| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 1995;25:507-510.)
© 1995 American Heart Association, Inc.
Articles |
Endothelin-1 in Rat Periaqueductal Gray Area Induces Hypertension Via Glutamatergic Receptors
From the Institute of Pharmacology and Toxicology, Faculty of Medicine and Surgery, II University of Naples (Italy).
Correspondence to Dr Liberato Berrino, Institute of Pharmacology and Toxicology, Faculty of Medicine and Surgery, II University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract We investigated the possible relationship between endothelin-1 injection into the dorsolateral periaqueductal gray area and the glutamatergic system in the control of cardiovascular function. Endothelin-1 was injected into the dorsolateral periaqueductal gray area of freely moving rats at doses ranging from 0.1 to 10 pmol. Endothelin-1 increased arterial blood pressure (from 7.0±1.6 to 55.0±4.1 mm Hg, mean±SEM) in a dose-dependent manner and induced characteristic behavioral changes such as longitudinal rolling of the body (barrel-rolling). DL-2-Amino-5-phosphonovaleric acid and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[D-
]cyclohepten-5,10-imine
hydrogen maleate, both selective
N-methyl-D-aspartate excitatory amino acid
receptor antagonists, but not 6-cyano-7-nitroquinoxaline-2,3-dione, a
non–N-methyl-D-aspartate excitatory amino
acid receptor antagonist, significantly decreased endothelin-1–induced
cardiovascular and behavioral changes (P<.01). Prazosin and
propranolol, adrenergic blocking agents, and reserpine, a depletor of
catecholamine stores, also prevented these effects. We propose that the
glutamatergic system may exert, via
N-methyl-D-aspartate receptors, a significant
influence on endothelin-1–induced cardiovascular and behavioral
effects after its injection into the periaqueductal area.
Key Words: receptors, N-methyl-D-aspartate • rats • endothelins • periaqueductal gray • hypertension, experimental
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Endothelin-1 (ET-1) is a novel, potent, and long-lasting vasoconstrictor peptide consisting of 21 amino acid residues.1 It has been isolated and characterized from the supernatant of cultured porcine endothelial cells.2 ET-1 induces pressor effects,2 stimulates the heart,3 inhibits platelet aggregation,4 and can modulate sympathetic neurotransmission at both vascular5 and nonvascular6 junctions. Moreover, ET-1 administered into the lateral cerebral ventricle of conscious rats was seen to provoke a dose-dependent increase in blood pressure; this pressor response was considered to be mediated in part by the stimulation of vascular
-adrenergic receptors.7
Koseki et al8 have suggested that, in addition to its
action on the cardiovascular system, ET-1 may act on the central
nervous system as a neurotransmitter or neuromodulator to control a
wide variety of organ functions. Specific high-affinity binding sites
for ET-1 are in the hypothalamus, thalamus, lateral ventricular region,
subfornical organ, globus pallidus, and caudate putamen.8
ET-1–like immunoreactivity has been demonstrated by
immunohistochemistry in the paraventricular and supraoptic nuclear
neurons of the pig and the rat hypothalamus.9 Our previous
study10 showed that the periaqueductal gray (PAG) area
plays an important role in the control of cardiovascular function by
excitatory amino acids. We have demonstrated that the activation of
glutamatergic neurons in the PAG area increased sympathetic tone and
the release of vasopressin. These effects were also observed after ET-1
administration in the rat lateral ventricle.11 Because of
the similarities of the mechanisms underlying the effects elicited by
glutamate and ET-1, we decided to investigate whether (1) ET-1
administration in the PAG area modifies cardiovascular function, (2)
the cardiovascular effects of ET-1 are related to excitatory amino acid
receptors in the PAG area, and (3) injection of ET-1 in the PAG area
also increases sympathetic outflow. |
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Male Sprague-Dawley rats (250 to 300 g) with free access to food and water were housed at constant temperature (21±1°C) and relative humidity (60%). The animals were maintained under a regular 12-hour light/dark schedule (light, 7 AM to 7 PM). Five animals were used in each group.
Surgical Preparation and Treatment
Two days before the experiments, the rats were placed in a
stereotaxic apparatus (David Kopf Instruments) under ketamine
anesthesia (100 mg/kg IP), and a stainless steel guide cannula was
implanted in the dorsolateral PAG area; dental zinc cement was used to
fix it to the skull. If in the course of the stereotaxic implantation
it was necessary to improve the state of the anesthesia, additional
ketamine was administered. The coordinates of the atlas of Paxinos and
Watson12 (measured in millimeters from the bregma:
posteriorly, -7.8; laterally, 0.8; vertically, 4.5) were used. The
intracerebral microinjections were carried out by a Hamilton 10-µl
syringe connected by means of a polyethylene tube to a stainless steel
fine cannula (0.6 mm OD), which was carefully inserted into the fixed
guide cannula. A control volume of 1 µL of 0.2 mol/L phosphate buffer
(pH 6.5) or the same volume of drug solution was injected over a period
of 10 seconds; every intracerebral injection delivered a total volume
of 1 µL at a rate of 1 µL/10 s. On the day of the experiment, a
catheter was inserted into a femoral artery of each rat under
conditions of 2% halothane anesthesia for later measurement of
arterial blood pressure in conscious freely moving rats by a pressure
transducer (Statham P23Db) connected to a polygraph (model 20601001,
Hellige). Another catheter was inserted into a jugular vein for
systemic administration of drugs. Both catheters were exteriorized
through the back of the neck. After the experiments, the stereotaxic
coordinates of the cannula were checked histologically. Five minutes
before the rat was killed, 100 nL methylene blue (0.2%) was injected
intracerebrally with a high dose of pentobarbital (200 mg/kg IV). Each
animal was perfused intracardially with 50 mL phosphate-buffered saline
followed by 50 mL of a 10% formalin solution in phosphate-buffered
saline. The brain was removed and immersed in saturated formalin for 24
hours. The injection site was verified using two consecutive sections
(40 µm), one stained with cresyl violet to identify nuclei and the
other unstained to determine the dye diffusion. Data from only those
rats whose microinjection site was in the dorsolateral PAG area were
used for computation. ET-1 was administered in the dorsolateral site of
the PAG area at doses from 0.1 to 10 pmol.
2-APV, CNQX, and MK 801 Pretreatments and ET-1 Pressor Effects
2-APV (DL-2-amino-5-phosphonovaleric acid; Sigma
Chemical Co), a selective antagonist of
N-methyl-D-aspartate (NMDA) receptors, and
CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; Research Biochemicals Int),
an antagonist of non-NMDA receptors, were administered in the same area
10 minutes before ET-1 administration at doses of 5 nmol (2-APV) and
0.04 nmol (CNQX). MK 801
[(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[D-]cyclohepten-5,10-imine
hydrogen maleate; Research Biochemicals Int], a noncompetitive NMDA
excitatory amino acid receptor antagonist, was administered
intraperitoneally 30 minutes before ET-1 at a dose of 3 µmol/kg.
Arterial blood pressure was monitored continuously.
Prazosin, Propranolol, and Reserpine Pretreatments and ET-1 Pressor
Effects
Prazosin and propranolol, - and ß-blocking adrenergic
agents, respectively, were administered intravenously (prazosin, 2.4
nmol/kg), intracerebrally (prazosin, 2.4 nmol), or intraperitoneally
(propranolol, 0.34 mmol/kg). Reserpine, a depletor of catecholamine
stores, was administered subcutaneously at a dose of 8 µmol/kg 24
hours before ET-1 administration. Arterial blood pressure was monitored
continuously.
Drugs
The following drugs were used: ET-1 (Novabiochem), prazosin,
propranolol, reserpine, 2-APV, MK 801, CNQX, ketamine hydrochloride (CH
Boehringer Ingelheim), and angiotensin II (Calbiochem Co). ET-1,
angiotensin II, and propranolol were solubilized in 0.9% NaCl. CNQX
and MK 801 were solubilized in 0.2 mol/L phosphate buffer (pH 6.5), and
the pH of the solution was adjusted to 7.2 with 12 mmol/L NaOH.
Prazosin was dissolved in 50% dimethyl sulfoxide and 50% saline;
reserpine was dissolved in a minimal quantity of acetic acid, and the
final solution was brought to volume with 0.9% NaCl. All drug
solutions were freshly prepared on the day of each experiment. Control
injections were carried out with the same amount of solvent in which
the drugs were solubilized and did not produce any changes in arterial
blood pressure or behavior.
Statistics
Basal pressures were obtained within the 5 minutes before drug
injection. The maximal change in blood pressure was compared with the
basal value. All results are expressed as mean±SEM, with a value of
P<.05 considered significant. Cardiovascular changes were
compared by ANOVA and the Newman-Keuls test for multiple
comparisons.13
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of ET-1 Injections Into the PAG Area
Microinjections (1 µL) of ET-1 (0.1, 1, and 10 pmol to the dorsolateral PAG area significantly increased arterial blood pressure in a dose-dependent manner (7.0±1.6, 41.0±5.4 [P<.01], and 55.0±4.1 mm Hg [P<.01], respectively; basal pressure, 94.7±7.1 mm Hg) (Fig 1). At all doses there was always a latency time (ranging from 95 to 105 seconds) for the onset of the ET-1 pressure effect. Moreover, the ET-1 hypertensive effect was always accompanied by behavioral changes such as head twitches, ataxia, and longitudinal rolling of the body, which Moser and Pelton14 and Nikolov et al15 have defined as barrel-rolling. These effects appeared within 2 to 3 minutes after the administration of ET-1 and lasted 2 to 40 minutes and were followed by immobility lasting approximately 2 to 4 minutes (data not shown).
|
ABP) in freely moving rats after microinjections (1 µL) of
endothelin-1 (ET-1) (from 0.1 to 10 pmol) or of vehicle (saline) into
the dorsolateral side of the periaqueductal gray area. Each point
represents the mean of five observations. Vertical bars
indicate SEM. Significant differences with respect to groups receiving
saline are shown by asterisks (**P<.01).
Effects of 2-APV, CNQX, and MK 801 Pretreatments
As shown in Fig 2, the administration in the PAG
area of 2-APV (5 nmol), a selective antagonist of NMDA excitatory amino
acid receptors, 10 minutes before ET-1 injection prevented the
ET-1–induced hypertension. On the contrary, the microinjection of CNQX
(0.04 nmol), a non-NMDA excitatory amino acid receptor antagonist,
administered in the same site 10 minutes before ET-1 injection, did not
modify the pressor effects induced by ET-1 (Fig 2). The systemic
pretreatment with MK 801 (3 µmol/kg IP), a noncompetitive NMDA
excitatory amino acid receptor antagonist, 30 minutes before ET-1
injection prevented the hypertension induced by ET-1
(P<.01) (Fig 2). 2-APV, MK 801, and CNQX treatments did not
affect the arterial blood pressure per se (-3.0±1.8, -5.0±2.3, and
2.0±1.7 mm Hg, respectively). The behavioral effects were also
prevented by 2-APV and MK 801 but not by CNQX (data not shown). 2-APV
pretreatment (5 nmol) did not affect the hypertension secondary to
angiotensin II administered into the dorsolateral PAG area at doses
from 1 to 20 pmol (data not shown).
|
Effects of Prazosin, Propranolol, and Reserpine
Pretreatments
Prazosin, a selective antagonist of 1-adrenergic
receptors, administered 10 minutes before ET-1 in the PAG area (2.4
nmol) or intravenously (2.4 nmol/kg); propranolol (0.34 mmol/kg IP), a
selective antagonist of ß-adrenergic receptors, administered 10
minutes before ET-1; and reserpine (8 µmol/kg SC 24 hours before ET-1
injection), a depletor of catecholamine stores, also prevented
ET-1–induced pressor effects (counteracting the effects of ET-1 by
89.1%, 95.6%, 100%, and 54.7%, respectively, for 10 pmol ET-1; for
0.1 and 1 pmol ET-1 the counteraction by prazosin, propranolol, and
reserpine was 100%) (Fig 3). Moreover, prazosin,
propranolol, and reserpine also prevented the barrel-rolling effect
induced by ET-1 (data not shown). Prazosin, propranolol, and reserpine
did not significantly modify arterial blood pressure.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our results confirm that ET-1 may induce significant pressor effects by acting on the central nervous system; in fact, ET-1 administered in the PAG area induces, in conscious rats, significant increases in arterial blood pressure and behavioral changes characterized by barrel-rolling.
In 1989 Ouchi et al7 and Kawano et al16 documented a pressor effect of ET-1 after intracerebroventricular administration, supporting a role of this neuropeptide in the central control of blood pressure. Matsumura et al11 confirmed the potent central hypertensive effect of ET-1, which was mediated by enhanced sympathoadrenal outflow and increased vasopressin plasma level. These studies raised the question of the physiopathological implications of this pressor effect. Later, Gulati and Rebello17 found a decreased binding of ET-1 in the hypothalamus and ventrolateral medulla of spontaneously hypertensive rats compared with normotensive rats. They suggested that the hypertensive state was due to increased levels of endothelin in the central nervous system that led to hypertension and downregulation of endothelin receptors. They concluded that central endothelin mechanisms might play an important role in hypertension. Our results, obtained with very low doses (not enough to induce ischemia in normotensive rats18 ) in a well-defined midbrain area important to the regulation of neurovegetative functions,19 are further evidence for the role of ET-1 in cardiovascular modifications. ET-1 also induced serious behavioral changes, characterized by head twitches, ataxia, and longitudinal rolling of the body, which has been defined as barrel-rolling.14 15
Moreover, the stimulation induced by ET-1 of the pressor neurons inside
the dorsolateral column of the PAG area produced an increase in
sympathetic tone. This effect is demonstrated by inhibition of
ET-1–induced hypertension after pretreatments with adrenergic blocking
agents (prazosin and propranolol) and with a depletor of catecholamine
stores (reserpine). Studies with ET-1 injected into cerebral
ventricles16 or specific areas regulating autonomic
functions20 show, as does the present study, that
endothelin may enhance sympathetic outflow. Furthermore, the inhibitory
effect of prazosin on the hypertension induced by ET-1 demonstrates
that this neuropeptide may also modulate the release of catecholamines
within the PAG matter that would activate
1-adrenoreceptors on PAG pressor neurons. Our data are
in agreement with those of Jones et al,21 who
demonstrated, using autoradiographic methods,
1-adrenoreceptors within the PAG matter.
An interesting observation made in this study is that pretreatments with competitive (2-APV) and noncompetitive (MK 801) antagonists of glutamate NMDA subtype receptors completely blocked the pressor effects of ET-1. In contrast, CNQX, a competitive antagonist of glutamate non-NMDA subtype receptors, did not inhibit ET-1–induced hypertension. These results indicate that the ET-1 pressor effect in the PAG area is linked to an activation of NMDA receptors, and this effect seems to be highly specific because an antagonist of non-NMDA receptors did not prevent it. Moreover, 2-APV does not antagonize the pressor effects of angiotensin II injected into the PAG. This further demonstrates the specificity of the involvement of NMDA receptors in ET-1–induced hypertension.
On the other hand, we previously demonstrated that glutamate in the midbrain PAG area may participate in the modulation of pressor neurons, with a relevant involvement of NMDA excitatory amino acid subtype receptors.10 These receptors thus appear to have an obligatory role in the ET-1–evoked pressor response.
Moreover, ET-1 injections into the PAG area induce barrel-rolling. This
effect may be a disturbance of motor coordination and, although ET-1
was injected into the PAG matter, barrel-rolling may be due, as
advocated by Moser and Pelton,14 to interference with the
cerebellar function. However, considering that ET-1 in other cerebral
areas also generates such a behavioral change, we suppose that
barrel-rolling may represent a specific and serious injury of
neuronal pathways regulating motor coordination. Therefore,
barrel-rolling may be a consequence of a late hyperstimulation of NMDA
glutamatergic receptors as well as of
1-adrenoreceptors.
In conclusion, this study provides the first in vivo evidence that stimulation of the PAG area by ET-1 activates the glutamatergic system: in particular, this peptide may act by means of selective involvement of glutamate NMDA subtype receptors but not non-NMDA subtype receptors. The precise role of this peptide in the cardiovascular modifications regulated by the PAG area remains to be defined.
|
Acknowledgments |
|---|
Financial support from MURST and CNR, Italy, is gratefully acknowledged. The authors wish to thank Adriana Palla for the excellent technical assistance.
Received July 28, 1994; first decision September 22, 1994; accepted November 16, 1994.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kimura S, Kasuya Y, Sawamura T, Shinmi O, Sugita Y, Yanagisawa M, Goto K, Masaki T. Structure-activity relationships of endothelin, importance of C-terminal moiety. Biochem Biophys Res Commun. 1988;156:1182-1186. [Medline] [Order article via Infotrieve]
2. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411-415. [Medline] [Order article via Infotrieve]
3.
Ishikawa T, Yanagisawa M, Kimura S, Goto K, Masaki T.
Positive inotropic action of novel vasoconstrictor peptide endothelin
on guinea pig atria. Am J Physiol. 1988;255:H970-H973.
4. Thiemermann C, Lidbury P, Thomas R, Vane JR. Endothelin inhibits ex-vivo platelet aggregation in the rabbit. Eur J Pharmacol. 1988;158:181-182. [Medline] [Order article via Infotrieve]
5. Rae GA, Calixto JB. Effects of endothelins on nerve mediating contractions of the mouse vas deferens. Life Sci. 1990;47: PL83-PL89.
6. D'Orleans-Juste P, Finet M, De Nucci G, Vane JR. Pharmacology of endothelin-1 in isolated vessels: effect of nicardipine, methylene blue, hemoglobin, and gossypol. J Cardiovasc Pharmacol. 1989;13(suppl 5):S19-S22.
7.
Ouchi Y, Kim S, Souza AC, Iijima S, Hattori A, Orimo H,
Yoshizumi M, Kurihara H, Yazaki Y. Central effects of endothelin on
blood pressure in conscious rats. Am J Physiol. 1989;256:H1747-H1751.
8.
Koseki C, Imai M, HirataY, Yanagisawa M, Masaki T.
Autoradiographic distribution in rat tissue of binding sites for
endothelin: a neuropeptide? Am J Physiol. 1989;256:R858-R866.
9.
Yoshizawa T, Shinmi O, Giaid A, Yanagisawa M, Gibson SJ,
Kimura S, Uchiyama Y, Polak JK, Masaki T, Kanazawa I. Endothelin: a
novel peptide in posterior pituitary system. Science. 1990;247:462-464.
10. Maione S, Berrino L, Vitagliano S, Leyva J, Rossi F. Interactive role of L-glutamate and vasopressin, at level of the PAG area, for cardiovascular tone and stereotyped behaviour. Brain Res. 1992;597:166-169. [Medline] [Order article via Infotrieve]
11.
Matsumura K, Abe I, Tsuchihashi T, Tominaga M, Kobayashi K,
Fujishima M. Central effect of endothelin on neurohormonal responses in
conscious rabbits. Hypertension. 1991;17:1192-1196.
12. Paxinos G, Watson C. The Rat Brain in the Stereotaxic Coordinates. 2nd ed. Sydney, Australia: Academic Press; 1986.
13. Tallarida RJ, Murray RB. Manual of Pharmacologic Calculations With Computer Programs. 2nd ed. Heidelberg, FRG: Springer-Verlag; 1987.
14. Moser R, Pelton JT. Behavioural effects of centrally administered endothelin in the rat. Br J Pharmacol. 1989;96:347P. Abstract.
15. Nikolov R, Maslarova J, Semkova I. Effects of intracerebroventricular endothelin-1 on CNS and cerebral hypoxia/ischemia and their modification by cinnarizine. Methods Find Exp Clin Pharmacol. 1992;14:577-583. [Medline] [Order article via Infotrieve]
16. Kawano Y, Yoshida K, Yoshimi H, Kuramochi M, Omae T. The cardiovascular effect of intracerebroventricular endothelin in rats. J Hypertens. 1989;7(suppl 6):S22-S23.
17. Gulati A, Rebello S. Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats. Neuropharmacology. 1992;31:243-250. [Medline] [Order article via Infotrieve]
18. Gross PM, Weaver DF. A new experimental model of epilepsy based on the intraventricular injection of endothelin. J Cardiovasc Pharmacol. 1993;22(suppl 8):S282-S287.
19. Bandler R, Carrive P, Depaulis A. Emerging principles of organization of the midbrain periaqueductal gray matter. In: Depaulis A, Bandler R, eds. The Midbrain Periaqueductal Gray Matter. New York, NY: Plenum Publishing Corp; 1991:1-10.
20. Kuwaki T, Koshiya N, Takahashi H, Terui N, Kumada M. Modulatory effects of rat endothelin on central cardiovascular control in rats. Jpn J Physiol. 1990;40:97-116. [Medline] [Order article via Infotrieve]
21. Jones LS, Gauger LL, Davis JN. Postnatal development of brain alpha 1-adrenergic receptors: in vitro autoradiography with [125I]HEAT in normal rats and rats treated with alpha-difluoromethylornitine, a specific, irreversible inhibitor of ornithine decarboxylase. Neuroscience. 1985;15:1195-1202. [Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  Y. Liu, E.-S. Ji, S. Xiang, R. Tamisier, J. Tong, J. Huang, and J. W. Weiss Exposure to cyclic intermittent hypoxia increases expression of functional NMDA receptors in the rat carotid body J Appl Physiol, January 1, 2009; 106(1): 259 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shihara, Y. Hirooka, N. Hori, I. Matsuo, T. Tagawa, S. Suzuki, N. Akaike, and A. Takeshita Endothelin-1 increases the neuronal activity and augments the responses to glutamate in the NTS Am J Physiol Regulatory Integrative Comp Physiol, August 1, 1998; 275(2): R658 - R665. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||