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(Hypertension. 1996;28:980-987.)
© 1996 American Heart Association, Inc.
Articles |
Antisense Inhibition of the Brain Kallikrein-Kinin System
Clinica Medica, University of Sassari (Italy) and Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston.
Correspondence to Paolo Madeddu, MD, Clinica Medica, University of Sassari, Viale S. Pietro 8, 07100 Sassari, Italy.
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Abstract |
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We used antisense oligodeoxynucleotide (ODN) strategy, based on interference of information flow from gene to protein, to determine the role of kininogen and bradykinin B2 receptor genes in the pathogenesis of genetic hypertension in rats. Mean blood pressure of 9-week-old spontaneously hypertensive rats (SHR) increased 4 hours after acute intracerebroventricular injection of synthetic 18-mer antisense ODNs targeting the translation initiation codon of kininogen mRNA (from 164±5 to 181±4 mm Hg, P<.01) or bradykinin B2 receptor mRNA (from 161±5 to 185±8 mm Hg, P<.01) and then returned to basal levels within 24 hours. Prolonged vasopressor effects were observed after repeated injections of antisense ODN targeting kininogen mRNA. Antisense ODNs to kininogen and B2 receptor mRNAs increased blood pressure of normotensive Wistar-Kyoto rats only slightly compared with SHR (from 116±3 to 124±1 and from 116±2 to 126±4 mm Hg, respectively; P<.05). Cardiovascular responses were confirmed by the use of antisense ODNs targeted to bind to different nonoverlapping regions of kininogen or B2 receptor mRNA. Microinjection of antisense ODN to B2 receptor mRNA into the nucleus tractus solitarii increased mean blood pressure in SHR and prevented the vasodepressor effect induced by intranuclear microinjection of bradykinin. No significant change in mean blood pressure was induced in either strain by intravenous injection of antisense ODNs or by central injection of sense or scrambled ODNs. A strong fluorescent signal was detected at the level of the hippocampus, thalamus, hypothalamus periventricularis, midbrain, and cerebrum 1 hour after central injection of fluorescein isothiocyanate–conjugated antisense ODNs. Kininogen levels were significantly lower in the brain of rats given intracerebroventricular antisense kininogen ODN compared with controls. Our results indicate that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure and suggest that this system may exert a protective action against further elevations of blood pressure levels in SHR.
Key Words: brain • hypertension, genetic • kallikrein • kinins • DNA • fluorescence
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Introduction |
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Kinins are local hormones cleaved by kallikrein from the substrate kininogen.1 2 They act on two bradykinin receptor subtypes, B1 and B2 (according to the classification proposed by Regoli and Barabe3 ). In the rat, most of the central and peripheral cardiovascular effects of bradykinin are mediated by the B2 receptor subtype. The observation that the components of the kallikrein-kinin system, together with kinin-metabolizing enzymes, are present in the brain suggests that this system has physiological relevance.4 5 6 7 8 9 10 Circumstantial evidence of a role for kinins in the central control of cardiovascular function was first provided through studies showing that administration of exogenous bradykinin into the cerebroventricular space caused increases in BP and HR in rats.11 12 13 14 However, studies using intranuclear injection of the peptide showed that bradykinin can produce opposite cardiovascular responses (ie, vasoconstriction and tachycardia or vasodilation and bradycardia) depending on the dose and injection site, possibly because of differential interactions with the sympathetic and parasympathetic components of the central nervous system.15 16 17 18
Pharmacological interventions that enhance the activity of the kallikrein-kinin system increase BP in rats.19 20 21 22 These acute effects are associated with the elevation of kinin levels in the brain and cerebrospinal fluid19 20 22 and are inhibited by a receptor antagonist of bradykinin.19 20 21 Thus, activation of the endogenous brain kallikrein-kinin system could lead to chronic, incremental increases in BP levels. Consistent with this possibility is the observation that this system is overexpressed in the SHR, as suggested by increased concentrations of bradykinin, kallikrein, and kininogen in various areas of the brain.23 24 In addition, exaggerated hypertensive responses reportedly occur in SHR after ICV injection of bradykinin or captopril, a kininase inhibitor.17 21 25 Nevertheless, it remains unknown whether the increased expression of the kallikrein-kinin system found in this strain represents a mere epiphenomenon or is functionally related to the elevated BP. Studies using chronic infusion of a potent and long-acting receptor antagonist of B2 receptors, D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (Hoe 140), failed to demonstrate any effect on BP although the antagonist altered the baroreflex control of HR.26 However, a limitation of these studies was that distribution of Hoe 140 was not evaluated. Thus, it is not clear whether the failure to affect BP is attributable to insufficient penetration of the antagonist into the brain.
Antisense ODNs offer the potential to block the expression of specific genes within cells.27 28 29 30 They are short strands of DNA or RNA, usually 12 to 18 bases in length, that are synthesized to bind to a target complementary mRNA sequence of a candidate gene. Once they enter the cells, they could bind to their target mRNA and block the translation of the cognate protein. In addition, they might provide substrate for RNase H, an enzyme that degrades the RNA-DNA duplex. The utility of this strategy has been demonstrated in cultured cell experiments and in recent in vivo studies that were aimed at addressing the role of peptidergic systems in the central regulation of BP and cerebral blood flow.28 31 32 The availability of antisense ODNs targeted to bind kininogen and B2 receptor mRNAs prompted us to apply this new approach in studies on the brain kallikrein-kinin system. Thus, we performed experiments to address the following questions: (1) Does the ICV injection of antisense ODNs alter the BP of normotensive and hypertensive rats? (2) where are antisense ODNs distributed after central injection? and (3) is the expression of targeted genes diminished in brain tissues where antisense ODNs are retained?
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Methods |
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Male WKY and SHR (Charles River, Milan, Italy) weighing between 220 and 260 g were housed at a constant room temperature with a 12-hour light/dark cycle and had free access to water and rat chow. The experimental protocol was approved by the local Animal Care and Use Committee. All procedures complied with the standards for the care and use of animal subjects as stated in Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Md).
Oligodeoxynucleotides
ODNs were synthesized as 18-mers targeted to bases 47 to 64 (sense ODN KG1: 5'-GTTTAGCGCAGGAAGAAG-3'; antisense ODN KG1: 5'-CTTCTTCCTGCGTAAAC-3'; scrambled ODN KG1: 5'-GCCCTACTATCCTTCGTA-3') or to bases 1132 to 1149 (sense ODN KG2: 5'-AGGCCTCCAGGATTTTCA-3'; antisense ODN KG2: 5'-TGAAAATCCTGGAGGCCT-3') of rat kininogen mRNA encompassing the translation initiation codon and exon 10 of the mRNA sequence, respectively.33 Eighteen-mer ODNs were synthesized to target to bases 2886 to 2913 (sense ODN BKR1: 5'-TGAAATGTTCAACATCAC-3'; antisense ODN BKR1: 5'-GTGATGTTGAACATTTCA-3'; scrambled ODN BKR1: 5'-TCTATTGTAGAGCTTAAG-3') or to bases 3406 to 3423 (sense ODN BKR2: 5'-TTCAGGACCATGAAGGAC-3'; antisense ODN BKR2: 5'-GTCCTTCATGGTCCTGAA-3') of rat B2 receptor mRNA encompassing the initiation codon and exon 3 of the mRNA sequence, respectively.34 ODNs were made resistant to nucleases by DNA backbone phosphorothioation. For analysis of brain distribution, antisense ODN KG1 and antisense ODN BKR1 were conjugated with FITC at both the 5' and 3' ends.
BP Effects of ICV ODNs
Rats were anesthetized with a cocktail of ketamine chloride (45 mg/kg body wt, Parke-Davis) and diazepam (5 mg/kg body wt, Roche). A 22-gauge stainless steel cannula fitted into a 3x4-mm membrane-valve plastic block (Umberto Danuso) was implanted stereotaxically into the left lateral cerebral ventricle (1.5 mm lateral and 1.0 mm posterior to the bregma; 4.5 mm below the skull surface). The plastic block was anchored to the skull with screws embedded in dental acrylic cement. The cannula was filled with sterile aCSF. Correct placement of the cannula was tested at the end of the experiment by determining the dipsogenic response to angiotensin II.
Five days after implantation of ICV cannulas, a polyethylene catheter (PE-10 connected to a PE-50, Clay Adams) filled with heparinized saline was inserted into the left femoral artery and advanced into the abdominal aorta of rats under ether anesthesia. The catheter was tunneled under the skin and exteriorized at the back of the neck. The following day, MBP and HR of freely moving, unanesthetized rats were measured with a Statham transducer (Gould Instruments) connected to the femoral catheter and were recorded on a recorder (Quartet, Ugo Basile). HR was determined with a counter triggered by the arterial pressure pulse. After a 15-minute stabilization period, a single 50-µg dose (in 5 µL aCSF) of ODNs was injected ICV. ODN injection was immediately followed by 5 µL aCSF to flush the cannula. Injections were made with a 25-µL syringe (Hamilton). Rats (n=7 per group) received only one antisense ODN or the respective sense or scrambled ODN. MBP and HR were recorded for 15 minutes at 1, 4, 8, and 24 hours after ODN injection. In some experiments, MBP was also measured at 48 hours.
In a separate set of experiments, SHR were injected ICV with 0, 50, and 150 µg antisense ODN KG1 (in 5 µL aCSF), and MBP and HR were recorded for 4 hours. Rats (n=6 per group) received only one dose of antisense ODN KG1. In addition, the chronic effect of ODN KG1 on systolic BP of SHR was tested by ICV injection of sense or antisense ODN KG1 (50 µg/d for 5 days, n=4 per group). Systolic BP was measured by tail-cuff plethysmography (Recorder 8002, Ugo Basile) in unanesthetized rats prewarmed for 10 minutes at 37°C. Measurements were performed under basal conditions, 1 and 5 days after ODN administrations, and on two occasions (days 1 and 5) after their discontinuation.
Effects of ICV ODNs on Bradykinin-Induced Changes in BP
The MBP of unanesthetized SHR, instrumented as described above, was measured before and after injection of a single dose (50 µg in 5 µL aCSF) of the sense or antisense ODN BKR1. Four hours later, the MBP effect induced by ICV injection of 380 pmol bradykinin (Phoenix Pharmaceuticals Inc) in 5 µL aCSF was tested in both groups (n=4 rats each). Additional experiments were performed for determination of whether ICV injections of ODNs (at the dose indicated above) alter the vasodepressor effect of intra-arterial injection of bradykinin (20, 50, and 200 pmol, in random order). Bradykinin was injected through a catheter inserted into the thoracic aorta via the left carotid artery. Each group consisted of four rats.
BP Effects of Intranuclear ODNs
SHR were anesthetized with urethane (1 g/kg IP), and a catheter was inserted into the abdominal aorta as described above. Then, the dorsal surface of the medulla oblongata was exposed by incision of the atlanto-occipital membrane, with the rat's head flexed downward at 45° in a stereotaxic frame. A single dose of sense or antisense ODN BKR1 (1 µg in 50 nL aCSF) was unilaterally microinjected into the medial NTS (0.4 mm and 0.3 mm lateral to the obex; 0.3 mm below the dorsal surface of the medulla) with a triple-barreled glass micropipette. One hour later, bradykinin (1 pmol in 50 nL aCSF) was injected into the NTS of either group (n=4 rats each). The substances were injected over 60 seconds. MBP was measured before ODN injection and then immediately before and for 15 minutes after bradykinin injection. The location of injections was determined by the use of 50 nL of Alcian blue dye and histological examination of brain sections.
BP Effects of Peripheral ODNs
SHR were instrumented with femoral catheters as described above. The following day, MBP was recorded for 15 minutes, and then a single dose (50 µg in 200 µL isotonic saline solution) of antisense ODN KG1 or antisense ODN BKR1 was injected intra-arterially (n=5 per group). The control groups consisted of SHR (n=5 per group) given the respective sense ODNs. MBP was recorded for 24 hours as described above.
Analysis of FITC-Conjugated Antisense ODN Distribution After ICV or Intra-arterial Administration
SHR were instrumented with an ICV cannula as described above. Five days later, they were injected ICV with FITC-conjugated antisense ODN KG1 or antisense ODN BKR1 (50 µg in 5 µL aCSF), followed by 5 µL aCSF to flush the cannula. One or 4 hours later, the rats were anesthetized with ether and then perfused transcardially with cold isotonic saline solution (30 mL), followed by 4% formaldehyde solution (30 mL) to fix the tissues. The brain was then removed and kept in 4% formaldehyde solution until cryostat sectioning. Sections (50 µm) were obtained from the injection site, the lateral and third ventricle regions, and the brain stem. They were mounted and observed by laser scanning confocal microscopy for determination of antisense ODN distribution and cellular uptake.
We carried out a separate set of experiments in SHR to determine the peripheral distribution of 50 µg FITC-conjugated antisense ODN BKR1. Tissues (liver, kidney, and heart) were examined 1 hour after ICV or intra-arterial ODN injection.
Effect of Antisense ODNs Targeted to Bind to Kininogen mRNA on Kininogen Levels in Specific Brain Regions
Two groups of SHR (n=9 per group), instrumented with brain cannulas as described above, were injected ICV with a single 50-µg dose of antisense or sense ODNs to kininogen. Four, 24, or 48 hours later, the rats were anesthetized and transcardially perfused with 20 mL isotonic saline solution to avoid blood contamination. Brains were quickly removed and placed on ice. Selected brain regions were homogenized in phosphate-buffered saline (0.2 g tissue/mL), pH 7.0, with a Polytron homogenizer. Samples were centrifuged at 600g for 15 minutes. The supernatants were treated with sodium deoxycholate (0.5% wt/vol) for 30 minutes at 20°C, sonicated for 10 minutes, and then centrifuged at 20 000g for 90 minutes. The final supernatants were divided into two aliquots for measurement of protein35 and kininogen36 concentrations. Fifty to 200 µL of the supernatants was added to 0.45 mL of 20 mmol/L Tris-HCl buffer, pH 8.0, boiled for 30 minutes to eliminate kininase activity, and then centrifuged for 5 minutes at 600g in a microcentrifuge. Aliquots (100 µL) of the supernatants were combined with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)–trypsin (40 µg, Sigma Chemical Co) or purified rat tissue kallikrein (40 µg) in 0.4 mL of 20 mmol/L Tris-HCl, pH 8.0, for release of the kinin moiety from kininogens. The samples were then incubated at 37°C for 30 minutes, and the reaction was stopped by boiling for 10 minutes. Kinin concentrations were measured by radioimmunoassay using rabbit antiserum against bradykinin and 125I-labeled Tyr-bradykinin.37 Kininogen levels in tissues were expressed as nanograms kinin equivalent per milligram protein.
Statistical Analysis
All data are expressed as mean±SE. Multivariate repeated measures ANOVA was performed to test interactions between time and grouping factors. Then, univariate ANOVA was used to test for differences between groups and over time. Differences within or between groups were determined by paired or unpaired t tests with the Bonferroni multiple comparison adjustment. A probability (P) value less than .05 was considered significant.
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BP Effects of ICV ODNs
In WKY, ICV administration of antisense ODNs targeting the translation initiation codon of kininogen or B2 receptor mRNA induced a significant increase in MBP (Fig 1
, top panels). This effect was highest between 4 and 8 hours after injection, and then MBP returned to baseline levels within 24 hours. By contrast, ICV injection of sense ODNs did not alter MBP levels in control groups.
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P<.05 vs time 0.
MBP was increased in SHR after administration of antisense ODN KG1 or antisense ODN BKR1 (from 164±5 to 181±4 mm Hg and from 161±5 to 185±8 at 4 hours, respectively; P<.01 for both comparisons) and then returned to basal levels within 24 hours (Fig 1
, bottom panels). No change in MBP was observed after ICV injection of the respective sense or scrambled ODNs in the control groups. The hypertensive effects observed after ICV antisense ODNs in SHR were greater than those of WKY (ODN KG1: 18±1 versus 7±2 mm Hg; ODN BKR1: 25±4 versus 10±2; P<.01 for both comparisons). Antisense and sense ODNs did not alter HR in either strain (data not shown).
In SHR, the magnitude of the MBP response recorded 2 hours after ICV injection of antisense ODN KG1 was increased in a dose-dependent manner (Fig 2). However, a similar plateau was reached with either 50 or 150 µg at 4 hours. As shown in Fig 3, pressor effects similar to those induced by ICV antisense ODNs targeted to translation initiation codons were observed in SHR after ICV injection of antisense ODNs targeted to bind to exon 10 of kininogen mRNA (left) or to exon 3 of B2 receptor mRNA (right), whereas the respective sense ODNs did not alter MBP.
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Daily ICV injections of 50 µg antisense ODN KG1 increased systolic BP of SHR from 187±4 to 224±8 mm Hg (P<.05), whereas sense ODN KG1 was not effective (from 187±5 to 181±3 mm Hg, P=NS). After discontinuation of ODN injections, systolic BP remained above basal levels in antisense-treated rats, and it was unchanged in controls (235±4 versus 180±5 mm Hg at day 1 and 209±3 versus 178±4 at day 5, P<.01 for both comparisons).
Effects of ICV ODNs on Bradykinin-Induced Changes in BP
ICV injections of bradykinin induced similar vasopressor effects in SHR given ICV sense or antisense ODN BKR1 (37±5 versus 40±4 mm Hg, P=NS). Prior ICV injection of antisense BKR1 did not alter the vasodepressor effect of intra-arterial bradykinin (data not shown).
BP Effects of Intranuclear ODNs
Microinjection of sense ODN BKR1 into the NTS did not affect the MBP of anesthetized SHR (from 153±5 to 154±3 mm Hg, P=NS), whereas antisense ODN increased MBP from 152±1 to 173±5 mm Hg (P<.01). Intranuclear injection of bradykinin decreased the MBP of SHR given intranuclear sense ODN BKR1 by 21±2 mm Hg (from 154±3 to 133±3 mm Hg, P<.01), whereas this effect was reduced in rats given antisense ODN BKR1 (from 173±5 to 170±3 mm Hg, P=NS).
BP Effects of Peripheral ODNs
No significant BP effect was observed in SHR after intra-arterial injection of antisense ODN KG1 (from 166±5 to 164±5, 162±4, 164±8, and 167±8 mm Hg at 1, 4, 8, and 24 hours, respectively; P=NS), antisense ODN BKR1 (from 170±5 to 176±4, 179±5, 169±7, and 161±11 mm Hg, respectively; P=NS), or their respective sense ODNs (ODN KG1: from 168±4 to 164±3, 165±4, 168±2, and 167±4 mm Hg; ODN BKR1: from 167±4 to 168±3, 169±2, 165±2, and 167±4, respectively; P=NS).
Analysis of FITC-Conjugated Antisense ODN Distribution After ICV or Intra-arterial Administration
In SHR, 1 hour after ICV injection of FITC-conjugated antisense ODN KG1, a strong fluorescent signal was detected around the lateral ventricles at the level of the hippocampus (Fig 4A) and around the third ventricle at the level of the nucleus paraventricularis rotundus cellularis of the thalamus (Figs 4A, 4B, and 4C) and the hypothalamus periventricularis. One hour after ICV injection of antisense ODN BKR1, a fluorescent signal was detected around the lateral ventricles at the level of the tegmentum and around the third ventricle at the level of the fasciculus longitudinalis dorsalis and the substantia grigia periventricularis of the pons and at the level of the nucleus dorsalis and nucleus arcuatus of the hypothalamus. Intranuclear uptake of antisense ODNs by individual cells was detected at greater magnifications. A weaker fluorescent signal was observed 4 hours after the injection of both ODNs in the brain of SHR (ODN KG1: thalamus>hypothalamus>midbrain>cerebrum; ODN BKR1: hypothalamus>thalamus>midbrain>cerebrum).
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FITC-conjugated ODNs were not detected in peripheral tissues 1 and 4 hours after ICV injection. A very weak fluorescent signal was detected in the liver 1 hour after intra-arterial injection of either of the antisense ODNs, whereas no signal was observed in the heart or kidneys.
Effect of Antisense ODNs Targeted to Bind to Kininogen mRNA on Kininogen Levels in Specific Brain Regions of SHR
Kininogen levels (measured in brain homogenates treated with trypsin) of SHR killed 4 hours after ICV injection of antisense ODN KG1 were significantly lower than those of rats given sense ODN KG1 (midbrain: 0.93±0.18 versus 2.24±0.60 ng bradykinin equivalent/mg protein; hippocampus: 0.81±0.12 versus 2.87±0.87; striatus: 1.23±0.36 versus 2.00±0.95; and hypothalamus: 0.72±0.11 versus 2.01±0.45; P<.05 for each comparison). Kininogen levels were lower in brain areas in which rat kallikrein was used instead of trypsin to hydrolyze kininogen. A between-group difference was detected in brain areas obtained at 4 hours after ODN injection (midbrain: 0.43±0.06 versus 1.03±0.11 ng bradykinin equivalent/mg protein; hippocampus: 0.40±0.02 versus 1.85±0.33; striatus: 0.13±0.06 versus 0.81±0.04; and hypothalamus: 0.25±0.03 versus 1.12±0.06; P<.05 for each comparison). No difference between groups was detected at 24 hours, a time period sufficient for MBP to return to baseline.
In rats given antisense ODN KG2, whose MBP remained elevated for a longer time, brain kininogen levels were still reduced at 24 hours; however, kininogen levels became similar to those of controls at 48 hours, when MBP returned to baseline (data not shown).
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One of the prerequisites for the in vivo use of antisense ODNs is that they must be relatively resistant to degradation. An advantage in the application of this approach to the central nervous system is that brain tissue has a relatively low capacity to degrade ODNs while its uptake efficiency is elevated.28 29 This explains why even unmodified ODNs are stable for up to 24 hours when incubated with rat cerebrospinal fluid. In the present study, we replaced the phosphodiester linkage of the DNA backbone with a nuclease-resistant entity to enhance stability. A single ICV injection of a phosphorothioated analogue targeted to the translation initiation codon of kininogen mRNA resulted in a prolonged increase of MBP. The increase was particularly evident in SHR, whereas HR remained unaltered. It is noteworthy that administration of a second antisense ODN targeted to bind the mRNA sequence encoding the bradykinin moiety produced an elevation of MBP lasting more than 24 hours, thus exceeding that of the probe targeting the translation initiation site. The latter approach has been used on the assumption that the initiation site of mRNA is important and easily accessible. However, recent studies have demonstrated the accessibility of other regions, except for those with strong secondary structure.27 Confirmation of the cardiovascular response by nonoverlapping antisense ODN sequence represents an important control for specificity that supplements the information derived by the lack of a cardiovascular effect of the respective sense and scrambled ODNs. Since MBP is determined by cardiac output and peripheral resistance, both could be responsible for the cardiovascular response to antisense ODNs. However, the contribution of cardiac output is not sustained by an increase in HR, since no tachycardic effect was observed.
The time course of the pressor response to antisense ODNs targeted to kininogen mRNA reflects their rapid uptake by brain tissue and interference with protein synthesis within a few hours. Decreased levels of kininogen were detected in specific brain areas involved in the central regulation of BP 4 hours after the ICV injection of antisense ODN KG1, whereas they were restored to normal after 24 hours when MBP also was returned to baseline. Similarly, changes in brain kininogen levels parallel changes in BP in rats given ICV antisense ODN KG2 although the duration of these effects was longer. These results are consistent with a kinetic model in which target mRNA is rapidly degraded and slowly translated and protein is rapidly degraded. Since maintenance of suppression and biological effects were limited to a few hours after a single injection of antisense ODN KG1, it was necessary to inject this ODN repeatedly to achieve prolonged hypertensive effects. A reduction in kininogen concentration could be relevant because this protein is overexpressed in the brain of SHR23 24 31 and the rate of generation of the biologically active peptides is substrate dependent. Therefore, the incomplete inhibition of the expression observed in this study might be sufficient to induce BP-altering effects. An additional explanation for the modest alteration in kininogen levels is that blocks of brain tissue were used for kininogen measurements and they may include nuclei or cells that were not affected by the antisense ODN. In addition, the effect of antisense ODNs on kininogen content may be less profound than on the rate of kininogen secretion. Previous studies have shown the presence of low molecular weight kininogen, high molecular weight kininogen, and T-kininogen in the rat brain.9 Since trypsin releases kinins from all kininogens, kinins thus generated represent total kininogens. Low molecular weight kininogen levels in brain homogenates were measured by treatment of the extracts with rat tissue kallikrein. The finding of a difference between groups given antisense or sense ODNs in brain samples treated with rat tissue kallikrein indicates that antisense ODNs affect kininogens other than T-kininogen.
Similar pressor responses were observed after the ICV administration of antisense ODNs targeted to bind B2 receptor mRNA. We could not detect a significant decrease in receptor mRNA content in the brain of rats given these ODNs (data not shown). According to the most accepted theory, antisense ODNs exert their effects by inducing translational arrest or by providing substrate for RNase H activity (an enzyme that degrades the RNA strand of an RNA-DNA duplex).27 28 29 30 However, it is not known whether RNase H plays an important role in mRNA degradation in mammalian neurons. Indeed, an antisense ODN targeted to bind to the initiation site of the N-methyl-D-aspartate glutamate receptor did not significantly affect the mRNA but reduced the protein concentration.28 Our results are consistent with the translational arrest model, which could cause a reduced translational rate but would not cause a decline in B2 receptor mRNA. A limitation of the present study is that the effectiveness of protein synthesis inhibition was not documented after administration of antisense ODN to bradykinin receptor mRNA. On the other hand, specificity of the cardiovascular response is confirmed with a second antisense ODN targeted to a different, nonoverlapping region of the B2 receptor mRNA.
Given their negative charge, antisense ODNs should not be able to cross the blood-brain barrier. Experiments were performed to rule out the possibility that the central effects were due to leakage of ODNs into the peripheral circulation. Since the same dose that was used for ICV injection proved to be ineffective intra-arterially, we concluded that the vasopressor effect is due to central inhibition of targeted genes.
Application of laser confocal microscopy for detection of FITC-conjugated ODNs allowed us to determine their distribution after central administration. As in previous studies with the use of an angiotensinogen probe,31 1 hour was sufficient for our antisense ODNs to be taken up by brain structures adjacent to the lateral and third ventricles, in particular, at the level of the hippocampus, hypothalamus, thalamus, and midbrain. Thus, distribution is consistent with the decrease in kininogen levels in these brain areas. Obviously, this does not allow us to relate fluorescence to the site of action.
When administered by the peripheral route, bradykinin is a potent vasodilator. A biphasic response occurs after ICV injection where an initial depressor effect is followed by a pressor response that becomes the predominant component in conscious rats.25 Our finding that the vasopressor effect of ICV injection is not altered by antisense ODN BKR1, at variance with effective antagonism achieved with B2 receptor antagonists,21 26 may implicate differences in the distribution, specificity, and degradation of these compounds. An important determinant for the direction of the cardiovascular response to bradykinin is its injection site. Microinjection of bradykinin into the nucleus hypothalamicus anterior (which controls parasympathetic tone) causes hypotension and bradycardia,16 whereas microinjection into the hypothalamus dorsalis and rostral ventrolateral medulla (which contain neurons regulating sympathetic tone) causes hypertension and tachycardia.16 17 Thus, the final response appears to depend on the selective interference of bradykinin with sympathetic or parasympathetic tone. In addition, a limitation of the majority of the studies reported above is that pharmacological doses may not reflect the physiological action of the endogenous peptide. In one of the few studies that examined a full dose-response curve starting with 1 fmol bradykinin, microinjection of the peptide into the NTS decreased MBP and HR in rats, an effect mediated by combined stimulation of vagal efferent activity and withdrawal of sympathetic tone.18 In the present study, we confirmed the vasodepressor effect of bradykinin at the level of the NTS. In addition, similar inhibitory effects on this vasodepressor action were observed in rats pretreated with a B2 receptor antagonist18 or antisense ODN BKR1. Since microinjection of the latter compound into the NTS increases rat BP, the hypertensive response to ICV ODN BKR1 might be related to diffusion from the lateral ventricle to the NTS. Unfortunately, examination of antisense ODN distribution was not extended to the medulla. Therefore, we cannot draw any conclusion about the possible distribution in this area after ICV injection. Alternatively, the pressor effect of ICV ODN BKR1 could be mediated by hypothalamic projections to the NTS. Taken together, these results suggest that bradykinin could act as a central vasodilating mechanism by activating B2 receptors in the NTS, an essential link in the reflex and tonic regulation of BP.
A different approach is to evaluate whether BP can be influenced by artificially increasing the levels of endogenous kinins. Actually, both melittin, a substance that solubilizes membrane-bound kallikrein, and captopril, a kininase inhibitor, are capable of producing short-term vasopressor effects, and these changes are prevented by the administration of a first-generation kinin receptor antagonist.19 20 21 However, it is unknown whether high brain kinin concentrations, similar to those reported after melittin administration, occur under physiological conditions. Certainly, the fact that the brain kallikrein-kinin system is overexpressed in SHR24 may suggest participation of the system in the pathogenesis of genetic hypertension. However, we should be cautious in attributing pathogenetic relevance to any biochemical patterns in the SHR model merely on the basis of quantitative differences from its normotensive counterpart. Increased levels of kinins38 and a kallikrein-like enzyme39 have been discovered recently in the vascular tissue of young SHR, but the meaning of this alteration remains to be elucidated.
Our studies using the methodology of translational arrest of gene expression by antisense ODNs deal primarily with the chronic action of the endogenous brain kallikrein-kinin system. Previous studies from our group demonstrated that neither acute nor chronic ICV administration of a potent and long-acting receptor antagonist of bradykinin (Hoe 140) is able to alter the BP of young SHR or to affect the progression toward severe hypertension.26 By contrast, antisense ODNs targeted to kininogen or bradykinin receptor mRNA produced an increase in BP that suggests a vasodilator action of the kallikrein-kinin system in the brain. This is not the first time that information inferred by the use of ODNs differs from that provided by receptor antagonists. Indeed, ICV administered angiotensin antagonists induced variable effects on the BP of SHR and were ineffective at low doses,40 41 produced unexpected increases of BP at high doses,42 and caused a hypotensive response only under conditions of salt loading.43 By contrast, antisense ODNs targeted to inhibit the expression of angiotensinogen or angiotensin receptors cause a consistent and prolonged vasodepressor response.31 32 These discrepancies might be related to differences in drug metabolism, distribution, and time of measurement.
In conclusion, inhibition of the brain kallikrein-kinin system by antisense ODNs causes a long-lasting pressor effect in rats. These results are consistent with the concept that this system may exert a systemic vasodilator action in young SHR. However, regions of the brain where kinins could exert predominant vasopressor effects might not be reached by ICV-delivered ODNs. Therefore, further studies are necessary to delineate the complex interactions that, at the level of different brain nuclei, underlie the actual role of the brain kallikrein-kinin system.
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Acknowledgments |
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This work was supported in part by a grant from the Minister of Universities and Scientific Research, a research grant from the National Research Council (CNR) targeted project "Prevention and Control of Disease Factors" No. 91.001173.41, and National Institutes of Health grants HL-29397 and HL-44083.
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TopAbstractIntroductionMethodsResultsDiscussionAppendix AppendixReferences |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix AppendixReferences |
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After acceptance of this article for publication, we got additional information regarding the specificity of the oligonucleotide sequences by screening known rat genes from the genomic databases of the National Center for Biological Information (NCBI) at the E-mail address blast@ncbi.nlm.nih.gov. These data indicate specificity of the sequences used in the present study and confirm their 100% homology with the endogenous genes. The sequences in the following table producing high-scoring segment pairs were obtained.
T-KG indicates T-kininogen; LMW, low molecular weight; and HMW, high molecular weight.
Received January 19, 1996;
first decision February 29, 1996; first decision July 12, 1996;
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