies (24,
30), but the effective sites were confined to within the LHA and the
effect was greatly attenuated or disappeared when the tips of cannulas
were located outside but in the vicinity of the LHA (Fig. 3). The rate
of administration (0.1 µl/min) was slow enough to avoid any
nonspecific effects. We did not observe any nonspecific effects such as
irritability, convulsion, or other aggressive behavior except for
digging behavior. The present results therefore suggest that the
hypocalcemic effect of the BM injection was induced by neural
activation of the LHA, not of other hypothalamic loci. In our
preliminary study, we found that a microinjection of a smaller volume
of BM into the VMH or PVN (0.3 or 0.2 µl, respectively) also induced
hypocalcemia. The effects were also greatly attenuated when the
injection sites were in the vicinity of, but outside, these areas
(unpublished observation).
In the previous studies in awake rats, we found that the stomach was
involved in blood calcium regulation during immobilization stress (3,
14) and that the hypocalcemic effects of electrical stimulation of the
LHA in anesthetized rats were abolished by a vagotomy of the gastric
branches but not by a vagotomy of the thyroid-parathyroid branches
(13). LHA stimulation has been shown to facilitate activities of
neurons of the dorsomotor nucleus of the vagus (17, 27) and gastric
vagal nerves (27). In the present study, the hypocalcemia induced by
the BM injection into the LHA was eliminated by a vagotomy of gastric
branches but not by that of the celiac or hepatic branches. Although a nonvagal mechanism cannot be ruled out, these findings suggest that the
gastric vagal nerves almost exclusively mediate the hypocalcemic effect
of LHA neuronal activation.
Atropinization has been shown to mimic the effect of a vagotomy in many
experimental conditions, and atropine methyl bromide, which did not
cross the blood-brain barrier, significantly suppressed the
hypocalcemic effect of LHA neuronal activation in the present study. This indicates that muscarinic receptors mediate, at least in
part, the hypocalcemic effect of an intra-LHA injection of BM through
the gastric vagal nerves. In a previous study, we found that atropine
methyl bromide at 0.6 mg/kg completely abolished the
immobilization-induced hypocalcemia (14). In the present study,
however, a slight decrease in the blood calcium levels was still
observed after the BM injection in rats pretreated with the same dose
of atropine methyl bromide. It is known that the gastric vagal nerves
contain not only cholinergic fibers but also catecholaminergic fibers
(12) and that a vagotomy reduces the catecholamine levels in the
stomach wall by 50% (7). Because nadolol, a peripherally acting
-adrenoceptive blocker, also suppressed the BM-induced hypocalcemia
in the present study, the hypocalcemia after the LHA neuronal
activation is mediated by -adrenergic receptor mechanisms as well as
muscarinic receptor mechanisms. It is worthwhile to note that atropine
methyl bromide suppressed both hypocalcemia and alkalosis, whereas
nadolol eliminated only the hypocalcemic response without affecting the
alkalosis. Therefore, different effects of cholinergic and adrenergic
inputs to gastric target cells may be involved in this phenomenon.
Gastrin and histamine, which are released from the stomach by vagal
stimulation, are known as hypocalcemic agents (10, 20). The
hypocalcemic effects of both agents are eliminated by gastrectomy (10,
20), thus indicating that the target site of these chemicals is the
stomach. Because a histamine H2 antagonist
(ranitidine) was shown to block the BM-induced hypocalcemia, the
release of histamine as a result of LHA neuronal activation may thus be
involved in hypocalcemia. In contrast, somatostatin and secretin,
inhibitors of gastrin release, failed to affect BM injection-induced
hypocalcemia. It has been reported that a -adrenergic agonist may
release gastrin from the stomach and that gastrin decreases the blood
calcium level through the release of thyrocalcitonin (4) and/or
the putative gastric hypocalcemic factor gastrocalcin (20). The present
results, however, suggest that gastrin is not an essential factor for
BM injection-induced hypocalcemia, even though an intra-LHA injection
of BM facilitates the release of gastrin.
Although homeostasis of blood calcium has long been thought to be
regulated by peripheral mechanisms, especially by calciotropic hormones, the present study demonstrates that LHA neurons also participate in the control of the blood calcium level by changes in the
activity of the gastric vagal nerves that involve the mechanisms of
muscarinic, -adrenergic, and histamine H2
receptors.
Perspectives
The physiological significance of the hypocalcemic action of the LHA
currently remains to be clarified. The LHA is well known to be involved
in the control of feeding behavior and the related visceral functions,
including secretion of gastric acid and insulin during the cephalic
phase (9). LHA neurons have been shown to respond to the sight, taste,
and/or smell of food (11, 25). The LHA contains a particular
group of neurons, designated as glucose-sensitive neurons, the firing
rate of which specifically decreases in response to glucose
administered either systemically or locally (18). The majority of
glucose-sensitive neurons respond to both taste and odor stimuli (11),
and the activation of these neurons promotes gastric acid secretion
(28). It has been reported that food intake never elicits hypercalcemia
but instead induces transient hypocalcemia (0.05 mM decrease) at an
early phase of food intake that may be mediated by the secretion of
gastrin and histamine (8). These findings together with the present
results thus support the hypothesis that the hypocalcemic mechanism of the LHA-gastric vagal axis plays a role in the cephalic control of
calcium homeostasis, thereby preventing a postprandial increase in
blood calcium concentration.
| |
ACKNOWLEDGEMENTS |
We thank Dr. B. T. Quinn for critical comments and help in
preparing this manuscript.
| |
FOOTNOTES |
This study was supported by Grants-In-Aid (05NP0101 and 07557008) for
Scientific Research from the Ministry of Education, Science, and
Culture of Japan (S. Aou) and a Research Grant for Nervous and Mental
Disorders from the Ministry of Health and Welfare of Japan (S. Aou).
Address reprint requests to S. Aou.
Received 29 October 1996; accepted in final form 10 July 1997.
| |
REFERENCES |
1.
Aou, S.,
J. Ma,
and
T. Hori.
Hypothalamic involvement in stress-induced hypocalcemia in rats.
Neurosci. Lett.
158:
197-200,
1993[Medline].
2.
Aou, S.,
J. Ma,
T. Hori,
and
N. Tashiro.
Hypothalamic linkage in stress-induced hypocalcemia, gastric damage, and emotional behavior in rats.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R38-R43,
1994[Abstract/Free Full Text].
3.
Aou, S.,
J. Ma,
K. Shiramine,
and
T. Hori.
The stomach is the etiologic organ for immobilization-induced hypocalcemia in rats.
Am. J. Physiol.
265 (Regulatory Integrative Comp. Physiol. 34):
R1376-R1379,
1993[Abstract/Free Full Text].
4.
Cooper, C. W.,
C. R. Biggerstaff,
C. W. Wiseman,
and
M. F. Carlone.
Hypocalcemic effect of pentagastrin and related gastrointestinal hormonal peptides in the rat.
Endocrinology
91:
1455-1461,
1972[Medline].
5.
Fogh-Andersen, N.
Ionized calcium analyzer with a built-in pH correction.
Clin. Chem.
27:
1264-1267,
1981[Abstract/Free Full Text].
6.
Goltzman, D.,
and
G. S. Tannenbaum.
Induction of hypocalcemia by intracerebroventricular injection of calcitonin: evidence for control of blood calcium by the nervous system.
Brain Res.
416:
1-6,
1987[Medline].
7.
Graffner, H.,
M. Ekelund,
R. Håkanson,
J. Oscarson,
E. Rosengren,
and
F. Sundler.
Effects of upper abdominal sympathectomy on gastric acid, serum gastrin, and catecholamines in the rat gut.
Scand. J. Gastroenterol.
19:
711-716,
1984[Medline].
8.
Håkanson, R.,
P. Persson,
and
J. Axelson.
Elevated serum gastrin after food intake or acid blockade evokes hypocalcemia.
Regul. Pept.
28:
131-136,
1990[Medline].
9.
Holmes, L. J.,
L. H. Storlien,
and
G. A. Smythe.
Hypothalamic monoamines associated with the cephalic phase insulin response.
Am. J. Physiol.
256 (Endocrinol. Metab. 19):
E236-E241,
1989[Abstract/Free Full Text].
10.
Kaplan, E. L.,
P. T. North,
H. P. Norberg,
J. A. Schulak,
and
B. J. Hill.
Evidence for a role of the stomach in serum calcium regulation.
J. Surg. Res.
22:
237-241,
1977[Medline].
11.
Karádi, Z.,
Y. Oomura,
H. Nishino,
T. R. Scott,
L. Lénárd,
and
S. Aou.
Responses of lateral hypothalamic glucose-sensitive and glucose-insensitive neurons to chemical stimuli in behaving rhesus monkeys.
J. Neurophysiol.
67:
389-400,
1992[Abstract/Free Full Text].
12.
Lundberg, J.,
H. Ahlman,
A. Dahlstrom,
and
J. Kewenter.
Catecholamine-containing nerve fibres in the human abdominal vagus.
Gastroenterology
70:
472-474,
1976[Medline].
13.
Ma, J.,
S. Aou,
and
T. Hori.
Hypothalamic stimulation induced vagally mediated hypocalcemia in the rat.
Brain Res. Bull.
33:
65-69,
1994[Medline].
14.
Ma, J.,
S. Aou,
H. Matsui,
and
T. Hori.
Gastric vagus mediates immobilization-induced hypocalcemia in rats.
Am. J. Physiol.
265 (Regulatory Integrative Comp. Physiol. 34):
R609-R614,
1993[Abstract/Free Full Text].
15.
Matsui, H.,
S. Aou,
J. Ma,
and
T. Hori.
Central actions of parathyroid hormone on blood calcium and hypothalamic neuronal activity in the rat.
Am. J. Physiol.
268 (Regulatory Integrative Comp. Physiol. 37):
R21-R27,
1995[Abstract/Free Full Text].
16.
Morimoto, S.,
A. Fausto,
S. J. Birge,
and
L. V. Avioli.
Effect of short- and long-term stress on plasma calcium and calcitonin in the rat.
Horm. Metab. Res.
18:
818-820,
1986[Medline].
17.
Nishimura, H.,
and
Y. Oomura.
Effects of hypothalamic stimulation on activity of dorsomedial medulla neurons that respond to subdiaphragmatic vagal stimulation.
J. Neurophysiol.
58:
655-675,
1987[Abstract/Free Full Text].
18.
Oomura, Y.,
H. Ooyama,
M. Sugimori,
T. Nakamura,
and
Y. Yamada.
Glucose inhibition of the glucose-sensitive neurons in the lateral hypothalamus.
Nature
247:
284-286,
1974[Medline].
19.
Paxinos, G.,
and
C. Watson.
The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1986.
20.
Persson, P.,
R. Håkanson,
J. Axelson,
and
F. Sundler.
Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach.
Proc. Natl. Acad. Sci. USA
86:
2834-2838,
1989[Abstract/Free Full Text].
21.
Peterson, N. A.,
G. A. Feigen,
and
J. M. Crismon.
Effect of pH on interaction of calcium ions with serum proteins.
Am. J. Physiol.
201:
386-392,
1961.
22.
Pomeranz, S.,
S. Constantini,
and
Z. H. Rappaport.
Hypokalemia in severe head trauma.
Acta Neurochir. (Wien)
97:
62-66,
1989[Medline].
23.
Rabinowitz, L.,
and
R. I. Aizman.
The central nervous system in potassium homeostasis.
Front. Neuroendocrinol.
14:
1-26,
1993[Medline].
24.
Roeling, T. A. P.,
M. R. Kruk,
R. Schuurmans,
and
J. G. Veening.
Behavioral responses of bicuculline methiodide injections into the ventral hypothalamus of freely moving, socially interacting rats.
Brain Res.
615:
121-127,
1993[Medline].
25.
Rolls, E. T.,
E. Murzi,
S. Yaxley,
S. J. Thorpe,
and
S. J. Simpson.
Sensory-specific satiety: food-specific reduction in responsiveness of ventral forebrain neurons after feeding in the monkey.
Brain Res.
368:
79-86,
1986[Medline].
26.
Shimizu, N.,
and
Y. Oomura.
Calcitonin-induced anorexia in rats: evidence for its inhibitory action on lateral hypothalamic chemosensitive neurons.
Brain Res.
367:
128-140,
1986[Medline].
27.
Shiraishi, T.
Effects of lateral hypothalamic stimulation on medulla oblongata and gastric vagal neural responses.
Brain Res. Bull.
5:
245-250,
1980[Medline].
28.
Shiraishi, T.
Hypothalamic control of gastric acid secretion.
Brain Res. Bull.
20:
791-797,
1988[Medline].
29.
Sigaard-Anderssen, O.
The Acid-Base Regulation: Its Physiology, Pathophysiology and the Interpretation of Blood Gas Analysis. Philadelphia, PA: Saunders, 1974.
30.
Soltis, R. P.,
and
J. A. DiMicco.
GABAA and excitatory amino acid receptors in dorsomedial hypothalamus and heart rate in rats.
Am. J. Physiol.
260 (Regulatory Integrative Comp. Physiol. 29):
R13-R20,
1991[Abstract/Free Full Text].
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Abstract
Introduction
![adjusted [Ca<SUP>2+</SUP>] = required [Ca<SUP>2+</SUP>]](/content/vol273/issue4/fulltext/R1492/img001.gif)
![⋅ [1 + 0.53 (required pH − adjusted pH)]](/content/vol273/issue4/fulltext/R1492/img002.gif)
