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(Hypertension. 2001;38:1075.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Effects of Antisense Oligodeoxynucleotide Targeting of the _2B-Adrenergic Receptor Messenger RNA in the Central Nervous System

Ekaterina Kintsurashvili; Irene Gavras; Conrado Johns; Haralambos Gavras

From the Hypertension and Atherosclerosis Section, Department of Medicine, Boston University School of Medicine, Boston, Mass.

Correspondence to Haralambos Gavras, MD, Chief, Hypertension & Atherosclerosis Section, Boston University School of Medicine, 715 Albany St, Boston, MA 02118. E-mail hgavras{at}bu.edu

	Abstract

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Abstract—— The results of previous studies with genetically engineered mice have suggested that an intact central _2B-adrenergic receptor (_2B-AR) subtype mediates the development and maintenance of salt-induced hypertension. In the present study, we sought to further define the role of this receptor by injecting antisense oligodeoxynucleotides (AS-ODNs), targeting a selected sequence of the _2B-AR mRNA, into the lateral cerebral ventricle of rats that had undergone prior subtotal nephrectomy and dietary salt loading. Cell culture studies showed that these AS-ODNs could block _2B-AR protein generation. Before AS-ODN injection, blood pressure (BP) averaged 133±5 mm Hg during the daytime and rose to 165±4 mm Hg during the nighttime activity hours (P<0.001 versus baseline average of 120±2 mm Hg). The injection of AS-ODNs during the early afternoon prevented the BP rise and was associated with a significant fall in heart rate (from 385±12 to 306±15 bpm, P<0.05) and symptoms of sedation that lasted for several hours, with a peak at 3 to 6 hours and full recovery by 24 hours. At that time, a second injection produced identical effects in all rats (n=9). Control rats (n=10) that received scrambled ODN injections had no changes in BP or heart rate patterns, and neither group had evidence of neurotoxicity, indicating that these effects are specifically due to translational inhibition of central _2B-AR. We conclude that a fully functional central _2B-AR is necessary for the induction of salt-dependent hypertension.

Key Words: hypertension, sodium dependent • genes • antihypertensive therapy

	Introduction

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The ₂-Adrenergic receptors (₂-ARs) are members of the superfamily of G protein–coupled receptors.¹ There are 3 members of the ₂-AR family: _2A/D, _2B, and _2C (on the basis of sequence homology, the _2D subtype is the rat equivalent to the human _2A-AR subtype).² All are known to inhibit adenylate cyclase and to be involved in both central and peripheral control of blood pressure (BP), behavior, insulin release, sedation, and the presynaptic regulation of neurotransmitter release.^3–6 ₂-AR subtypes differ in their structure, patterns of tissue expression, and pharmacological profile.^7–11

Traditional pharmacological methods have a limited capacity to determine the functional role of receptor subtypes. In many cases, agents that display high selectivity within a family in vitro fail to maintain selectivity in vivo and can even interact with different classes of G protein–coupled receptors.^12,13 Alternative approaches to defining the functional role of receptor subtypes include the generation of transgenic animals, which overexpress or lack the gene for a certain receptor or signaling component, and gene treatment. The latter involves amplification of a gene product via extra gene copies or inhibition of gene expression via antisense techniques.

Recently, genetically engineered mice with modified genes for any 1 of the ₂-AR subtypes became available.^4,5,14 This has already helped in the assignment of subtype-specific functions and should help in the design of new drugs. Studies with such animals have shown that the _2A-AR is primarily responsible for the centrally mediated and clinically beneficial hypotensive effect of ₂-AR stimulation by agonists, such as clonidine, whereas the _2B-AR is the primary mediator of the hypertensive effects of ₂-AR stimulation.^4,5 Recent data from our laboratory have shown that the _2B-AR subtype is necessary in the hypertensive response to salt loading and suggested that the pressor effect is a function of _2B-AR located in the central nervous system (CNS),^15,16 not in the periphery, as proposed by other investigators.⁵ To further assess the role of this subtype in salt-induced hypertension, we attempted to selectively suppress its expression in the CNS using antisense technology, which provides a highly specific and reversible means for the inhibition of protein expression. To this aim, we injected antisense oligodeoxynucleotides (AS-ODNs) targeted to rat _2B-AR mRNA into the lateral cerebral ventricle of rats with salt-induced hypertension under constant monitoring of BP. We hypothesized that inhibition of the generation of the _2B-AR would decrease BP in these animals.

	Methods

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ODN Synthesis and Use
AS-ODNs were synthesized as phosphorothioated 18-mers targeted to bases 12 to 29 of the rat _2B-AR coding sequence according to the RNG ₂ sequence of Zeng et al.¹⁷ The antisense sequence chosen was 5'-CTCCTGATGGTCCATGGT-3'. As a control, we used scrambled ODNs, the sequence of which was 5'-TGACGTCTCCTCGGTGTA-3'. Fluorescein isothiocyanate (FITC)-conjugated ODNs were composed of phosphorothioated sequence with FITC conjugation at the 5' ends. The phosphorothioated AS-ODNs and scrambled ODNs were synthesized at Gemini Biotech. All oligomers were purified by HPLC.

In Vitro Testing of Rat ₂-AR Inhibition by AS-ODNs
The capacity of AS-ODNs to inhibit rat _2B-AR gene expression was tested on mouse neuroblastomaxrat glioma NG108-15 hybrid cells. This cell line is known to express ₂-AR.¹⁸ reverse transcription–polymerase chain reaction (RT-PCR) was performed to ascertain rat-specific expression of the _2B-AR.

Uptake of AS-ODNs by cells expressing rat _2B-AR was shown by the addition of 2 µmol/L FITC-labeled AS-ODNs into the culture medium. After 24 hours of incubation, the cells were fixed and visualized using a fluorescent microscope.

The culture media with 2 µmol/L phosphorothioated AS-ODNs, scrambled ODNs, or PBS were changed twice a day for 3 days. After 72 hours, the NG108-15 cells were harvested, and membranes from cells were isolated as described by Phillips et al.¹⁹ Protein content was assayed according to Lowry et al.²⁰ Proteins were subjected to Western blot analysis for rat _2B-AR protein level assessment.

Animals and Procedures
Twenty-two male Wistar rats, each weighing 237 to 396 g (Charles River Laboratories Inc, Wilmington, Mass), were used in these experiments, which were conducted in accordance with the guidelines for the Care and Use of Animals approved by the Boston University Medical Center.

For subtotal nephrectomy,²¹ rats first had both poles of the left kidney excised while under anesthesia. Five to 7 days later, a 24-gauge guide cannula (Plastic One) was implanted stereotaxically into the left lateral ventricle. Five to 7 days after that, a PA-C40 radiotelemetry BP transmitter probe (Data Sciences International) was implanted in the aorta. Five to 7 days later, rats had the right kidney removed. After baseline measurements were taken for 5 days, rats were placed on 1% NaCl as drinking water.

On the day of the AS-ODN injection, a 31-gauge inner cannula (Plastic One) was lowered into the guide cannula, projecting 1 mm below the end (4.5 mm), and attached to a Harvard pump. Injections were made with a 25-µL Hamilton syringe. Then, 10 µL of 5 nmol AS-ODNs or scrambled ODNs in 0.9% saline was infused over 15 minutes followed by 10 µL of 0.9% saline flush over 15 minutes. (In preliminary dose-finding experiments, we determined that a 10 nmol dose produced results identical to those of the 5 nmol dose, which was then chosen for subsequent experiments.) BP and heart rate were continuously measured for 30-second periods at 2-minute intervals for the entire 8-day period of the experiments. Behavioral changes in each rat were assessed on the basis of the righting reflex. At the end of each experiment, the location of the cannula within the lateral ventricle and the integrity of surrounding tissues were confirmed histologically.

Distribution of FITC-Conjugated AS-ODNs in the Brain
Next, 10 µL of 5 nmol FITC-conjugated AS-ODNs was infused over 15 minutes in the left lateral brain ventricle of rats (n=3). One hour later, the animals were anesthetized and killed. Frozen brain was cryostat-sectioned into 30-µm-thick sections and viewed with a microscope.

An expanded Methods section can be found in an online data supplement available at http://www.hypertensionaha.org.

	Results

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Detection of Rat _2B-AR Gene in NG108-15 Cells by RT-PCR and Cellular Uptake
The rat-specific expression of the _2B-AR gene in hybrid NG108-15 cells was ascertained by RT-PCR. With mouse-specific primers, no product was detected, whereas an expected 365-bp fragment was detected with primers recognizing both mouse and rat sequences, indicating that the NG108-15 cells express a rat _2B-AR transcript.

The uptake and intracellular localization of FITC-labeled AS-ODNs by NG108-15 cells after 24-hour incubation in culture were visualized using a fluorescent microscope.

Inhibition of Rat _2B-AR Gene in NG108-15 Cells In Vitro
After 3-day AS-ODN treatment of NG108-15 cells, there was a clear reduction in immunodetectable rat _2B-AR protein as shown by Western blot analysis with _2B-AR–specific antibodies. Thus, the rat _2B-AR protein level was significantly reduced in NG108-15 cells treated with rat _2B-AR AS-ODNs (by 52%, P<0.05), compared with cells treated with the PBS control or scrambled ODNs, indicating the efficacy of the AS-ODNs for inhibition of _2B-AR expression.

Inhibition of Rat _{2 B}-AR Gene In Vivo
The effect of intracerebroventricular AS-ODNs (n=9) or scrambled ODNs (n=10) on BP in rats that underwent subtotal nephrectomy and received 1% NaCl as drinking water is shown in Figure 1. After 3 days of baseline BP recording, the animals underwent 3 days of salt loading, during which systolic BP rose significantly in all animals during the nighttime activity hours, with lesser increases during daytime sleep hours.

View larger version (27K): [in this window] [in a new window]   Figure 1. BP effect of intracerebroventricular injection of AS-ODNs or scrambled ODNs against the 2B-AR gene in subtotally nephrectomized salt-fed rats. Systolic BP is normal during the first 3 baseline days and increases significantly in the next 5 days of dietary salt loading with a large diurnal variation. Treatment with injection of AS-ODN (n=9) prevents the BP rise for at least 6 hours, but the effect gradually wears off. Repeat injection at 24 hours produces the same BP effect. Injection of scrambled ODNs (n=10) produces no BP changes. *P<0.01 vs BP at same times during first 3 days of saline loading.

The average 24-hour baseline BP for both groups was 120±2 mm Hg, with minimal daytime/nighttime variability. During the 3 days of salt loading, the nighttime BP was 163±4 mm Hg (P<0.001 from baseline), whereas daytime BP was 133±5 mm Hg (not significantly different from baseline). The injection of AS-ODNs in the early afternoon prevented the expected BP rise during the next several hours, with the greatest effect apparent at 3 and 6 hours postinjection, when the rats receiving scrambled ODNs were already exhibiting a marked BP elevation. By 24 hours, there was no difference between the rats that had received AS-ODNs and those that had received the scrambled ODNs. The second injection of AS-ODNs or scrambled ODNs was given at that time and produced essentially the same effect as the first one. Figure 2 shows detailed individual BP recordings of 1 AS-ODN–injected and 1 scrambled ODN–injected rat.

View larger version (87K): [in this window] [in a new window]   Figure 2. Representative individual BP recordings during the entire experiment in 1 AS-ODN–treated rat (top) and 1 scrambled ODN–treated rat (bottom). BP is normal with minimal variability during the first 3 baseline days and starts increasing, with a large diurnal variation, after initiation of dietary salt loading with saline as drinking water. On days 6 and 7, the rats receive treatment with intracerebroventricular injection. There is a delayed rise and lower peak BP in the AS-ODN–treated rat (top), whereas BP progressively increased in the control rat (bottom).

Heart rate was not altered by salt loading but significantly decreased after AS-ODN injections and returned to control level by 24 hours (388±12.9 bpm at baseline, 385±11.9 bpm during salt loading, and 306±15.4 bpm at 3 to 6 hours postinjection; P<0.05 versus both control and salt-loading periods). The second AS-ODN injection had a similar effect on heart rate as the first injection. Scrambled ODN injections caused no significant changes in heart rates (402±6.0, 405±10.5, 384±14.9, and 388±11.7 bpm at baseline, salt loading, 3 to 6 hours postinjection, and 24 hours postinjection, respectively).

Behavioral Changes
Within 30 minutes from the AS-ODN injection, the rats displayed signs of sedation (ie, they were unable to right themselves for several seconds after being placed on their back, had difficulty raising their head from the floor of the cage, and appeared to lose balance). These behaviors peaked at 4 hours and wore off gradually. By the next day, the rats had fully recovered and again responded in the same manner to the second AS-ODN injection. The scrambled ODNs produced no behavioral changes.

FITC-Conjugated AS-ODN Distribution in the Brain
To confirm the location of AS-ODNs delivered through the cannula, FITC-conjugated AS-ODNs were injected as described in Methods. Fluorescent signal was detected in the brain areas adjacent to the third and fourth cerebral ventricles. The highest concentrations of the fluorescent signal were found in the tissues bordering the ventricles and the cerebellum. High concentrations of fluorescent deposits were noted in the nucleus tractus solitarii, locus ceruleus, dorsal raphe nucleus, ventral parabrachial nucleus, and central gray pons. The optic nerve also displayed a strong fluorescent signal. Figure 3 shows representative brain sections.

View larger version (118K): [in this window] [in a new window]   Figure 3. Photomicrographs of brain sections showing the distribution of FITC-conjugated AS-ODNs targeted to 2B-AR mRNA. Sections were examined by fluorescent microscope (magnification: A, B, and C x40; D x100). Lighter areas indicate intense fluorescence. A, Section of lateral ventricle. Top arrow indicates injection site; bottom arrow, lateral ventricle. There is no evidence of tissue damage, such as neuronal loss, to suggest nonspecific neurotoxicity. B, Ventral section of third ventricle and surrounding tissues with foci of high uptake of AS-ODNs. C, Fourth ventricle and surrounding tissues. D, Area of the nucleus tractus solitarii with foci of high uptake of AS-ODNs.

	Discussion

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In this study, we demonstrated that when AS-ODNs targeted to a complementary sequence of rat _2B-AR mRNA are injected directly into the CNS of rats, they produce a significant attenuation of salt-induced hypertension accompanied by a fall in heart rate. The specificity of this effect is shown by the fact that scrambled ODNs administered into the third cerebral ventricle of subtotally nephrectomized, salt-fed hypertensive rats in the same manner produced no changes in the BP or heart rate patterns. The data indicate that translational inhibition of the central _2B-AR can prevent the salt-induced BP rise in this typical experimental model of salt-dependent hypertension.

These results are consistent with previous studies by other investigators, who have shown in genetically engineered animals that the _2B-AR has a hypertensive function.^4,5 Furthermore, our recent studies showed that the induction of hypertension via short- or long-term salt loading requires a full complement of functional _2B-AR.^15,16 We also presented evidence that this is a function of presynaptic centrally located _2B-AR. Indeed, the only peripheral postsynaptic ₂-AR subtype capable of direct vasoconstriction in response to catecholamines (under ₁-AR blockade) is _2A-AR,²² and _2B-AR mRNA could not be detected in arterial wall tissues via in situ hybridization,^23,24 whereas it was abundantly present in CNS nuclei, along with mRNA for the other 2 ₂-AR subtypes.²⁵

Experimental gene treatment of hypertension has been attempted via various methods in recent years.²⁶ Approaches include the delivery of extra copies of candidate genes whose products contribute to vasodilation and the inhibition of expression of genes whose products contribute to vasoconstriction. Inhibition of gene expression can be achieved through the administration of AS-ODNs against a targeted mRNA sequence, either directly or via viral vectors. For the present study, we chose an AS-ODN sequence that was located downstream of the initiation site, because the region that encompasses the codon AUG of rat _2B-AR mRNA includes 4 consecutive C nucleotides and the corresponding antisense sequence would contain a G quartet, which may cause nonspecific toxic effects.²⁷ The specificity and efficacy of the AS-ODNs against rat _2B-AR mRNA were confirmed in vitro on NG108-15 cells, which are known to express the rat _2B-AR subtype.²⁸ Indeed, we demonstrated that when AS-ODNs were added to the culture medium of these cells, they resulted in markedly decreased production of _2B-AR protein in vitro. Therefore, the attenuation of BP rise in vivo represents a specific effect of inhibition of the expression of these receptors in the CNS areas adjacent to the site of intracerebroventricular injection, as shown by the fluorescent signal of AS-ODN distribution in those areas. Scrambled ODNs had no effect on _2B-AR protein production in vitro or the heart rate and BP pattern in vivo, arguing against a nonspecific effect. The lack of tissue damage around the ventricles suggests the absence of neurotoxicity.

Our results are at variance with those of Nunes,²⁹ who found no BP effect with the central administration of AS-ODNs directed to a different region of the rat _2B-AR transcript. Because he found a hypertensive effect from AS-ODN inhibition of the _2A-AR, he concluded that only the _2A-AR subtype has a role on central BP regulation, a conclusion that is contrary not only to our findings but also to those of others who have used genetically engineered mice and found a hypertensive influence of _2B-AR.^4,5

The magnitude of the antihypertensive action of our current approach is comparable to that achieved by other investigators who used AS-ODNs against other candidate genes. Examples include AS-ODNs directed against the mRNA of various components of the renin-angiotensin system, such as angiotensinogen, angiotensin II type 1 receptor, ACE, and the renin gene. The onset and duration of the BP-lowering effect vary and have been reported to occur within 3 to 9 hours and to last for 3 to 7 days.²⁶ It is unclear at this point whether the time frame is determined mainly by the kinetic characteristics of the targeted proteins or whether the mode of delivery (eg, choice of vector, encapsulation in liposomes, and so on) can enhance the efficiency or prolong the half-life of AS-ODNs. In the present study, the onset of behavioral changes occurred at 30 minutes after the injection of AS-ODNs and peaked at 4 hours, whereas the attenuation of BP rise was most apparent between 3 and 6 hours postinjection and lasted <24 hours. These observations imply a rather rapid turnover of _2B-AR. The half-life of the _2B-AR has not yet been fully elucidated and varies widely in different reports, with the shortest being 1.2 hours,³⁰ which is consistent with our findings. It is also possible that the turnover of _2B-AR differs among various tissues and even across areas of the CNS or that a more functionally active pool of receptors has a more rapid turnover rate.³¹

Interestingly, in addition to the antihypertensive effect, the CNS injection of AS-ODNs against _2B-AR mRNA produced symptoms of sedation and loss of equilibrium. The latter symptom is consistent with the fact that _2B-AR mRNA was found to be abundantly present in cerebellar structures.²⁵ The sedation was unexpected, because the sedative, anesthetic, and sympatholytic effects of pharmacological nonselective ₂-AR agonists have been generally attributed to the _2A-AR or _2C-AR subtype in studies with genetically engineered mice,^32,33 and these results have been corroborated by the use of AS-ODNs against _2A-AR.³⁴ _2B-AR is believed to have mainly a hypertensive role^5,15,16 and a poorly defined developmental function,³⁵ whereas _2C-AR has been implicated mostly in behavioral alterations.^33,35 However, the present findings indicate that it is difficult to separate the hemodynamic from the behavioral aspects of sympathetic interventions. The use of pharmacological agents with central ₂-AR agonistic properties, such as clonidine, for the treatment of hypertension is limited by side effects such as drowsiness that have been attributed to indiscriminate binding of these agents with all ₂-AR subtypes. It is, however, possible that this difficulty is not due to nonselectivity of the drugs but that it may indicate that sympathetic suppression per se is inevitably accompanied by such symptoms.

In conclusion, our data indicate that the inhibition of central _2B-AR gene expression through the specific targeting of mRNA with AS-ODNs injected into the lateral cerebral ventricle can prevent for several hours the expected BP rise in subtotally nephrectomized rats that underwent dietary salt loading, thus further corroborating the notion that a fully functional central _2B-AR is a necessary mediator of salt-induced hypertension. The specificity of this effect is shown by the fact that scrambled ODNs have no influence on BP or heart rate patterns.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grant P50-HL-55001.

Received April 5, 2001; first decision April 16, 2001; accepted May 7, 2001.

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