INTRODUCTION
revious studies have shown that nitric oxide (NO), as
endothelium-derived relaxing factor, is synthesized from L-arginine in the
central nervous system (CNS) by NOS (NO synthase) and acts through cGMP
formation [1]. NOS is distributed in brain regions related to the regulation of
cardiovascular functions [2].
The
caudal zone of the nucleus tractus solitarii (NTS) is the site of the first
synapse of baroreceptor fibers where NTS interacts with the nucleus reticularis
lateralis (NRL) located in the rosteroventrolateral part of the medulla (RVLM)
via a nitric oxidergic pathway [3, 4]. The basal production of NO in the NTS is
153 nmol/L [5], its sources within the NTS are peripheral afferent inputs to
the nucleus, local interneuron within the NTS, and inputs from other central
sites to the NTS [6, 7]. It is revealed that NO plays a key role in glucose and
cardiovascular homeostasis and drugs releasing NO may represent potential new
treatment for insulin resistance [8].
It was
reported that microinjection of L-arginine and sodium nitroprusside (SNP) into
the NTS decreased blood pressure (BP) but L-NAME (NO synthase inhibitor)
increased BP [9-12]. Many neurons in the caudal NTS recorded in vivo
respond to moderate glycemic fluctuations [13]. The NO plasma levels and
soluble guanylyl cyclase (SGC) levels in diabetes are controversial. In some
studies, the endothelial form of NOS was upregulated in diabetes mellitus and
also plasma concentration of NO in diabetes increased [14, 15], whereas in
other studies activity of vascular SGC diminished in diabetes [16]. Since
glucose infusion would yield a remarkably greater increase in arterial pressure
during NO synthesis blocking [13], in this study, we decided to determine the
effect of hyperglycemia on BP in diabetes during NO synthesis inhibiting or
exciting in NTS, and then to find a way for hypertension treatment in diabetes.
MATERIALS
AND METHODS
Male
Wistar rats (n = 18), weighing 250-300 g, were obtained from the Pasteur
Institute of Iran, Tehran. The rats were kept in individual cage in a room with
controlled light 12 h on/12 h off, and the temperature was maintained at
23°C-24°C with free access to water and food for one week. After 24 hours
fasting, diabetes was induced by a single intraperitoneal injection of 0.2 to
0.3 ml 50 mM of sodium citrate solution (pH 4.5) containing stereptozotocin (65
mg/kg body weight.) After injection, animals were fasted for a further 24 h,
after which plasma glucose levels were checked and diabetes was confirmed
(glucose level > 250 mg/dl) [17].
After about one week, rats were anesthetized with
urethane (1.0 g/kg i.p. and 300 mg/kg i.v.), and then were placed in a
stereotaxic instrument. For NTS
microinjection, a 22-gauge stainless steel guide cannula was implanted
stereotaxically into the NTS with the anterior-posterior coordinates 13.3 mm;
medio-lateral, 0.6 mm; and dorsoventral, 8 mm [18], through which a 28-gauge
stainless steel injection cannula was inserted into the NTS. BP was measured
directly through a cannula placed in the femoral artery and connected to a pressure
transducer (Gould p23 ID) and polygraph (NARCO) [19]. BP (mean, systolic and
diastolic BP) was measured before every injection. Then, the injection cannula
of NTS was connected to a Hamilton microsyringe by polyvinyl tubing and was
filled with L-glutamate (L-Glu, 78 pmol/60 nL) to functionally identify
the NTS. After decrement of BP due to Glu injection, and >60 minutes
recovery, we divide 18 rats into two groups:
In the
first group of diabetic rats, lidocaine (78 pmol/60 nL) was injected to functionally
inhibit the NTS and in the second group, SNP was injected (100 mM, as a
NO-donor). In this group, >60 minutes after SNP microinjection, Glu was
injected for second time and BP change was compared with response to the first
Glu injection.
At the
beginning of experiments, the NTS injection sites were confirmed by
responsiveness to L-Glu administration. A specific decrease in BP
(at least -35 mm Hg) has been demonstrated after microinjection of
2.3 nmol L-Glu in the NTS. The response is restricted to the
intermediate one third of the NTS, and the administration of the
same dose of L-Glu in adjacent areas to the NTS fails to elicit
the response [9]. Injections were given over 10 seconds by air pressure
generated by a hand-held syringe while the pipette tip was positioned in the
NTS. For NTS microinjection, the drugs were dissolved in a sterile saline to
the final concentrations in a volume not exceeding 60 nL. For each drug, only
60 nL was pressure-microinjected into the NTS.
After the
completion of the experiment, ink was injected through the cannula, and the
rats were perfused with saline, followed by a solution of 4% formaldehyde, and
finally with 30% sucrose solution. Section (40
) of the
brainstem was stained with cresyl violet, and proper placement of the pipette
tip in the NTS was verified with histological sections under the microscope.
RESULTS
Our
results indicated that mean blood pressure (MBP) decreased from 106 ± 8 mmHg
(basal level) to 86
12 mmHg (P<0.001) and systolic BP decreased form
1198.02 mmHg to 9314.989 mmHg (P<0.05) by unilateral microinjection
of L-Glu in NTS of diabetic rats (Table 1 and Fig. 1), and it was revealed that
the injection site is appropriate. So, L-Glu injection in diabetic rats caused
hypotension.
Fig.
1. Effect of unilateral
injection of L-glutamate (Glu, 2.3 nmol) into the NTS on blood pressure in
anesthetized diabetic rats. Data are represented by mean SEM. Vertical bars
represent SEM change from baseline values. Each bar represents the average data
from 18 rats. Ps, systolic pressure; Pd, diastolic pressure; Pm, mean blood
pressure; *, P<0.05 and **, P<0.001
significantly different from baseline value.
In the
first group, ten minutes after unilateral lidocaine microinjection in NTS, MBP
increased from 86 ± 6.7
(basal level) to 97 ± 6.9 mmHg
significantly (P<0.01, Table 1). At times of 20, 40 and 60 minutes
after lidocaine injection, it was no significant difference with respect to
basal level. Then, NTS inhibition by lidocaine in diabetic rats caused
hypertension (Fig. 2).
To test
whether NO system was involved in the cardiovascular effects of Glu, SNP was
used in second group. SNP (NO donor) decreased MBP from 89 ± 8.2 (basal level) to 73 ± 10.1 mmHg
Fig.
2. Effect of unilateral injection
of lidocaine (0.5l) into the NTS on blood pressure in anesthetized diabetic
rats. Data are represented by mean SEM. Vertical bars represent SEM change from baseline values.
Each bar represents the average data from 9 rats. Ps, systolic pressure; Pd, diastolic
pressure; Pm, mean blood pressure; *, P<0.01 significantly different
from baseline value.
and diastolic
BP from 83
8.6 to 67 ± 10.07 significantly (P<0.05,
Table 1). Therefore, SNP injection in NTS decreases BP in diabetic rats (Fig.
3).
Finally,
we compared the effect of Glu and SNP on MBP of diabetic rats (88.6 9.7 and 100.6618.31, respectively) that was significantly (P<0.05,
Fig. 4).
In all of the groups, in diabetic rats, BP was
measured before (basal level) and after drug microinjection and so basal level
of BP was as control group.
Fig.
3. Effect of unilateral
injection of SNP (100 mM) into the NTS on blood pressure in anesthetized
diabetic rats. Data are represented by
Mean SEM. Vertical bars represent SEM change from baseline values.
Each bar represents the average data from 9 rats. Ps, systolic pressure; Pd,
diastolic pressure; Pm, mean blood pressure; SNP, sodium nitroprusside;
*, P<0.05 significantly differents form baseline value.
DISCUSSION
The aforementioned studies support the potential in
the CNS system for integration of Glu and NO. Direct and integrative effects of
glutamatergic and nitroxidergic neurons on transmission of cardio-vascular
reflex signals within the NTS were observed [20, 21]. So, decreases of MBP by
L-Glu microinjection demonstrated that the injection site was correctly
selected. To confirm this finding, after lidocaine microinjection into this depressor
area and inhibiting NO neurons, a significant, immediate and reversible
decrease in MBP was seen. This fast
acting local anesthetic agent blocks sodium channels, thus inhibiting neuronal
electrical activity in the affected area. The 1-ml injection of a 2% lidocaine solution has an
effective duration of 10-15 min. This makes the microinjection of
lidocaine an excellent
technique for interrupting
Fig.
4. Comparative effect of
L-glutamate (Glu, 2.3 nmol) and sodium nitroprusside (SNP) on mean blood
pressure. Each bar represents the average data from 9 rats. *, P<0.05
significantly different from baseline value.
local
neuronal activity without permanently altering the system.
In the
previous studies, microinjection of L-Arg into the NTS elicited dose-dependent
depressor and bradycardic effects. This suggests that L-Arg was transferred
into NO by NOS present in the NTS [22, 23]. In this study, SNP was used for
detection of nitric oxidergic neurons role in BP regulation. According to the
results, it was observed that SNP injection into NTS decreases diabetic rats’
MBP. The reason is that NO (released from SNP) diffused into presynaptic
terminals to activate guanylate cyclase. Resultant CGMP can increase the firing
rate of the calcium channels. Thus, increased calcium influx activates the
neurons and releases excitatory amino acids including Glu [9].
The
comparison of results of L-Glu and SNP microinjection demonstrated that the
attenuation effect of SNP on MBP was more potent than that of L-Glu (P<0.01).
Glu as an excitatory nouro-transmitter causes excitation of all kinds of
neurons including intrinsic nitric oxidergic neurons of NTS, but SNP increases
only NO level in the nucleus. BP was similarly affected by both drugs but SNP
was higher than the other, therefore it can be postulated that in diabetes,
intrinsic nitric oxidergic neurons of NTS were somehow inhibited. With respect
to sensitivity of many neurons in NTS to glycemic fluctuation and presence of
hypertension in diabetes, it can be postulated that diabetes has probably
inhibited either intrinsic nitric oxidergic neurons or has an excitatory effect
on nitric oxidergic inhibitory neurons in NTS. It is concluded that in
diabetes, hypertension is caused by nitric oxidergic neurons inhibition which
is caused by high level of glucose. The results of this study can be used for
mechanism of BP control in diabetes.
REFERENCES
1.
Lo, W.C., Jan, C.R., Wu, S.N. and
Tesng, C.J. (1998) Cardiovascular effects of nitric oxide and adenosine in the
nucleus tractus solitarii of rats. Hypertension 32: 1034-1038.
2.
Maeda, M., Inoue, M., Takao, S. and
Nakai, M. (1999) Central control mechanisms of circulation in the medulla
oblongata by nitric oxide. Jpn. J.
Physiol. 49 (6): 467-478.
3.
Esteves, F.O., McWilliam, P.N.
and Batten, T.F. (2000) Nitric oxide producing neurons in the rat medulla
oblongata that project to nucleus tractus solitarii. J. Chem. Neuroanat. 20 (2): 185-197.
4.
Sya, G.Y., Beubana, V., Bousquet,
P.A. and Feldmana, J. (2002) Nitric oxide discriminates the sites and
mechanisms of action of centrally
acting anti-hypertensive drugs in rabbits. Neuropharmacology 43 (8): 1330-1338.
5.
Dobrucki, L.W., Cabrera, C.L.,
Bohr, D.F. and Malinski, T. (2001) Central hypotensive action of clonidine
requires nitric oxide. Circulation 104 (16): 1884-1886.
6.
Lo, W.C., Lin, H.C., Ger, L.P.,
Tung, C.S. and Tseng, C.J. (1997) Cardiovascular effects of nitric oxide and
N-methyl- D- aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30: 1499-1503.
7.
Carolina, A., Dias1, R.,
Colombari, E. and Mifflin, S.W. (2003) Effect of nitric oxide on excitatory
amino acid-evoked discharge of neurons in NTS.
Am. J. Physiol. Heart Circ. Physiol.
284: H234-H240.
8.
Jayel, P.Y., Thalmann, S., Cook,
S., Duplain, H., Sartorl, C., Vollenwelder, P. and Scherrer, U. (2004) Nitric
oxide donors, a new treatment for insulin resistance, metabolic syndrome and
diabetes. Rev. Med. Suisse. Romande.
124 (10): 642-644.
9.
Tseng, C.J., Liu, H.Y., Lin,
H.C., Ger, L.P., Tung, C.S. and Yen, M.H. (1996) Cardiovascular effects of
nitric oxide in the brainstem nuclei of rats.
Hypertension 27: 36-42.
10.
Lo, W.J., Liu, H.W., Lin, H.C.,
Ger, L.P., Tung, C.S. and Tseng, C.J. (1996) Modulatory effects of nitric oxide
on baroreflex activation in the brainstem nuclei of rats. Chin. J. Physiol. 39: 57-62.
11.
Wua, C.W., Wangb, Y., Kaoc, L.S.,
Tangc, F.I. and Chai, C.Y. (2002) Nitric oxide reduces blood pressure in the
nucleus tractus solitarius: a real time electrochemical study. Brain Res. Bull. 15: 171-177.
12.
Vitagliano, S., Berrino, L.,
Damico, M., Maione, S., De Novellis, V. and Rossi, F. (1996) Involvement of
nitric oxide in cardiorespiratory regulation in the nucleus tractus
solitarii. Neuropharmacology 32 (5): 625-631.
13.
Dallaporta, M., Himmi, T.,
Perrin, J. and Orsini, J.C. (1999) Solitarii tract nucleus sensitivity to
moderate changes in glucose level. Neuroreport 10 (12): 2657-2660.
14.
Liorens, S. and Nava, E. (2003)
Cardiovascular diseases and the nitric oxide pathway. Crit. Vasc. Pharmacol. (3): 3335-3346.
15.
Chien, W.Y., Yang, K.D., Eng,
H.L., Hu, Y.H., Lee, R.Y., Wang, S.T. and Wang, P.W. (2005) Increased plasma
concentration of nitric oxide in type 2 diabetes but not in nondiabetic
individuals with insulin resistance. Diabetes
Metab. 31 (1): 63-68.
16.
Witte, K., Hachenberger, J.,
Castell, M.F., Vahi, C.F. and Haller, C. (2004) Nitric oxide- sensitive soluble
guanylyl cyclase activity is preserved in internal mammary artery of type 2
diabetic patients. Diabetes 53 (10): 2640-2444.
17.
Shimomura, I., Bashmakov, Y.,
Ikemoto, S., Horton, J.D., Brown, M.S. and Goldstein, J.L. (1999) Insuline
selectively increases SREBP-1c m RAN in the livers of rats with
stereptozotocin-induced diabetes. Proc.
Natl. Acad. Sci. USA 96 (24):
13656-13661.
18.
Pexinos, G. and Watson, C. (1986)
The rat brain in stereotaxi coordinates.
Academic press, San Diego, USA.
19.
Rosa, F., Vasouez, J. and Lupi,
J. (1997) Pharmacological modulation of the cardiovascular response to
hypertonic NACL injection in the anteroventral area of the brain third
ventricle. Pharmacology 45 (2): 98.
20.
Talman, W.T., Dragon, D.N., Otha,
H. and Lin, L.H. (2001) Nitroxidergic influences on cardiovascular control by
NTS: a link with glutamate. Ann.
NY Acad. Sci. 940: 169-178.
21.
Dias, A.C., Vitela, M.,
Colombari, E. and Mifflin, S.W. (2005) Nitric oxide modulation of
glutamatergic, baroreflex, and cardiopulmonary transmission in the nucleus of
the solitary tract. Am. J. Physiol. Heart Circ. Physiol. 288 (1): H256-H262.
22.
Matsumura, K., Tsuchihashi, T.,
Kagiyama, S., Abe, I. and Fujishima, M. (1998) Role of nitric oxide in the
nucleus of the solitary tract of rats. Brain
Res. 798 (1-2): 232-238.
23.
Ma, S., Abboud, F.M. and Felder,
R.B. (1995) Effects of L-arginine-derived nitric oxide synthesis on neuronal
activity in nucleus tractus solitarius. Am.
J. Physiol. 268: R487-R491.
