Endothelin-1 in Rat Periaqueductal Gray Area Induces Hypertension Via Glutamatergic Receptors
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-α]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.
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 α-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.
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-α]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, α- and β-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.
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.
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
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).
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 α1-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 β-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.
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 α1-adrenoreceptors on PAG pressor neurons. Our data are in agreement with those of Jones et al,21 who demonstrated, using autoradiographic methods, α1-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 α1-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.
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.
- Revision received September 22, 1994.
- Accepted November 16, 1994.
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