Upregulation of tachykinin NK-1 and NK-3 receptor binding sites in the spinal cord of spontaneously hypertensive rat: impact on the autonomic control of blood pressure

Effects of intrathecally (i.t.) injected tachykinin NK-1 and -3 receptor agonists and antagonists were measured on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR,15-week-old) and age-matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiography was also performed on the lower thoracic spinal cord of both strains and Wistar rats using specific radioligands for NK-1 receptor ([125I]HPP[Arg3,Sar9,Met(O2)11]SP (3–11)) and NK-3 receptor ([125I]HPP-Asp-Asp-Phe-N-MePhe-Gly-Leu-Met-NH2). The NK-1 agonist [Sar9,Met(O2)11]SP (650 and 6500 pmol) decreased MAP and increased HR in WKY. The fall in MAP was blunted in SHR and substituted by increases in MAP (65–6500 pmol) and more sustained tachycardia. The NK-3 agonist senktide (6.5–65 pmol) evoked marked increases in MAP and HR (SHR>>>WKY), yet this response was rapidly desensitized. Cardiovascular effects of [Sar9,Met(O2)11]SP (650 pmol) and senktide (6.5 pmol) were selectively blocked by the prior i.t. injection of LY303870 (NK-1 antagonist, 65 nmol) and SB235375 (NK-3 antagonist, 6.5 nmol), respectively. Antagonists had no direct effect on MAP and HR in both strains. Densities of NK-1 and -3 receptor binding sites were significantly increased in all laminae of the spinal cord in SHR when compared to control WKY and Wistar rats. The dissociation constant was however not affected in SHR for both NK-1 (Kd=2.5 nM) and NK-3 (Kd=5 nM) receptors. Data highlight an upregulation of NK-1 and -3 receptor binding sites in the thoracic spinal cord of SHR that may contribute to the hypersensitivity of the pressor response to agonists and to the greater sympathetic activity seen in this model of arterial hypertension. Keywords: Tachykinins, senktide, [Sar9,Met(O2)11]SP, NK-1 and NK-3 receptors, spinal cord, blood pressure, hypertension, autoradiography Introduction Tachykinins belong to a family of neuropeptides including substance P (SP), neurokinin A and neurokinin B whose biological effects are mediated via the activation of three transmembrane G-protein-coupled receptors denoted as NK-1, -2 and -3. These receptors are widely distributed in the central nervous system (for a review see Ribeiro-Da-Silva et al., 2000) and thought to play a role in the autonomic control of blood pressure (Unger et al., 1988; Couture et al., 1995; Culman et al., 1995; 1997; Cellier et al., 1997; 1999; Lessard & Couture, 2001; Brouillette & Couture, 2002; Lessard et al., 2003; 2004; Deschamps & Couture, 2005). Cerebral activation of tachykinin receptors with various natural and selective agonists leads to increases in mean arterial blood pressure (MAP) and heart rate (HR) through the activation of the sympathetic nervous system (Unger et al., 1981; Takano et al., 1990; Deschamps & Couture, 2005) and the release of vasopressin (NK-3 receptors) (Polidori et al., 1989; Massi et al., 1991; Nakayama et al., 1992; Yuan & Couture, 1997). Cardiovascular effects evoked by intrathecally (i.t.) injected tachykinin agonists were mediated by NK-1 receptors and sympatho-adrenal activation (Hassessian & Couture, 1989; Hassessian et al., 1988; 1990; Couture et al., 1995). In normotensive rats, intracerebral or i.t. injection of tachykinin receptor antagonists failed to affect baseline arterial blood pressure and HR, suggesting that these peptides do not exert a tonic control of cardiovascular function although they could play a role as neuromodulators. Pharmacological and anatomical findings suggest that the function and expression of central tachykinins and their receptors are altered during arterial hypertension. For instance, hypersensitivity of the pressor response to intracerebroventricular (i.c.v.) SP was reported in SHR (Unger et al., 1980) and both SP binding sites and SP-like immunoreactivity were higher in several brain nuclei of adult SHR (Shigematsu et al., 1987; Chen et al., 1990). More recently, injection of tachykinin NK-3 receptor antagonists in the substantia nigra or i.c.v. decreased systemic arterial blood pressure in SHR but not in WKY (Lessard et al., 2003; 2004). In the latter studies, NK-1 and -2 receptor antagonists failed to affect systemic blood pressure in both strains. These observations suggest a role for brain tachykinin NK-3 receptors in the maintenance of arterial blood pressure in SHR. At the spinal cord level, previous studies have reported significant differences between SHR and WKY with regard to NK-1 receptors. Takano et al. (1985) have shown increased number of [3H]SP binding sites and SP-like immunoreactivity in the intermediolateral cell column (IML) in the upper thoracic spinal cord of 16-week-old SHR. In the same study, the i.t. injection of the SP antagonist [D-Pro4, D-Trp7,9]SP (4–11), caused similar dose-dependent decreases in MAP and HR in anaesthetized SHR and WKY. However, the latter results are misleading as the first generation of SP antagonists was found nonspecific for SP receptors and toxic in the rat spinal cord (Couture et al., 1987a, 1987b). Although Solomon et al. (1999) reported that the nonpeptide NK-1 receptor antagonist (RP 67580) also caused the same fall in blood pressure in both strains, this antagonist did not affect the biphasic MAP response induced by the selective NK-1 receptor agonist, GR 73632, in SHR. All these studies were performed with the use of anaesthetics which interfere with sympathetic nervous activity and baroreceptors function. Further systematic studies with selective NK-1 and -3 receptor agonists and antagonists, particularly in un-anaesthetized animals, are needed to define the putative role of tachykinin receptors in the autonomic control of blood pressure and HR at the level of the spinal cord in arterial hypertension. The present study was undertaken to re-evaluate the role of spinal NK-1 receptors and to test the hypothesis that NK-3 receptors might also participate to the spinal autonomic control of MAP and HR by tachykinins in a model of hypertension. Two experimental approaches were used: (i) in vivo pharmacological experiments in freely behaving SHR (15-weeks-old) and age-matched WKY to determine the cardiovascular effects of selective NK-1 and -3 receptor agonists and antagonists i.t. injected to the ninth thoracic vertebra; (ii) quantitative autoradiography on lower thoracic spinal cord sections with newly developed selective radioligands to compare the density and distribution of NK-1 and -3 receptor binding sites in both strains and Wistar rats.