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Am J Physiol Heart Circ Physiol 284: H1949-H1958, 2003. First published February 13, 2003; doi:10.1152/ajpheart.01113.2002
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Vol. 284, Issue 6, H1949-H1958, June 2003

SPECIAL TOPICS
Regulation of Cardiovascular Signaling by Kinins and Products of Similar Converting Enzyme Systems
Chronic effects of angiotensin-converting enzyme inhibition on kinin receptor binding sites in the rat spinal cord

Brice Ongali1, Hudson de Sousa Buck1, Frank Cloutier1, Francine Legault2, Domenico Regoli3, Chantal Lambert2, Gaétan Thibault4, and Réjean Couture1

Departments of 1 Physiology, 2 Pharmacology, and 4 Clinical Research Institute, Université de Montréal, Montréal, Québec H3C 3J7; Department of 3 Pharmacology, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

With the use of in vitro receptor autoradiography, this study aims at determining whether the higher level of kinin B2 receptor density in the spinal cord of the spontaneously hypertensive rat (SHR) is secondary to arterial hypertension and whether chronic treatment with angiotensin I-converting enzyme inhibitors (ACEI) can regulate neuronal B1 and B2 receptors. SHR received, from the age of 4 wk, one of the two ACEI (lisinopril or zofenopril, 10 mg · kg-1 · day-1) or for comparison, the selective AT1 antagonist (losartan, 20 mg · kg-1 · day-1) in their drinking water for a period of 4, 12, and 20 wk. Age-matched untreated SHR and Wistar-Kyoto rats (WKY) were used as controls. B2 receptor binding sites in most laminae were higher in SHR than in WKY from the age of 8 to 24 wk. Whereas B1 receptor binding sites were significantly present in young SHR and WKY, they were barely detectable in adult rats. ACEI (16 and 24 wk) and AT1 antagonist (24 wk) enhanced the number of B2 without changing B1 receptor binding sites. However, at 8 wk the three treatments significantly increased B1 and decreased B2 receptors in lamina I. It is concluded that 1) the higher density of B2 receptors in the spinal cord of SHR is not due to hypertension, 2) kinin receptors are regulated differently by ACEI in neuronal and vascular tissues, and 3) aging may have a profound impact on levels of B1 and B2 receptors in the rat spinal cord.

bradykinin; B1 receptor; B2 receptor; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

KININ-RELATED PEPTIDES referring mainly to bradykinin (BK) and kallidin (Lys-BK) were identified as neuromediators in the central control of arterial blood pressure and nociceptive information (7, 8). Kinins act on two transmembrane G protein-coupled receptors denoted as B1 and B2 receptors (38, 39). The widely distributed B2 receptor is constitutive and mediates most of the biological effects of kinins. Whereas the B1 receptor is absent or underexpressed under physiological conditions, this receptor is induced and upregulated during tissue injury or in the presence of cytokines (27). The induction of B1 receptor by cytokines involves the transcriptional nuclear factor kappa B and the mitogen-activated protein kinase (35, 40, 46).

Kinins are metabolized by a group of carboxypeptidases named kininases I and II. Kininase I removes the COOH-terminal arginine from the parent molecules to yield the active metabolites des-Arg9-BK and des-Arg10-kallidine, which act as potent B1 receptor agonists (39). Kininase II, also known as angiotensin I-converting enzyme (ACE), is responsible for the inactivation of kinins and the generation of angiotensin II (14). Breakdown inhibition of vasoactive kinins is believed to contribute to the therapeutic effects of ACE inhibitors (ACEI) in the treatment of hypertension and other cardiovascular diseases (21, 22). A recent study also reported an upregulation of B1 receptors at both mRNA and functional levels in vascular and renal tissues from normotensive rats and mice under chronic treatment with ramipril, an ACEI (29).

Recent work suggests a putative role for central kinin receptors in arterial hypertension. For instance, the increased number and expression of B2 receptors have been shown in the cardiovascular centers of the human medulla from hypertensive donors (12) and in the hypothalamus and cardiovascular medullary nuclei of spontaneously hypertensive rats (SHR) (8, 36). Higher density of B2 receptor binding sites in the thoracic spinal cord, an important center of autonomic control of blood pressure, was correlated with a greater cardiovascular response to intrathecal injection of BK in 16-wk-old SHR (6). However, there is no evidence so far that the upregulation of B2 receptors in the spinal cord and brain of SHR is causal or secondary to arterial hypertension.

Therefore, the aims of this study were to determine whether 1) the higher density of spinal B2 receptors in SHR is secondary to arterial hypertension or is related to a genetic feature of the strain, and 2) ACEI can regulate the expression of B1 receptors in the thoracic spinal cord of SHR as observed in peripheral vascular and renal tissues. These issues were addressed by measuring the effects of three antihypertensive agents, including two unrelated classes of ACEI [lisinopril without sulfhydryl (SH) group and zofenopril with SH group] and one antagonist of angiotensin AT1 receptor (losartan), which is commonly used in the treatment of human hypertension (17), on the density of B1 and B2 receptor binding sites in the thoracic spinal cord (T9-T10) of SHR by in vitro autoradiography. The effects of ACEI and losartan on kinin receptor densities were evaluated in young SHR at the onset of hypertension (8 wk old, after 4 wk of treatment) and in adult SHR during the established phase of hypertension (16 and 24 wk old, after 12 and 20 wk of treatment). Data were compared with age-matched untreated SHR and normotensive Wistar-Kyoto rats (WKY), which also allowed the determination of the effect of aging on the level of receptor binding sites.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and materials. HPP-desArg10-HOE 140 (3-4 hydroxyphenyl-propionyl-desArg9-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]-BK) and HPP-HOE 140 (3-4 hydroxyphenyl-propionyl-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]-BK) were developed from the selective B1 receptor antagonist desArg10-HOE 140 (44) and the B2 receptor antagonist HOE 140 or Icatibant (18), respectively. They were synthesized in the laboratory of D. Regoli (Department of Pharmacology, Université de Sherbrooke). Autoradiographic 125I-labeled microscales (20 µm) and 3H Hyperfilm (single-coated, 24 × 30 cm) were purchased from Amersham Pharmacia Biotech Canada. Losartan (Cozaar tablet), lisinopril, PIPES, 1,10-phenanthroline, dithiothreitol, bacitracin, captopril, and BSA (protease free) were purchased from Sigma-Aldrich Canada, and zofenopril was a gift from Menarini Ricerche Sireneze in Italy.

Peptide iodination. Iodination of HPP-desArg10-HOE 140 and HPP-HOE 140 was performed according to the chloramine T method (19). Briefly, 5 µg of peptide were incubated in 0.05 M phosphate buffer for 30 s in the presence of 0.5 mCi (18.5 MBq) of 125I-labeled Na and 220 nmol of chloramine T in a total volume of 85 µl. The monoiodinated peptide was then immediately purified by high pressure liquid chromatography on a C4 Vydac column (0.4 × 250 mm) (The Separations Group, Hesperia, CA) with 0.1% trifluoroacetic acid and acetonitrile as mobile phases. The specific activity of the iodinated peptides corresponds to 2,000 counts · min-1 · fmol-1 or 1,212 Ci/mmol.

Animal source and care. Male SHR (n = 48) and WKY (n = 12) were purchased from Charles River (St-Constant, Québec, Canada). They were individually housed in wire-bottom cages, in rooms under controlled temperature (23°C), humidity (50%), and lighting (12:12-h light-dark cycle) with food (Charles River Rodent) and tap water available ad libitum. All animal procedures were in strict compliance with the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and approved by the Animal Care Committee of our University.

Treatments of SHR. SHR received from the age of 4 wk, one of the two ACEI, lisinopril or zofenopril (10 mg · kg-1 · day-1), or the selective AT1 receptor antagonist, losartan (20 mg · kg-1 · day-1), in their drinking water for a period of 4, 12, and 20 wk. To ascertain that the animals took the expected dose of the drug, the daily water intake and body weight were taken into account and adjusted accordingly. Control age-matched SHR and WKY had no treatment during the same periods. Equiactive oral dose of zofenopril and lisinopril was selected, based on an earlier study using ex vivo inhibition of tissue ACE in SHR (9). The dose of losartan selected was found to be effective in chronic studies in SHR (16, 42, 43). Before euthanasia, mean arterial blood pressure (MAP) was measured in awake animals at the age of 8, 16, and 24 wk with a catheter implanted 24 h earlier into the abdominal aorta through the femoral artery and exteriorized at the back of the neck. The latter surgery was made under anesthesia with pentobarbital sodium (65 mg/kg ip). Body weight of animals was measured daily from the onset of treatments.

Tissue preparation for autoradiography. Rats were euthanized at the age of 8, 16, and 24 wk by asphyxia by respiratory CO2 inhalation and subjected to dorsal laminectomy. Spinal cords (segments T8-T11) were immediately removed after careful incision of the dura mater and frozen in 2-methylbutane cooled at -45 to -55°C with liquid nitrogen and then stored at -80°C until use. Matched spinal cord segments (T9 to T10) of the four rats from the same experimental group were mounted together in a gelatin block and serially cut into 20-µm-thick coronal sections with a cryostat fixed at temperatures varied between -11 and -13°C. Thus each section of the cryostat was from four spinal cords. A total of eight sections per slide were then alternatively thaw-mounted on 0.2% gelatin-0.033% chromium potassium sulfate-coated slides. Three slides were taken for the total binding and two slides (adjacent sections) for the nonspecific binding. A total of 50 slides (1,600 sections) were obtained for each group studied and kept at -80°C until use.

In vitro receptor autoradiography. Sections were thawed, preincubated for 30 s in 25 mM PIPES buffer (pH 7.4; 4°C), and incubated at room temperature for 90 min in the same buffer containing 1 mM 1,10-phenanthroline, 1 mM dithiothreitol, 0.014% bacitracin, 0.1 mM captopril, 0.2% BSA (protease free), and 7.5 mM magnesium chloride in the presence of 150 pM 125I-labeled HPP-desArg10-HOE 140 ([125I]HPP-desArg10-HOE 140) (for B1 receptor) or 200 pM 125I-labeled HPP-HOE 140 ([125I]HPP-HOE 140) (for B2 receptor). The concentrations of radioligands chosen yielded maximal specific binding (Bmax) on the saturation curves in the spinal dorsal horn of SHR and WKY (6). The dissociation constant (Kd) of [125I]HPP-HOE 140 was identical in SHR and WKY (Kd = 30 pM), whereas that of [125I]HPP-desArg10-HOE 140 was calculated at 27 pM in SHR. The nonspecific binding was determined in the presence of 1 µM of unlabeled ligands (HPP-desArg10-HOE 140 for B1 receptor and HPP-HOE 140 for B2 receptor). To ascertain the specificity of the labeled B2 radioligand, the same concentration of unlabeled B1 ligand was added to the solution. Likewise, the same concentration of the unlabeled B2 ligand was added to the labeled B1 ligand solution. At the end of the incubation period, slides were transferred sequentially through four rinses of 4 min each in 25 mM PIPES (pH 7.4; 4°C), dipped for 15 s in distilled water (4°C) to remove the excess of salts, and then air-dried. 3H Hyperfilm was juxtaposed onto the slides in the presence of 125I microscales and exposed at room temperature for 3 days (B1 ligand) or 2 days (B2 ligand). The films were developed in D-19 (Kodak developer) and fixed in Kodak Ektaflo. Autoradiograms were quantified by densitometry using an image analysis system (MCID Imaging Research; Ontario, Canada). Standard curve from 125I microscales was used to convert density levels into fentomoles per milligram of tissue. Specific binding was determined by subtracting superimposed digitalized images of nonspecific labeling from total binding. The anatomic structures with the corresponding nomenclature are depicted in Fig. 1 and adapted from the Atlas of Paxinos and Watson (33).


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  		Fig. 1.   Schematic representation of anatomic structures of a coronal section of lower thoracic spinal cord (left) along with the autoradiographic distribution of B2 receptor binding sites in 8-wk-old spontaneously hypertensive rats (SHR) (right). I-X, number of spinal cord laminae; D, dorsal nucleus; DH (LI, LII, LIII), dorsal horn; IML, intermediolateral cell column; IMM, intermediomedial cell column; Py, pyramidal tract.

Statistical analysis of data. Results represent the means ± SE of four animals per group. Quantification was performed on 400 sections for each spinal cord (T9-T10) on both sides. Statistical analysis of data was performed with the Graph-Pad Prism computer program, and the statistical significance between SHR and WKY was determined with a Student's t-test for unpaired samples. For multiple comparisons to the same control group (untreated SHR), a one-way analysis of variance (ANOVA) followed by the test of Dunnett was employed. A one-way ANOVA in conjunction with Bonferroni confidence intervals was used for multiple comparisons between WKY and SHR. Only P values <0.05 were considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Body weight and blood pressure. Body weight and baseline MAP in SHR and WKY are shown in Fig. 2. At the onset of the treatment (4 wk) and at 24 wk, no statistical difference was found in body weight between SHR and WKY in all groups. However, the body weight of SHR at 8 and 16 wk was significantly higher than in age-matched WKY. Chronic treatment with zofenopril and lisinopril (16 wk) prevented excessive body weight gain in SHR. At 8 wk, the difference in body weight between strains was no more significant with lisinopril.


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  		Fig. 2.   Body weight and mean arterial pressure (MAP) of SHR and Wistar-Kyoto rats (WKY) used in the study. Values represent the means ± SE of 12 rats (4 wk) and 4 rats (8, 16, and 24 wk) per group. Statistical comparison to WKY (dagger ) or untreated SHR (*) is indicated by *dagger P < 0.05, **dagger dagger P < 0.01, and ***dagger dagger dagger P < 0.001 (one-way ANOVA with a post hoc Bonferroni test).

MAP was significantly augmented in SHR at 8, 16, and 24 wk compared with age-matched WKY. Whereas zofenopril and losartan prevented the development of hypertension from 8 to 24 wk in SHR, the reduction of MAP produced by lisinopril was greater and caused significant hypotension throughout the treatment.

B1 receptor binding sites. Representative distribution of B1 receptor binding sites in the lower thoracic spinal cord of 8-, 16-, and 24-wk-old WKY and SHR is depicted in Fig. 3, and the corresponding quantitative values are shown in Fig. 4. Discrete distribution of [125I]HPP-desArg10-HOE 140 labeling was detected in the dorsal horn lamina I of which its density was significantly higher at 8 wk in SHR (2 fmol/mg tissue) than in age-matched WKY (1.14 fmol/mg tissue). The other structures of the spinal cord gray matter of 8-wk-old SHR and WKY had lower specific densities of B1 receptors (around 1.0 fmol/mg tissue) with no statistical difference between strains (Fig. 4). The addition of 1 µM of unlabeled HPP-desArg10-HOE 140 to the incubation medium completely eliminated the labeling in all laminae (Fig. 3). However, a background of very low specific labeling to B1 receptor was found in all laminae (values <= 0.5 fmol/mg tissue) in 16- and 24-wk-old WKY and SHR (data not shown).


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  		Fig. 3.   Autoradiographic distribution of 125I-labeled HPP-desArg10-HOE 140 binding sites in the thoracic spinal cord of 8-, 16-, and 24-wk-old WKY (W), SHR without treatment (S), and SHR treated with losartan (L), zofenopril (Z), or lisinopril (Li). Note the high level of specific B1 receptor binding sites in the dorsal horn of both strains (SHR > WKY) at 8 wk only. Nonspecific binding (NS) in the presence of 1 µM of HPP-desArg10-HOE 140 is also shown.



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  		Fig. 4.   Quantification of specific B1 receptor binding sites in the various laminae of the thoracic spinal cord of WKY and SHR at the age of 8 wk (4 wk of treatment). Values represent the means ± SE of 4 rats per group. SE are too small to be illustrated. Statistical comparison to WKY (dagger ) or untreated SHR (*) is indicated by **P < 0.01 (one-way ANOVA with a post hoc Dunnett test) and dagger dagger dagger P < 0.001 (Student's t-test).

B2 receptor binding sites. The distribution of B2 receptor binding sites and their relative densities in each lamina of the spinal cord of 8-, 16-, and 24-wk-old WKY and SHR are shown in Figs. 5 and 6 and Table 1. The labeling of [125I]HPP-HOE 140 was observed all over the structures of the gray matter, with discrete definition in the dorsal horn of all studied animals. The addition of 1 µM of unlabeled HPP-HOE 140 to the incubation medium largely eliminated the total labeling.


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  		Fig. 5.   Autoradiographic distribution of 125I-labeled HPP-HOE 140 binding sites in the thoracic spinal cord of 8-, 16-, and 24-wk-old WKY (W), SHR without treatment (S), and SHR treated with losartan (L), zofenopril (Z), or lisinopril (Li). Note the high level of specific B2 receptor binding sites in the dorsal horn of both strains and the greater intensity of labeling in SHR. Nonspecific binding (NS) in the presence of 1 µM of HPP-HOE 140 is also shown.



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  		Fig. 6.   Quantification of specific B2 receptor binding sites in the superficial laminae (LI and LII) of the thoracic spinal cord of WKY and SHR at the age of 8, 16, and 24 wk (4, 12, 20 wk of treatment, respectively). Values represent the means ± SE of 4 rats per group. SE are too small to be illustrated. Statistical comparison to WKY (dagger ) or untreated SHR (*) is indicated by **P < 0.01 (one-way ANOVA with a post hoc Dunnett test) and dagger dagger dagger P < 0.001 (Student's t-test).


                              
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  		Table 1.   Densities of specific B2 receptor binding sites in various thoracic spinal cord laminae in WKY and SHR

Densities of B2 receptors were higher in the dorsal horn (lamina I > lamina II), and one- to eightfold greater in SHR. Also, values in lamina I were significantly higher at 16 wk than at 8 and 24 wk in SHR (P < 0.01), whereas values were lower at 8 wk (P < 0.01) and similar at 16 and 24 wk in WKY. The corresponding densities in lamina I were as follows: 3.35, 9.90, and 9.42 fmol/mg tissue for WKY and 20.14, 47.11 and 24.29 fmol/mg tissue for SHR at the age of 8, 16, and 24 wk, respectively. Although densities of specific binding sites were relatively low in most other laminae, most values were generally significantly higher in SHR than in age-matched WKY at 16 and 24 wk old (Table 1).

Effects of long-term treatment with antihypertensive drugs. Effects of chronic treatments with ACEI (zofenopril or lisinopril) and a selective AT1 receptor antagonist (losartan) were assessed on the densities of both kinin receptors in the spinal cord of SHR. The density of B1 receptors in lamina I was significantly increased after 4 wk of treatment with lisinopril (+16%, P < 0.01), losartan (+6%, P < 0.01), and zofenopril (+36%, P < 0.01), yet no significant changes were measured in the other laminae. In contrast, the density of B2 receptors was markedly decreased in lamina I by the three treatments (-57% to -65%, P < 0.01) (Figs. 5 and 6). B2 receptor binding values in other laminae (except lamina II) were too small to discriminate a real effect with the drugs, and therefore they were not considered for the remainder of the study.

Contrary to results obtained at 8 wk, a longer period of treatment (16- and 24-wk-old SHR) with either ACEI produced a marked increase of B2 receptor binding sites in laminae I and II of the spinal cord (Fig. 6). Whereas the reduction of B2 receptor binding sites seen with losartan at 8 wk persisted at 16 wk in SHR, the latter treatment caused a significant increase of B2 receptor binding sites in laminae I and II at 24 wk (Fig. 6). The latter augmentation of B2 receptor binding sites was similar to that occurring with ACEI. However, at 16 and 24 wk, ACEI and losartan had no significant effect on B1 receptor binding sites, which remained barely detectable in the spinal cord of SHR (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The main findings of this study are 1) the greater density of B2 receptor binding sites in the thoracic spinal cord of SHR is unlikely secondary to arterial hypertension because it subsisted in SHR subjected to antihypertensive therapy with ACEI or losartan; 2) aging has an opposite effect on the level of B1 and B2 receptors densities in the spinal cord because adult SHR displayed greater density of B2 but scarce density of B1 receptor binding sites compared with young SHR, which displayed the highest density of B1 and the lowest density of B2 receptor binding sites; 3) ACEI enhanced the density of spinal B1 receptor binding sites as in vascular and renal organs, yet this occurred only after 4 wk of treatment in young SHR and not in adult SHR exposed to a longer period of antihypertensive therapy; 4) ACEI has an opposite influence on B1 and B2 receptors because B2 receptor binding sites were decreased after 4 wk of treatment in young SHR and increased after a longer period (8 and 16 wk) of antihypertensive therapy in adult SHR; and 5) the effect of ACEI on the expression of B2 receptors is not shared by losartan after 12 wk of treatment (16 wk old SHR), and therefore a dissociation could be established between the changes of receptors and the antihypertensive effect of ACEI.

Distribution and density of B1 receptors in the thoracic spinal cord of SHR and WKY. Aging had a profound influence on the density of [125I]HPP-desArg10-HOE 140 binding to B1 receptors in the spinal cord of WKY and SHR as it was present at 8 wk (SHR >> WKY) and severely decreased in both strains at the age of 16 and 24 wk. Notwithstanding, this limited life span of B1 receptors, a low but detectable density of specific binding sites persisted in all laminae in both strains, suggesting the presence of a basal expression of B1 receptors in the spinal cord of adult SHR and WKY. This finding is in agreement with the basal B1 receptor expression (mRNA) and its immunohistochemical detection in rat and human spinal cord dorsal horn (26, 45). The significance of the higher level of B1 receptor expression in young SHR (lamina I) and its decline in all laminae in adult is not understood at the present time, yet the present finding is congruent with an earlier study that concluded that B1 receptors are not involved in spinal cardiovascular regulation in adult SHR and WKY (6). Because B1 receptors are detectable only in young SHR, it is unlikely that they are responsible for the maintenance of high blood pressure in adult SHR.

Distribution and density of B2 receptors in the thoracic spinal cord of SHR and WKY. The distribution of [125I]HPP-HOE 140 binding sites in the spinal cord of WKY and SHR is in agreement with previous observations made in the rat, guinea pig, and sheep spinal cord with [125I-Tyr8]BK or [125I]HPP-HOE 140 as the radioligand (24, 25, 31). The highest density of specific binding sites was found in superficial layers of the dorsal horn, whereas moderate to lower specific B2 receptor binding sites were detected in other laminae with no evidence of labeling in the white matter. However, contrary to the Wistar rat, which displayed the highest density of 125I-labeled [Tyr8]BK binding in lamina II (substantia gelatinosa) (24), the highest level of binding with [125I]HPP-HOE 140 was located in lamina I in both SHR and WKY. This discrepancy is likely due to the strain difference and not to the radioligand because the use of 125I-labeled HPP-HOE 140 confirms the highest concentration of B2 receptor binding sites in lamina II in the Wistar rat (data not shown). This strain difference remains unknown at this time but may indicate a differential expression of B2 receptors on Adelta - and C-fiber primary sensory neurons. B2 receptors are predominantly located on terminals of capsaicin-sensitive primary sensory C-fibers and of bulbospinal noradrenergic neurons in the spinal dorsal horn of Wistar rats (24). Activation of these receptors on sensory and noradrenergic terminals led to nociceptive and antinociceptive responses, respectively, in the rat tail-flick test (7, 20).

A small population of B2 receptors is also present in deeper laminae, including the intermediomedial and intermediolateral cell columns, the location of cell bodies, and dendrites of preganglionic sympathetic fibers, which are involved in the autonomic control of blood pressure (8). Thus our data suggest that B2 receptors are extensively distributed throughout sensory and autonomic areas in the spinal cord of WKY and SHR, where they may be implicated in the modulation of nociceptive information and in the spinal control of blood pressure (8).

Effect of ACEI and AT1 receptor antagonist on spinal kinin receptors. Apart from their distinctive pharmacokinetic and pharmacodynamic features, the therapeutic benefits of ACEI in the treatment of hypertension are thought to be class effects (41). Two major groups of ACEI have been documented: those containing a SH group as captopril and zofenopril and those without a SH group represented by lisinopril (4). Lisinopril and zofenopril can pass the blood-brain barrier to produce significant inhibition of brain ACE activity after oral administration in SHR (9, 37). However, in our study, while zofenopril normalized blood pressure and lisinopril caused hypotension, both ACEI produced similar changes in the density of B1 and B2 receptors in the spinal cord of SHR, suggesting that these changes are not associated with or without the presence of SH group of ACEI. Pharmacodynamic differences between zofenopril and lisinopril may however explain their distinct antihypertensive profile. Lisinopril was also more effective than zofenopril in reducing the body weight of overweight SHR. Because losartan was less effective in reducing body weight increase in SHR, the antiobesity effect of ACEI cannot be entirely ascribed to inhibition of the hypertrophic feature of angiotensin II. Thus a role for endogenous kinins cannot be excluded in this additional beneficial effect of ACEI on body weight.

In addition, pretreatment for 4 wk with ACEI or losartan, which prevented the development of hypertension in SHR, caused a further increase of B1 receptors in lamina I yet a decrease of B2 receptors. Upregulation of B2 receptors occurred only after a longer treatment with ACEI (16 and 24 wk) or losartan (24 wk). These changes of B1 and B2 receptor densities in SHR are unlikely attributable to the antihypertensive effect of the treatment, because the prevention of hypertension with losartan did not reproduce ACEI-induced B2 receptor upregulation at 16 wk. Although both ACEI and losartan reduced the number of B2 receptors in SHR after 4 wk of treatment (8 wk old), the levels of B2 receptors were still significantly higher in the dorsal horn of SHR compared with age-matched WKY. This evidence suggests that the higher densities of B2 receptor binding sites in most laminae in SHR are related to a genetic feature of the strain and not to hypertension.

Whereas it is uncertain whether the greater number of B2 receptors found in the spinal cord of SHR, particularly between the age of 8 and 16 wk, contributes to the pathogenesis of hypertension, the higher density of B2 receptor binding sites was ascribed as the likely mechanism to explain the hypersensitivity of the pressor response to BK injected in the spinal cord of awake SHR (6). Higher concentrations of kinins, kininogen, kallikrein, and kininase II (ACE) activity were also reported in the cerebrospinal fluid of adult SHR compared with their normotensive controls, suggesting a hyperactive kallikrein-kinin system in the brain and spinal cord of SHR (for a review, see Ref. 8). Because the activation of B2 receptors in the spinal cord leads to increases of blood pressure through the stimulation of the sympathoadrenal system and the peripheral release of catecholamines (23), increased sensitivity to the pressor action of BK along with increased number of receptors and endogenous ligands may contribute to the higher sympathetic tone generally reported in different models of hypertension, including SHR (10, 11). This hypothesis remains to be challenged with a prolonged infusion of the entire spinal cord with specific kinin receptor antagonists because acute blockade of B1 and B2 receptors at the T-9 spinal cord level in SHR was insufficient to alter baseline blood pressure (6). It is also possible that kinins act chiefly as neuromodulators and not as primary mediators of spinal autonomic functions (8).

Reciprocal regulation of B1 and B2 receptors during aging and antihypertensive therapy. Interestingly, the downregulation of B1 receptors from 8 to 24 wk was accompanied by an upregulation of B2 receptors, suggesting an age-dependent regulation of kinin receptors in WKY and SHR. Moreover, the upregulation of B1 receptors induced by the two ACEI and losartan at 8 wk was accompanied by a downregulation of B2 receptors, suggesting again that these receptors are regulated in an opposite way by ACEI and losartan in SHR. This is in keeping with the phenomenon described previously where complete desensitization of B2 receptors in inflammatory models or its deletion in B2 receptor gene knockout mice led to overexpression of B1 receptors (5, 13, 29).

Conflicting data have been reported regarding the regulation of kinin receptors by ACEI. An early pharmacological study has reported an upregulation of B1 receptors in the rabbit vascular system both in vivo and in vitro following acute treatment (18 h) with ACEI (32). More recently, acute inhibition of ACE (48 h) failed to upregulate the B1 receptor (mRNA expression and function) in vascular and nonvascular peripheral organs of rabbits (28). However, a 6-wk treatment with ramipril is associated with functional induction of vascular and renal B1 receptor in normotensive rats, wild-type and B2 receptor gene knockout mice (29). Our results at 8 wk support an upregulation of B1 receptors after 4 wk of treatment with ACEI, although a longer period of treatment (12 and 20 wk) had no effect. It is therefore apparent that this phenomenon occurs within a specific time window, and different conclusions can be drawn depending on the duration of the treatment. It is also noteworthy that contrary to B1 receptors, vascular and renal B2 receptor mRNA was not affected by ACEI in rats (29), suggesting that kinin receptor expression is regulated differently in peripheral and central nervous tissues.

Possible mechanisms underlying the effects of ACEI and losartan on kinin receptors. The mechanism of downregulation and upregulation of neuronal B2 receptors and upregulation of B1 receptors by ACEI is still unknown. We can exclude that the radioligand binds to ACE or to other proteins as captopril was present throughout the autoradiographic procedure in all experimental groups and B2 receptor binding sites seen with the same radioligand in the spinal cord of wild-type mice were gone in B2 receptor gene knockout mice (8).

Although ACEI do not act directly on B2 receptors (14), they can interfere with the sequestration and internalization of B2 receptors within the cellular membrane in native porcine aortic endothelial cells (3) and in Chinese hamster ovary cells (30). An interaction with the process of B2 receptor dimerization, which is directly involved in the endogenous and recycling of receptors, may represent another putative mechanism by which ACEI could affect B2 receptor binding (2). Also, the formation of stable heterodimers between B2 and AT1 receptors, which change the endocytotic pathway of both receptors and enhance G protein activation (1), may provide a possible mechanism by which losartan can affect the expression of B2 receptors in our paradigm. On the other hand, the B1 receptor is not subjected to internalization and resensitization; therefore, its regulation is more likely to occur at the transcriptional and mRNA levels (15, 46). Because B2 and B1 receptors are largely synthesized outside the spinal cord in dorsal root ganglia (24, 34, 45), one cannot exclude the possibility that the site of regulation of kinin receptors by ACEI and losartan is at the level of the cell body of primary sensory fibers.


    ACKNOWLEDGEMENTS

The authors acknowledge G. Lapalme (Clinical Research Institute of Montréal) for technical assistance in the iodination of ligands and Dr. Jean-Guy Chabot (Department of Psychiatry, Douglas Hospital Research Center, McGill University) for conceding the use of the Image Analysis System (MCID, Imaging Research, Ontario, Canada).


    FOOTNOTES

This project was supported by Grant-In-Aid Canadian Institutes of Health Research MOP-14379. B. Ongali holds a Studentship from the Republic of Gabon, and H. S. Buck was the recipient of a Postdoctoral Fellowship from the Fundação de Amparo a Pesquisa do Estado de São Paolo (São Paolo, Brazil).

Address for reprint requests and other correspondence: R. Couture, Université de Montréal, Pavillon Paul-G.-Desmarais, 2960, Chemin de la Tour, Montréal, Québec, Canada H3T 1J4 (E-mail: couturer{at}physio.umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 13, 2003;10.1152/ajpheart.01113.2002

Received 19 December 2002; accepted in final form 7 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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